ALBERT

Description: The CRIPSR-Cas9 system consists of a site-specific, targetable DNA nuclease that holds great potential in gene editing and genome-wide screening applications. In order to apply the CRISPR-Cas9 system to these assays successfully, the rate at which Cas9 induces DNA breaks at undesired loci must be understood. We characterised the rate of Cas9 off-target activity in typical Cas9 experiments in two human and one mouse cell lines. We analysed the Cas9 cutting activity of 12 gRNAs in both their targeted sites and ∼90 predicted off-target sites per gRNA. In a Cas9-based knock-out experiment, gRNAs induced detectable Cas9 cutting activity in all on-target sites and in only a few off-target sites genome-wide in human 293FT, human iPS cells and mouse ES cells. Both the cutting rates and DNA repair patterns were highly correlated between the two human cell lines in both on-target and off-target sites. In clonal Cas9 cutting analysis in mouse ES cells, bi-allelic Cas9 cutting was observed with low off-target activity. Our results show that off-target activity of Cas9 is low and predictable by the degree of sequence identity between the gRNA and a potential off-target site. Off-target Cas9 activity can be minimized by selecting gRNAs with few off-target sites of near-complementarity. genesis 00:00–00, 2014. © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT The recent advances on ascidian pigment sensory organ development and function represent a fascinating platform to get insight on the basic programs of chordate eye formation. This review aims to summarize current knowledge, at the structural and molecular levels, on the two main building blocks of ascidian light sensory organs, i.e . pigment cells and photoreceptor cells. The unique features of these structures (e.g. simplicity and well characterized cell lineage) are indeed making it possible to dissect the developmental programs at single cell resolution and will soon provide a panel of molecular tools to be exploited for a deep developmental and comparative-evolutionary analysis. © 2014 Wiley Periodicals, Inc.

Description: Temporally controlled induction of gene expression is a useful technique for analyzing gene function. To make such a technique possible in Ciona intestinalis embryos, we employed the cis -regulatory region of the heat-shock protein 70 (HSP70) gene Ci-HSPA1/6/7-like for heat-inducible gene expression in C. intestinalis embryos. We showed that Ci-HSPA1/6/7-like becomes heat shock-inducible by the 32-cell stage during embryogenesis. The 5'-upstream region of Ci-HSPA1/6/7-like , which contains heat-shock elements indispensable for heat-inducible gene expression, induces the heat shock-dependent expression of a reporter gene in the whole embryo from the 32-cell to the middle gastrula stages and in progressively restricted areas of embryos in subsequent stages. We assessed the effects of heat-shock treatments in different conditions on the normality of embryos and induction of transgene expression. We evaluated the usefulness of this technique through overexpression experiments on the well-characterized, developmentally relevant gene, Ci-Bra , and showed that this technique is applicable for inferring the gene function in C. intestinalis . © 2014 Wiley Periodicals, Inc.

Description: Since the discovery of brain asymmetry in a wide range of vertebrate species, it has become possible to study development and expression of lateralized behavior accurately in well-controlled experiments. Several species have emerged as useful models for investigating aspects of lateralization. Discussed here are: (1) the influence of exposure to light during embryonic development on lateralization, (2) effects of steroid hormones on lateralization, (3) developmental changes in which hemisphere is controlling behavior, and (4) asymmetry in memory formation and recall. The findings have bearing on understanding the development of hemispheric specialization in humans and are likely to provide insight into dysfunctional behavior associated with weak or absent lateralization and impaired interhemispheric communication (e.g., autism, schizophrenia, dyslexia). This review features research on chicks, pigeons and zebrafish, with the addition of some recent evidence of lateralization in bees. Discoveries made using these species have highlighted the interaction between experience, hormones and genetic factors during development, and have provided some of the first clear evidence of the advantage of having a lateralized brain. © 2014 Wiley Periodicals, Inc.

Description: Summary : The radiate sunflower inflorescence is composed by zygomorphic ray flowers and actinomorphic disk flowers. Studies performed on mutants identify HaCYC2c , a CYCLOIDEA ( CYC ) -like gene, as one of the key players controlling flower symmetry in sunflower. turf and tub mutants are characterized by a shift from zygomorphic to actinomorphic ray flowers, caused by insertion of transposable elements (TEs) in HaCYC2c gene. In dbl or Chry mutants, an insertion upstream the coding region of HaCYC2c causes the ectopic expression of the gene and the shift from actinomorphic to zygomorphic disk flowers. We focused on Chry2 mutant: a 1034 bp insertion placed 558 bp before the start codon of HaCYC2c was identified. The insertion is a truncated version of a CACTA TE. Unexpectedly, phenotypic and genetic co-segregation analysis in F 2 and F 3 progenies derived from the crosses Chry2 × turf and turf × Chry2 demonstrated that CACTA insertion is not always sufficient to alter the expression of HaCYC2c gene and generate Chry2 phenotype. F 3 plants homozygous for the CACTA insertion displayed either HaCYC2c transcription pattern identical to wild type plants or a normal heterogamous inflorescence. Stated these results, we conclude that a much more complex regulatory system stays behind the Chry2 phenotype. © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Targeted mutagenesis of genes-of-interest, or gene- knockout, is a powerful method to address the functions of genes. Engineered nucleases have enabled this approach in various organisms because of their ease of use. The ascidian Ciona intestinalis is an excellent organism to analyze gene functions by means of genetic technologies. In our previous study, we reported mutagenesis of Ciona somatic cells with TALE nucleases ( TALENs ) by electroporating expression constructs. In this study, we report germ cell mutagenesis of Ciona by microinjecting mRNAs encoding TALENs. TALEN mRNAs introduced mutations to target genes in both somatic and germ cells. TALEN-mediated mutations in the germ cell genome were inherited by the next generation. We conclude that knockout lines of Ciona that have disrupted target genes can be established through TALEN-mediated germ cell mutagenesis. © 2014 Wiley Periodicals, Inc.

Description: As neural stem cells differentiate into neurons during neurogenesis, the proteome of the cells is restructured by de novo expression and selective removal of regulatory proteins. The control of neurogenesis at the level of gene regulation is well documented and the regulation of protein abundance through protein degradation via the Ubiquitin/26S proteasome pathway is a rapidly developing field. This review describes our current understanding of role of the proteasome pathway in neurogenesis. Collectively, the studies show that targeted protein degradation is an important regulatory mechanism in the generation of new neurons. © 2014 Wiley Periodicals, Inc.

Description: This review summarizes and integrates our current understanding of how sea stars make gametes. Although little is known of the mechanism of germ line formation in these animals, recent results point to specific cells and to cohorts of molecules in the embryos and larvae that may lay the ground work for future research efforts. A coelomic outpocketing forms in the posterior of the gut in larvae, referred to as the posterior enterocoel (PE), that when removed, significantly reduces the number of germ cell later in larval growth. This same PE structure also selectively accumulates several germ-line associated factors – vasa, nanos, piwi – and excludes factors involved in somatic cell fate. Since its formation is relatively late in development, these germ cells may form by inductive mechanisms. When integrated into the morphological observations of germ cells and gonad development in larvae, juveniles, and adults, the field of germ line determination appears to have a good model system to study inductive germ line determination to complement the recent work on the molecular mechanisms in mice. We hope this review will also guide investigators interested in germ line determination and regulation of the germ line in how these animals can help in this research field. The review is not intended to be comprehensive – sea star reproduction has been studied over 100 years and many reviews are comprehensive in their coverage of, for example, seasonal growth of the gonads in response to light, nutrient, and temperature. Rather the intent of this review is to help the reader focus on new experimental results attached to the historical underpinnings of how the germ cell functions in sea stars with particular emphasis to clarify the important areas of priority for future research. © 2014 Wiley Periodicals, Inc.

Description: Insufficiency of surfactants is a core factor in respiratory distress syndrome (RDS), which causes apnea and neonatal death, particularly in preterm infants. Surfactant proteins are secreted by alveolar type II cells in the lung epithelium, the differentiation of which is regulated by Fgf10 elaborated by the adjacent mesenchyme. However, the molecular regulation of mesenchymal Fgf10 during lung development has not been fully understood. Here we show that Pbx1, a homeodomain transcription factor, is required in the lung mesenchyme for the expression of Fgf10 . Mouse embryos lacking Pbx1 in the lung mesenchyme show compact terminal saccules and perinatal lethality with failure of postnatal alveolar expansion. Mutant embryos had severely reduced expression of Fgf10 and surfactant genes ( Spa , Spb , Spc , and Spd ) that are essential for alveolar expansion for gas exchange at birth. Molecularly, Pbx1 directly binds to the Fgf10 promoter and cooperates with Meis and Hox proteins to transcriptionally activate Fgf10 . Our results thus show how Pbx1 controls Fgf10 in the developing lung. © 2014 Wiley Periodicals, Inc.

Description: Drosophila is a classical model to study body patterning, however Left-Right (L/R) asymmetry had remained unexplored, until recently. The discovery of the conserved myosin ID gene as a major determinant of L/R asymmetry has revealed a novel L/R pathway involving the actin cytoskeleton and the adherens junction. In this process, the HOX gene Abdominal-B plays a major role through the control of myosin ID expression and therefore symmetry breaking. In this review, we present organs and markers showing L/R asymmetry in Drosophila and discuss our current understanding of the underlying molecular genetic mechanisms. Drosophila represents a valuable model system revealing novel strategies to establish L/R asymmetry in invertebrates and providing an evolutionary perspective to the problem of laterality in bilateria. © 2014 Wiley Periodicals, Inc.

Description: Magoh encodes a core component of the exon junction complex (EJC), which binds mRNA and regulates mRNA metabolism. Magoh is highly expressed in proliferative tissues during development. EJC components have been implicated in several developmental disorders including TAR syndrome, Richieri-Costa-Pereira Syndrome, and intellectual disability. Existing germline null Magoh mice are embryonic lethal as homozygotes and perinatal lethal as heterozygotes, precluding detailed analysis of embryonic and postnatal functions. Here we report the generation of a new genetic tool to dissect temporal and tissue specific roles for Magoh in development and adult homeostasis. This Magoh conditional allele has two LoxP sites flanking the second exon. Ubiquitous Cre-mediated deletion of the floxed allele in a heterozygous mouse ( Magoh del /+ ) causes 50% reduction of both Magoh mRNA and protein. Magoh del /+ mice exhibit both microcephaly and hypopigmentation, thus phenocopying germline haploinsufficient Magoh mice. Using Emx1 - Cre , we further show that conditional Magoh deletion in neural progenitors during embryonic development also causes microcephaly. We anticipate this novel conditional allele will be a valuable tool for assessing tissue specific roles for Magoh in mammalian development and postnatal processes. © 2014 Wiley Periodicals, Inc.

Description: Anatomical left-right (L/R) handedness asymmetry in C. elegans is established in the four-cell embryo as a result of anteroposterior skewing of transverse mitotic spindles with a defined handedness. This event creates a chiral embryo and ultimately an adult body plan with fixed L/R positioning of internal organs and components of the nervous system. While this “dextral” configuration is invariant in hermaphrodites, it can be reversed by physical manipulation of the early embryo or by mutations that interfere with mitotic spindle orientation, which leads to viable, mirror-reversed (sinistral) animals. During normal development of the C. elegans male, the gonad develops on the right of the midline, with the gut bilaterally apposed on the left. However, we found that in males of the laboratory N2 strain and Hawaiian (“Hw”) wild isolate, the gut/gonad asymmetry is frequently reversed in a temperature-dependent manner, independent of normal embryonic chirality. We also observed sporadic errors in gonad migration occurring naturally during early larval stages of these and other wild strains; however, the incidence of such errors does not correlate with the frequency of L/R gut/gonad reversals in these strains. Analysis of N2/Hw hybrids and recombinant inbred advanced intercross lines (RIAILs) indicate that the L/R organ reversals are likely to result from recessively acting variations in multiple genes. Thus, unlike the highly reproducible L/R asymmetries of most structures in hermaphrodites, the L/R asymmetry of the male C. elegans body plan is less rigidly determined and subject to natural variation that is influenced by a multiplicity of genes. © 2014 Wiley Periodicals, Inc.

Description: The primitive node is the ‘hub' of early left-right patterning in the chick embryo: (1) it undergoes asymmetrical morphogenesis immediately after its appearance at stage 4; (2) it is closely linked to the emerging asymmetrical expression of nodal and shh at stage 5; and (3) its asymmetry is spatio-temporally related to the emerging notochord, the midline barrier maintaining molecular left-right patterning from stage 6 onwards. Here, we study the correlation of node asymmetry to notochord marker expression using high-resolution histology, and we test pharmacological inhibition of shh signaling using cyclopamine at stages 4 and 5. Just as noggin expression mirrors an intriguing structural continuity between the right node shoulder and the notochord, shh expression in the left node shoulder confirms a similar continuity with the future floor plate. Shh inhibition at stage 4 or 5 suppressed nodal in both its paraxial or lateral plate mesoderm domains, respectively, and resulted in randomized heart looping. Thus, the ‘primordial' paraxial nodal asymmetry at stage 4/5 (1) appears to be dependent on, but not instructed by, shh signaling, and (2) may be fixed by asymmetrical roots of the notochord and the floor plate, thereby adding further twists to the node's pivotal role during left-right patterning. © 2014 Wiley Periodicals, Inc.

Description: One of the central concerns of Evolutionary Developmental biology is to understand how the specification of cell types can change during evolution. In the last decade, developmental biology has progressed towards a systems level understanding of cell specification processes. In particular, the focus has been on determining the regulatory interactions of the repertoire of genes that make up gene regulatory networks (GRNs). Echinoderms provide an extraordinary model system for determining how GRNs evolve. This review highlights the comparative GRN analyses arising from the echinoderm system. This work shows that certain types of GRN sub-circuits or motifs, i.e. those involving positive feedback, tend to be conserved and may provide a constraint on development. This conservation may be due to a required arrangement of transcription factor binding sites in cis regulatory modules. The review will also discuss ways in which novelty may arise, in particular through the co-option of regulatory genes and sub-circuits. The development of the sea urchin larval skeleton, a novel feature that arose in echinoderms, has provided a model for study of co-option mechanisms. Finally, the types of GRNs that can permit the great diversity in the patterns of ciliary bands and their associated neurons found among these taxa are discussed. The availability of genomic resources is rapidly expanding for echinoderms, including genome sequences not only for multiple species of sea urchins, but also a species of sea star, sea cucumber, and brittle star. This will enable echinoderms to become a particularly powerful system for understanding how developmental GRNs evolve. © 2014 Wiley Periodicals, Inc.

Description: Photodynamic therapy (PDT) utilizes light, a photosensitizer, and molecular oxygen as a treatment modality for a variety of cancers. We have recently combined ruthenium(II) polypyridyl groups with a zinc(II) centered porphyrin as a new photosensitizer for the treatment of melanoma. In-vitro studies have indicated that this photosensitizer is toxic to melanoma cells when irradiated with low energy light; however, it is non-toxic to normal cells under similar conditions. To determine the toxicity and cell viability of this compound in-vivo we present herein a study using Drosophila melanogaster. In the absence of light the new photosensitizer shows no discernible effects to fly larvae at various concentrations of compound and stages of larval development. When the larvae were fed the photosensitizer it was observed, by fluorescence microscopy, that the compound passes through the cell membrane and localizes in the cytosol at lower concentrations and the nucleus at slightly higher concentrations indicating that the compound is not immediately metabolized. © 2014 Wiley Periodicals, Inc.

Description: Directional Left/Right (LR) asymmetries, in which there are consistent, heritable differences in morphology between the left and right sides of bilaterally symmetrical organisms, are found in animals across the Bilateria. For many years we have lacked evidence for shared mechanisms underlying their development. This led to the supposition that the mechanisms driving establishment of LR asymmetries, and consequently the asymmetries themselves, had evolved separately in the three major Superphyla that constitute the Bilateria. The recent discovery that the TGFβ ligand Nodal plays a role in the regulation of LR asymmetry in both Deuterostomia and Lophotrochozoa has reignited debate in this field, as it suggests that at least this aspect of the development of the LR axis is conserved. In this review, we discuss evidence for shared mechanisms of LR asymmetry establishment across the bilaterian tree of life, and consider how these mechanisms might have diverged across the Metazoa over the last 500 million years or so of evolution. As well as the likelihood that Nodal is an ancestral mechanism for regulating LR asymmetry, we reemphasize cytoskeletal architecture as a potential shared mechanism underlying symmetry breaking. However convergent evolution remains a distinct possibility and study of a wider diversity of species will be needed to distinguish between conserved and lineage specific mechanisms. © 2014 Wiley Periodicals, Inc.

Description: The evolution of various body plans results from the acquisition of novel structures, as well as the loss of existing structures. Some novel structures necessitate multiple evolutionary steps, requiring organisms to overcome the intermediate steps, which might be less adaptive or neutral. To examine this issue, echinoderms might provide an ideal experimental system. A larval skeleton is acquired in some echinoderm lineages, such as sea urchins, probably via the co-option of the skeletogenic machinery that was already established to produce the adult skeleton. The acquisition of a larval skeleton was found to require multiple steps and so provides a model experimental system for reproducing intermediate evolutionary stages. The fact that echinoderm embryology has been studied with various natural populations also presents an advantage. © 2014 Wiley Periodicals, Inc.

Description: It is a challenge to understand how the information encoded in DNA is used to build a three dimensional structure. To explore how this works the assembly of a relatively simple skeleton has been examined at multiple control levels. The skeleton of the sea urchin embryo consists of a number of calcite rods produced by 64 skeletogenic cells. The ectoderm supplies spatial cues for patterning, essentially telling the skeletogenic cells where to position themselves and providing the factors for skeletal growth. Here we describe the information known about how this works. First the ectoderm must be patterned so that the signaling cues are released from precise positions. The skeletogenic cells respond by initiating skeletogenesis immediately beneath two regions (one on the right and the other on the left side). Growth of the skeletal rods requires additional signaling from defined ectodermal locations, and the skeletogenic cells respond to produce a membrane-bound template in which the calcite crystal grows. Important in this process are three signals, FGF, VEGF, and Wnt5. Each is necessary for explicit tasks in skeleton production. © 2014 Wiley Periodicals, Inc.

Description: Senescence-associated β-galactosidase (SA-β-gal) activity is widely used as a marker of cellular senescence and as an indicator of organismal aging. Here we report that SA-β-gal activity is present in the visceral endoderm layer of early post-implantation mouse embryos in predictable patterns that vary as the embryo progresses in development. However, determination of the mitotic index and analysis of the expression of Cdkn1a (p21), a marker of senescent cells, do not indicate cellular senescence. Instead, analysis of embryos in culture revealed the presence of SA-β-gal activity in apical vacuoles of visceral endoderm cells likely a reflection of acidic β-galactosidase function in these organelles. This feature serves as a practical marker of the dynamics of the visceral endoderm that can be applied to developmental as well as functional studies of early mammalian embryos. © 2014 Wiley Periodicals, Inc.

Description: Many different types of molecules have essential roles in patterning the left-right axis and directing asymmetric morphogenesis. In particular, the relationship between signalling molecules and transcription factors has been explored extensively. Another group of proteins implicated in left-right patterning are components of the extracellular matrix, apical junctions and cilia. These structural molecules have the potential to participate in the conversion of morphogenetic cues from the extracellular environment into morphogenetic patterning via their interactions with the actin cytoskeleton. While it has been relatively easy to temporally position these proteins within the hierarchy of the left-right patterning pathway, it has been more difficult to define how they mechanistically fit into these pathways. Consequently, our understanding of how these factors impart patterning information to influence the establishment of the left-right axis remains limited. In this review we will discuss those structural molecules that have been implicated in early phases of left-right axis development. © 2014 Wiley Periodicals, Inc.

Description: Epithelial morphogenesis is an essential process in all metazoans during both normal development and pathological processes such as wound healing. The coordinated regulation of cell shape, cell size, and cell adhesion during the migration of epithelial sheets ultimately gives rise to the diversity of body plans among different organisms as well as the diversity of cellular structures and tissues within an organism. Metamorphosis of the Drosophila pupa is an excellent system to study these transformative events. During pupal development the cells of the wing imaginal discs migrate dorsally and fuse to form the adult thorax. Here I show centralspindlin, a protein complex well known for its role in cytokinesis, is essential for migration of wing disc cells and proper thorax closure. I show the subcellular localization of centralspindlin is important for its function in thorax development. This study demonstrates the emerging role of centralspindlin in regulating cell migration and cell adhesion in addition to its previously known function during cytokinesis. © 2014 Wiley Periodicals, Inc.

Description: Motile cilia are an essential component of the mouse, zebrafish and Xenopus laevis Left Right Organisers, generating nodal flow and allowing the reception and transduction of mechanosensory signals. Non-motile primary cilia are also an important component of the Left Right Organiser's chemosensory mechanism. It has been proposed in the chicken that signalling in Hensen's node, the Left Right Organiser of the chicken, is independent of cilia, based on a lack of evidence of motile cilia or nodal flow. It is speculated that the talpid 3 chicken mutant, which has normal left-right patterning despite lacking cilia at many stages of development, is proof of this hypothesis. Here we examine the evidence for cilia in Hensen's node and find that although cilia are present, they are likely to be immotile and incapable of generating nodal flow. Furthermore we find that early planar cell polarity patterning and ciliogenesis is normal in early talpid 3 chicken embryos. We conclude that patterning and development of the early talpid 3 chicken is normal, but not necessarily independent of cilia. While it appears that Hensen's node does not require motile cilia or the generation of motile flow, there may remain a requirement for cilia in the transduction of SHH signalling. Results • FOXJ1 is expressed at low levels in the chicken node incompatible with motile cilia generation • Short cilia are present in the mesodermal cells of the chicken node • Talpid 3 chicken embryos have normal VANGL2 localisation early in development • Talpid 3 chicken embryos have primary cilia early in development © 2014 Wiley Periodicals, Inc.

Description: The transforming acidic coiled-coil containing protein 2 ( Tacc2) gene and its paralogs, Tacc1 and Tacc3 encode proteins that are associated with the centrosome and involved in microtubule assembly during the cell cycle. Tacc2 produces several splice variants, which are poorly characterized, especially in the rat. Characterization of the temporal/spatial expression patterns of these isoforms would be useful in understanding their distinct and over-lapping functions. By comparative sequence analyses of Tacc2 in multiple species, we identified a third splice variant in rat, which is much shorter in size (1021 aa) than the longest isoform (2834 aa). This newly identified Tacc2 splice variant (isoform 3) uses a distinct first exon and generates a different Open Reading Frame (ORF). While isoform 3 is expressed predominantly during developmental stages, the long Tacc2 isoform (isoform 1) is distributed mainly in adult tissues. Multiple protein sequence analyses revealed that Tacc2 isoform 3 could be the ancient form, as it is conserved in mammals, birds and amphibians; whereas the long Tacc2 isoforms may have evolved in the mammalian lineage by adding exons towards the 5’ region of the ancient isoform. © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT The spleen is a lymphoid organ that serves as a unique niche for immune reactions, extramedullary hematopoiesis, and the removal of aged erythrocytes form the circulation. While much is known about the immunological functions of the spleen, the mechanisms governing the development and organization of its stromal microenvironment remain poorly understood. Here we report the generation and analysis of a Tlx1 CreER-Venus knock-in mouse strain engineered to simultaneously express tamoxifen-inducible CreER T2 and Venus fluorescent protein under the control of regulatory elements of the Tlx1 gene, which encodes a transcription factor essential for spleen development. We demonstrated that Venus as well as CreER expression recapitulates endogenous Tlx1 transcription within the spleen microenvironment. When Tlx1 CreER-Venus mice were crossed with the Cre-inducible reporter strain, Tlx1 -expressing cells as well as their descendants were specifically labelled following tamoxifen administration. We also showed by cell lineage tracing that asplenia caused by Tlx1 deficiency is attributable to altered contribution of mesenchymal cells in the spleen anlage to the pancreatic mesenchyme. Thus, Tlx1 CreER-Venus mice represent a new tool for lineage tracing and conditional gene manipulation of spleen mesenchymal cells, essential approaches for understanding the molecular mechanisms of spleen development. © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Transgenic mice were generated using a heat shock protein 2 ( Hspa2 ) gene promoter to express green fluorescent protein (GFP) at the beginning of meiotic prophase I in spermatocytes. Expression was confirmed in four lines by in situ fluorescence, immunohistochemistry, western blotting, and PCR assays. The expression and distribution of the GFP and HSPA2 proteins co-localized in spermatocytes and spermatids in three lines, but GFP expression was variegated in one line (F46), being present in some clones of meiotic and post-meiotic germ cells and not in others. Fluorescence-activated cell-sorting (FACS) was used to isolate purified populations of spermatocytes and spermatids. Although bisulfite sequencing revealed differences in the DNA methylation patterns in the promoter regions of the transgene of the variegated expressing GFP line, a uniformly expressing GFP reporter line, and the Hspa2 gene, these differences did not correlate with variegated expression. The Hspa2 -GFP reporter mice provide a novel tool for studies of meiosis by allowing detection of GFP in situ and in isolated spermatogenic cells. They will allow sorting of meiotic and post-meiotic germ cells for characterization of molecular features and correlation of expression of GFP with stage-specific spermatogenic cell proteins and developmental events. © 2014 Wiley Periodicals, Inc.

Description: The molecular events that control cell fate determination in cardiac and smooth muscle lineages remain elusive. Myocardin is an important transcription co-factor that regulates cell proliferation, differentiation and development of the cardiovascular system. Here, we describe the construction and analysis of a dual Cre and Enhanced Green Fluorescent Protein ( EGFP ) knock-in mouse line in the Myocardin locus ( Myocd KI ). We report that the Myocd KI allele expresses the Cre enzyme and the EGFP in a manner that recapitulates endogenous Myocardin expression patterns. We show that Myocardin expression marks the earliest cardiac and smooth muscle lineages. Furthermore, this genetic model allows for the identification of a cardiac cell population which maintains both Myocardin and Isl-1 expression, in E7.75 - E8.0 embryos, highlighting the contributions and merge of the first and second heart fields during cardiogenesis. Therefore, the Myocd KI allele is a unique tool for studying cardiovascular development and lineage-specific gene manipulation. © 2014 Wiley Periodicals, Inc.

Description: Meis1 is a highly conserved transcription factor that is activated in a regionally restricted manner from early stages of development. Meis1 belongs to the three amino acid loop extension (TALE) homeodomain family. Together with Pbx1, Meis1 plays a major role as a Hox cofactor and therefore plays an essential role in the development of several embryonic organs and systems, including limbs, heart, blood and vasculature. In addition Meis1 is required for the development of Hox-free embryonic regions and interacts with non-Hox homeodomain and non-homeodomain transcription factors. During postnatal life Meis1 is involved in adult cardiomyocyte homeostasis and has been associated with predisposition to human neural and cardiac pathologies. Given the relevance of this transcription factor, we have developed two new Meis1 gene knockin models; a direct ECFP knockin insertion that allows the direct identification of Meis1 -expressing cells in living tissues, and a CreERT2 insertion that allows the inducible genetic tracing of Meis1 -expressing cells in a time-controlled manner. Importantly, these two alleles represent the first Meis1 mutations in which Meis1 protein production is completely eliminated. These newly targeted Meis1 alleles will be valuable tools to further our understanding of the role of this critical transcription factor during development and disease. © 2014 Wiley Periodicals, Inc.

Description: Insulin plays an extensively characterized role in the control of sugar metabolism, growth and homeostasis in a wide range of organisms. In vertebrate chordates, insulin is mainly produced by the beta cells of the endocrine pancreas, while in non-chordate animals insulin-producing cells are mainly found in the nervous system and/or scattered along the digestive tract. However, recent studies have indicated the notochord, the defining feature of the chordate phylum, as an additional site of expression of insulin-like peptides. Here we show that two of the three insulin-like genes identified in Ciona intestinalis , an invertebrate chordate with a dual life cycle, are first expressed in the developing notochord during embryogenesis and transition to distinct areas of the adult digestive tract after metamorphosis. In addition, we present data suggesting that the transcription factor Ciona Brachyury is involved in the control of notochord expression of at least one of these genes, Ciona insulin-like 2 . Lastly, we review the information currently available on insulin-producing cells in ascidians and on pancreas-related transcription factors that might control their expression. genesis 00:00–00, 2014. © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Focal Adhesion Kinase is a non-receptor protein tyrosine kinase with signalling functions downstream of integrins and growth factor receptors. In addition to its role in adhesion, migration and proliferation it also has non-kinase scaffolding functions in the nucleus. FAK activation involves the following: (1) ligand bound growth factors or clustered integrins activate FAK kinase domain; (2) FAK autophosphorylates Tyrosine (Y) 397; (3) Src binds pY397 and phosphorylates FAK at various other sites including Y861; (4) downstream signalling of activated FAK elicits changes in cellular behaviour. Although many studies have demonstrated roles for the kinase domain, Y397 and Y861 sites in vitro much less is known about their functions in vivo . Here we report the generation of a series of FAK-mutant knockin mice where mutant FAK, either kinase dead (KD), non-phosphorylatable mutants Y397F and Y861F or mutant Y397E – containing a phosphomimetic site that results in a constitutive active Y397, can be expressed in a Cre inducible fashion driven by the ROSA26 promoter. In future studies, intercrossing these mice with FAKflox/flox mice and inducible cre-expressing mice will enable the in vivo study of mutant FAK function in the absence of endogenous FAK in a spatially and temporally regulated fashion within the whole organism. © 2014 Wiley Periodicals, Inc.

Description: The phox2b gene encodes a transcription factor that is expressed in the developing enteric nervous system (ENS). An enhancer element has been identified in the zebrafish phox2b locus that can drive tissue specific expression of reporter genes in enteric neuron precursor cells. We have generated a transgenic zebrafish line in which the Kaede fluorescent protein is under the control of this phox2b enhancer. This line has stable expression of the Kaede protein in enteric neuron precursor cells over 3 generations. To demonstrate the utility of this line we compared the migration and division rates of enteric neuron precursor cells in wild type and the zebrafish ENS mutant lessen . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT The Hairy-related transcription factor family of Notch- and ALK1-downstream transcriptional repressors, called Hrt/Hey/Hesr/Chf/Herp/Gridlock, has complementary and indispensable functions for vascular development. While mouse embryos null for either Hrt1/Hey1 or Hrt2/Hey2 did not show early vascular phenotypes, Hrt1/Hey1; Hrt2/Hey2 double null mice (H1 ko /H2 ko ) showed embryonic lethality with severe impairment of vascular morphogenesis. It remained unclear, however, whether Hrt/Hey functions are required in endothelial cells or vascular smooth muscle cells. In this study, we demonstrate that mice with endothelial-specific deletion of Hrt2/Hey2 combined with global Hrt1/Hey1 deletion (H1 ko /H2 eko ) show abnormal vascular morphogenesis and embryonic lethality. Their defects were characterized by the failure of vascular network formation in the yolk sac, abnormalities of embryonic vascular structures and impaired smooth muscle cell recruitment, and were virtually identical to the H1 ko /H2 ko phenotypes. Among signaling molecules implicated in vascular development, Robo4 expression was significantly increased and activation of Src family kinases was suppressed in endothelial cells of H1 ko /H2 eko embryos. The present study indicates an important role of Hrt1/Hey1 and Hrt2/Hey2 in endothelial cells during early vascular development, and further suggests involvement of Robo4 and Src family kinases in the mechanisms of embryonic vascular defects caused by the Hrt/Hey deficiency. © 2014 Wiley Periodicals, Inc.

Description: Developmental biology relies heavily on microscopy to image the finely-controlled cell behaviors that drive embryonic development. Most embryos are large enough that a field of view with the resolution and magnification needed to resolve single cells will not span more than a small region of the embryo. Ascidian embryos, however, are sufficiently small that they can be imaged in toto with fine subcellular detail using conventional microscopes and objectives. Unlike other model organisms with particularly small embryos, ascidians have a chordate embryonic body plan that includes a notochord, hollow dorsal neural tube, heart primordium and numerous other anatomical details conserved with the vertebrates. Here we compare the size and anatomy of ascidian embryos with those of more traditional model organisms, and relate these features to the capabilities of both conventional and exotic imaging methods. We review the emergence of Ciona and related ascidian species as model organisms for a new era of image-based developmental systems biology. We conclude by discussing some important challenges in ascidian imaging and image analysis that remain to be solved. © 2014 Wiley Periodicals, Inc.

Description: Chromatin remodeling via incorporation of histone variants plays a key role in the regulation of embryonic development. The histone variant H3.3 has been associated with a number of early events including formation of the paternal pronucleus upon fertilization. The small number of amino acid differences between H3.3 and its canonical counterparts (H3.1 and H3.2) has limited studies of the developmental significance of H3.3 deposition into chromatin due to difficulties in distinguishing the H3 isoforms. To this end, we used zinc-finger nuclease (ZFN) mediated gene editing to introduce a small C-terminal hemagglutinin (HA) tag to the endogenous H3.3B locus in mouse embryonic stem cells (ESCs), along with an internal ribosome entry site (IRES) and a separately translated fluorescent reporter of expression. This system will allow detection of expression driven by the reporter in cells, animals, and embryos, and will facilitate investigation of differential roles of paternal and maternal H3.3 protein during embryogenesis that would not be possible using variant-specific antibodies. Further, the ability to monitor endogenous H3.3 protein in various cell lineages will enhance our understanding of the dynamics of this histone variant over the course of development. © 2014 Wiley Periodicals, Inc.

Description: Homologous recombination in embryonic stem cells (ESCs) is widely utilized in genome engineering, particularly in the generation of gene targeted mice. However, genome engineering is often plagued by the problem of low homologous recombination efficiency. In this study, we developed a novel method to increase the efficiency of homologous recombination in ESCs by changing its culture conditions. By comparing the efficiency of different ESCs in various culture conditions, we determined that chemicals that inhibit the MEK and GSK3β pathways (2i condition) enhance homologous recombination and eliminate differences in efficiencies among cell lines. Analysis of gene expression patterns in ESCs maintained in different culture conditions has identified several homologous recombination-related candidates, including the pluripotent markers Eras and Tbx3. The results of this study suggest that homologous recombination is associated with ESC pluripotency . © 2014 Wiley Periodicals, Inc.

Description: As a group closely related to chordates, hemichordate acorn worms are in a key phylogenic position for addressing hypotheses of chordate origins. The stomochord of acorn worms is an anterior outgrowth of the pharynx endoderm into the proboscis. In 1886 Bateson proposed homology of this organ to the chordate notochord, crowning this animal group “hemichordates”. Although this proposal has been debated for over a century, the question still remains unresolved. Here we review recent progress related to this question. First, the developmental mode of the stomochord completely differs from that of the notochord. Second, comparison of expression profiles of genes including Brachyury , a key regulator of notochord formation in chordates, does not support the stomochord/notochord homology. Third, FoxE that is expressed in the stomochord-forming region in acorn worm juveniles is expressed in the club-shaped gland and in the endostyle of amphioxus, in the endostyle of ascidians, and in the thyroid gland of vertebrates. Based on these findings, together with the anterior endodermal location of the stomochord, we propose that the stomochord has evolutionary relatedness to chordate organs deriving from the anterior pharynx rather than to the notochord. © 2014 Wiley Periodicals, Inc.

Description: We report the generation of five mouse strains with the tamoxifen-inducible Cre ( Cre-ER T2 ; CE ) gene cassette knocked into the endogenous loci of Pax3 , Myod1 , Myog , Myf6 , and Myl1 , collectively as a resource for the skeletal muscle research community. We characterized these CE strains using the Cre reporter mice, R26R LacZ , during embryogenesis and show that they direct tightly controlled tamoxifen-inducible reporter expression within the expected cell lineage determined by each myogenic gene. We also examined a few selected adult skeletal muscle groups for tamoxifen-inducible reporter expression. None of these new CE alleles direct reporter expression in the cardiac muscle. All these alleles follow the same knock-in strategy by replacing the first exon of each gene with the CE cassette, rendering them null alleles of the endogenous gene. Advantages and disadvantages of this design are discussed. While we describe potential immediate use of these strains, their utility likely extends beyond foreseeable questions in skeletal muscle biology. © 2014 Wiley Periodicals, Inc.

Description: The genome sequencing and the development of RNAi knockdown technologies in the urochordate Oikopleura dioica are making this organism an attractive emergent model in the field of EvoDevo. To succeed as a new animal model, however, an organism needs to be easily and affordably cultured in the laboratory. Nowadays, there are only two facilities in the world capable to indefinitely maintain Oikopleura dioica , one in the SARS institute (Bergen, Norway) and the other in the Osaka University (Japan). Here, we describe the setup of a new facility in the University of Barcelona (Spain), in which we have modified previously published husbandry protocols to optimize the weekly production of thousands of embryos and hundreds of mature animals using the minimum amount of space, human resources and technical equipment. This optimization includes novel protocols of cryopreservation and solid cultures for long-term maintenance of microalgal stocks - Chaetoceros calcitrans , Isochrysis sp. , Rhinomonas reticulata and Synechococcus sp.- needed for Oikopleura dioica feeding. Our culture system maintains laboratory partially inbred lines healthy with similar characteristics to wild animals, and it is easily expandable to satisfy on demand the needs of any laboratory that may wish to use Oikopleura dioica as a model organism. © 2014 Wiley Periodicals, Inc.

Description: Urchin embryos continue to prove useful as a means of studying embryonic signaling and gene regulatory networks, which together control early development. Recent progress in understanding the molecular mechanisms underlying the patterning of ectoderm has renewed interest in urchin neurogenesis. We have employed an emerging model of neurogenesis that appears to be broadly shared by metazoans as a framework for this review. We use the model to provide context and summarize what is known about neurogenesis in urchin embryos. We review morphological features of the differentiation phase of neurogenesis and summarize current understanding of neural specification and regulation of proneural networks. Delta-Notch signaling is a common feature of metazoan neurogenesis that produces committed progenitors and it appears to be a critical phase of neurogenesis in urchin embryos. Descriptions of the differentiation phase of neurogenesis indicate a stereotypic sequence of neural differentiation and patterns of axonal growth. Features of neural differentiation are consistent with localized signals guiding growth cones with trophic, adhesive, and tropic cues. Urchins are a facile, post-genomic model with the potential of revealing many shared and derived features of deuterostome neurogenesis. © 2014 Wiley Periodicals, Inc.

Description: The transcription factor Foxj1 is expressed by cells destined to differentiate into epithelial cells projecting motile cilia into fluid- or air- filled cavities. Here we report the generation of an inducible knock-in Foxj1 CreERT2::GFP mouse which we show reliably induces Cre-mediated recombination for genetic studies in epithelial cells with motile cilia throughout embryonic and postnatal development. Induction during embryonic stages revealed efficient recombination in the epithelial component of the choroid plexus in the developing brain as early as E12.5. Induction during late embryonic stages showed confined recombination not only in the choroid plexus, but also in the ventricular walls of the brain. Recombination induced during postnatal periods expanded to include epithelia of the lungs, testis, oviduct, and brain. Using these mice, we confirmed our recent discovery of a perinatally derived neuronal population in the mouse olfactory bulbs which is derived from the Foxj1 lineage. Our Foxj1 CreERT2::GFP knock-in mouse will be a powerful tool for studying molecular mechanisms associated with the continuum of cells that form the Foxj1 lineage, and for assessing their physiological significance during development and aging. © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT WNT signaling is, in all multicellular animals, an essential intercellular communication pathway that is critical for shaping the embryo. At the molecular level, WNT signals can be transmitted by several transduction cascades, all activated commonly by the binding of WNT ligands to receptors of the FRIZZLED family. The first step in assessing the biological functions of WNT signaling during embryogenesis is thus the establishment of the spatiotemporal expression profiles of wnt and frizzled genes during development. To this end, using qPCR, Northern blot and in situ hybridization assays, we report here the comprehensive expression patterns of all eleven wnt and four frizzled genes present in the genome of the sea urchin Paracentrotus lividus throughout its embryogenesis. Our findings indicate that the expression of these wnt ligands and frizzled receptors is highly dynamic in both time and space. We further establish that all wnt genes are chiefly transcribed in the vegetal hemisphere of the embryo, whereas expression of the frizzled genes is distributed more widely across the embryonic territories. Thus, in P. lividus , WNT ligands might act both as short- and long-range signaling molecules that may operate in all cell lineages and tissues to control various developmental processes throughout embryogenesis. © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Migration and anchorage of nuclei within developing and adult tissues rely on Linkers of the Nucleoskeleton to the Cytoskeleton (LINC complexes). These macromolecular assemblies span the nuclear envelope and physically couple chromatin and nuclear lamina to cytoplasmic cytoskeletal networks. LINC complexes assemble within the perinuclear space through direct interactions between the respective evolutionary-conserved SUN and KASH domains of Sun proteins, which reside within the inner nuclear membrane, and Nesprins, which reside within the outer nuclear membrane. Here, we describe and validate a dominant-negative transgenic strategy allowing for the disruption of endogenous SUN/KASH interactions through the inducible expression of a recombinant KASH domain. Our approach, which is based on the Cre/Lox system, allows for the targeted disruption of LINC complexes in a wide array of mouse tissues or specific cell types thereof and bypasses the perinatal lethality and potential cell nonautonomous effects of current mouse models based on germline inactivation of genes encoding Sun proteins and Nesprins. For these reasons, this mouse model provides a useful tool to evaluate the physiological relevance of LINC complexes integrity during development and homeostasis in a wide array of mammalian tissues. © 2014 Wiley Periodicals, Inc.

Description: Myelinogenesis is a complex process that involves substantial and dynamic changes in plasma membrane architecture and myelin interaction with axons. Highly ramified processes of oligodendrocytes in the central nervous system (CNS) make axonal contact and then extrapolate to wrap around axons and form multilayer compact myelin sheathes. Currently, the mechanisms governing myelin sheath assembly and axon selection by myelinating cells are not fully understood. Here, we generated a transgenic mouse line expressing the membrane-anchored green fluorescent protein (mEGFP) in myelinating cells, which allow live imaging of details of myelinogenesis and cellular behaviors in the nervous systems. mEGFP expression is driven by the promoter of 2'-3'-cyclic nucleotide 3'-phosphodiesterase (CNP) that is expressed in the myelinating cell lineage. Robust mEGFP signals appear in the membrane processes of oligodendrocytes in the CNS and Schwann cells in the peripheral nervous system (PNS), wherein mEGFP expression defines the inner layers of myelin sheaths and Schmidt-Lanterman incisures in adult sciatic nerves. In addition, mEGFP expression can be used to track the extent of remyelination after demyelinating injury in a toxin-induced demyelination animal model. Taken together, the membrane-anchored mEGFP expression in the new transgenic line would facilitate direct visualization of dynamic myelin membrane formation and assembly during development and process remodeling during remyelination after various demyelinating injuries. © 2014 Wiley Periodicals, Inc.

Description: The sea urchin is a penta-radial marine invertebrate of the phylum Echinodermata, yet sea urchins develop initially as bilaterally symmetric embryos and become penta-radial secondarily during development of the adult. Late in embryogenesis indirect developing larvae produce molecular asymmetries that dictate the positioning and formation of the adult rudiment on the left side. The rudiment gives rise to the radially symmetric adult during metamorphosis. The mechanism of L-R establishment in sea urchin involves highly conserved signaling pathways including Nodal, BMP, Notch and perhaps Hedgehog. Thus, L-R symmetry-breaking in the sea urchin appears to utilize a mechanism that is conserved among deuterostomes. At the same time establishment of L-R symmetry in the sea urchin deploys a number of features that are mechanistically unique. Here we review this mechanism and the uncertainties that remain. © 2014 Wiley Periodicals, Inc.

Description: By the 6-cell stage, embryos of C. elegans are morphologically L-R asymmetric with an invariant handedness that persists throughout development. We used intracellular markers to ask whether breaking of L-R symmetry could be observed at earlier stages. Observation of 2-3-cell embryos carrying intracellular markers indicated that L-R symmetry is broken concomitantly with establishment of D-V axis polarity during division of the anterior AB cell. The AB cleavage furrow initiates asymmetrically and always from the left, suggesting L-R differences in the AB cell cortex. An invariantly handed cortical rotation observed earlier during first cleavage implies that the 1-cell embryo has an intrinsic chirality. We propose that L-R differences in the cortex could result from mechanical forces on asymmetric components of a chiral cortical network during the off-axis elongation of the AB-cell spindle prior to AB cleavage. © 2014 Wiley Periodicals, Inc.

Description: ADAMTS9 is the most conserved member of a large family of secreted metalloproteases having diverse functions. Adamts9 null mice die before gastrulation, precluding investigations of its roles later in embryogenesis, in adult mice or disease models. We therefore generated a floxed Adamts9 allele to bypass embryonic lethality. In this mutant, unidirectional loxP sites flank exons 5 through 8, which encode the catalytic domain, including the protease active site. Mice homozygous for the floxed allele were viable, lacked an overt phenotype, and were fertile. Conversely, mice homozygous for a germ-line deletion produced from the floxed allele by Cre -lox recombination did not survive past gastrulation. Hemizygosity of the deleted Adamts9 in combination with mutant Adamts20 led to cleft palate and severe white spotting as previously described. Previously, Adamts9 haploinsufficiency combined with either Adamts20 or Adamts5 nullizygosity suggested a cooperative role in interdigital web regression, but the outcome of deletion of Adamts9 alone remained unknown. Here, Adamts9 was conditionally deleted in limb mesoderm using Prx1 -Cre mice. Unlike other ADAMTS single knockouts, limb-specific Adamts9 deletion resulted in soft-tissue syndactyly (STS) with 100% penetrance and concurrent deletion of Adamts5 increased the severity of STS. Thus, Adamts9 has both non-redundant and cooperative roles in ensuring interdigital web regression. This new allele will be useful for investigating other biological functions of ADAMTS9. © 2014 Wiley Periodicals, Inc.

Description: Vertebrates display asymmetric arrangements of inner organs such as heart and stomach. The Nodal signaling cascade in the left lateral plate mesoderm in all cases directs asymmetric morphogenesis and placement during organogenesis. Mechanisms that lead up to left-asymmetric Nodal induction seem to differ between the vertebrates. Cilia produce a leftward extracellular fluid flow in zebrafish, medaka, mouse, rabbit and Xenopus embryos during neurulation. In Xenopus, earlier asymmetric cues were described. Some, such as Rab11, apparently act in the zygote. Others were efficiently manipulated in ventral-right cells at the 4-cell stage, a lineage presumably independent of the ciliated gastrocoel roof plate (GRP) during neurulation. Here we show that 1- and 4-cell manipulations of Rab11 showed equal low efficiencies of left-right disturbances. We also re-evaluated the lineage of the GRP. By tracing back future ciliated cells from the gastrula to the 4-cell stage, we show that ventral cells contribute to ciliated sensory cells at the border of the GRP. Knockdown of the Nodal inhibitor Coco in the ventral right lineage resulted in embryos with ectopic right-sided Nodal and Pitx2c expression. Together, these experiments support a cilia-based mechanism of symmetry breakage in the frog Xenopus. © 2014 Wiley Periodicals, Inc.

Description: The first molecular herald of organ asymmetry during murine embryogenesis is found at the periphery of the node in early-somite stage embryos. Asymmetric gene expression and calcium accumulation at the node occurs in response to a left-ward flow of extracellular fluid across the node, generated by motile cilia within the pit of the node and likely sensed by immotile cilia in the periphery of the node. The ciliation of node cells is controlled by a cascade of node-restricted transcription factor activity during mid-late gastrulation. Mutation of the murine Zic2 transcription factor is associated with random cardiac situs and a loss of asymmetric gene expression at the early-somite node and in the lateral plate. Zic2 is not expressed in these regions but is transiently expressed in the mid-late gastrula node at the time of ciliogenesis. The cilia of the node are overtly abnormal in Zic2 mutant embryos being dysmorphic and short relative to wild-type littermates. The expression of the Noto , Rfx3 and Foxj1 transcription factors known to regulate ciliogenesis is greatly depleted in the mid-gastrula node of mutants, as is the expression of the Pkd1l1 gene required for cilia function. Zic2 appears to be a component of the gene regulatory network that drives ciliation of node cells during gastrulation. © 2014 Wiley Periodicals, Inc.

Description: Sphingomyelin phosphodiesterase 3 (SMPD3) is a pleiotropic lipid metabolizing enzyme involved in multiple physiological processes. A deletion mutation in the murine Smpd3 gene called fragilitas ossium (fro ) leads to severe skeletal abnormalities in the developing fro/fro embryos and high rate of perinatal lethality in these homozygous mutants. Although fro/fro mouse can be a useful tool to study many different aspects of SMPD3 functions, the perinatal lethality makes it difficult to generate sufficient number of mice for controlled studies. In fact, on the C57BL/6 genetic background none of the fro/fro mice survive beyond the perinatal stage. In the current study, we used the ‘Tet-On’ inducible gene expression system to express Smpd3 transiently in fro/fro;ROSA-rtTA;TRE-Smpd3 embryos on the C57BL/6 background. This induced Smpd3 expression corrected all the skeletal abnormalities in these embryos and prevented the perinatal lethality. However, induction of Smpd3 expression in the adolescent fro/fro;ROSA-rtTA;TRE-Smpd3 mice was not sufficient to correct the defects in trabecular bone mineralization and the impaired growth of the long bones. This novel mouse model will be a useful tool to study SMPD3 biology in vivo . © 2014 Wiley Periodicals, Inc.

Description: Epidermal growth factor-like domain 7 ( Egfl7 ) expression in the developing embryo is largely restricted to sites of mesodermal progenitors of angioblasts/hemangioblasts and the vascular endothelium. We hypothesize that Egfl7 marks the endothelial lineage during embryonic development, and can be used to define the emergence of endothelial progenitor cells, as well as to visualize newly forming vasculature in the embryo and during the processes of physiological and pathological angiogenesis in the adult. We have generated a transgenic mouse strain that expresses enhanced green fluorescent protein (eGFP) under the control of a minimal Egfl7 regulatory sequence (Egfl7:eGFP). Expression of the transgene recapitulated that of endogenous Egfl7 at sites of vasculogenesis and angiogenesis in the allantois, yolk sac, and in the embryo proper. The transgene was not expressed in the quiescent endothelium of most adult organs. However, the uterus and ovary, which undergo vascular growth and remodeling throughout the estrus cycle, expressed high levels of Egfl7:eGFP. Importantly, expression of the Egfl7:eGFP transgene was induced in adult neovasculature. We also found that increased Egfl7 expression contributed to pathological revascularization in the mouse retina. To our knowledge, this is first mouse model that enables monitoring endothelial cells at sites of active vasculogenesis and angiogenesis. This model also facilitated the isolation and characterization of EGFL7 + endothelial cell populations by fluorescence activated cell sorting (FACS). Together, our results demonstrate that the Egfl7:eGFP reporter mouse is a valuable tool that can be used to elucidate the mechanisms by which blood vessels form during development and under pathologic circumstances. © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT The role of the habenular nuclei in modulating fear and reward pathways has sparked a renewed interest in this conserved forebrain region. The bilaterally paired habenular nuclei, each consisting of a medial/dorsal and lateral/ventral nucleus, can be further divided into discrete subdomains whose neuronal populations, precise connectivity and specific functions are not well understood. An added complexity is that the left and right habenulae show pronounced morphological differences in many non-mammalian species. Notably, the dorsal habenulae of larval zebrafish provide a vertebrate genetic model to probe the development and functional significance of brain asymmetry. Previous reports have described a number of genes that are expressed in the zebrafish habenulae, either in bilaterally symmetric patterns or more extensively on one side of the brain than the other. The goal of our study was to generate a comprehensive map of the zebrafish dorsal habenular nuclei, by delineating the relationship between gene expression domains, comparing the extent of left-right asymmetry at larval and adult stages, and identifying potentially functional subnuclear regions as defined by neurotransmitter phenotype. While many aspects of habenular organization appear conserved with rodents, the zebrafish habenulae also possess unique properties that may underlie lateralization of their functions. © 2014 Wiley Periodicals, Inc.

Description: Much of the gnathostome (jawed vertebrate) evolutionary radiation was dependent upon the ability to sense and interpret the environment and subsequently act upon this information through utilization of a specialized mode of feeding involving the jaws. While the gnathostome skull, reflective of the vertebrate baüplan, typically is bilaterally symmetric with right (dextral) and left (sinistral) halves essentially representing mirror images along the midline, both adaptive and abnormal asymmetries have appeared. Herein we provide a basic primer on studies of the asymmetric development of the gnathostome skull, touching briefly on asymmetry as a field of study, then describing the nature of cranial development and finally underscoring evolutionary and functional aspects of left-right asymmetric cephalic development. © 2014 Wiley Periodicals, Inc.

Description: Some tropical ascidians of the family Didemnidae invariably harbor cyanobacterial cells in the common cloacal cavities and/or tunic. This is the only lifelong, obligate photosymbiosis reported in chordates. Photosymbionts are transferred from the maternal colony to embryos or pre-hatching larvae brooded in the colonies. Here we review the diversity of modes of cyanobacterial transmission to shed a light on the evolutionary history of ascidian photosymbiosis. Ascidian species exhibit several modes of cyanobacterial transmission. In Diplosoma species, the mode is constrained phylogenetically; all photosymbiotic Diplosoma have the same mode of transmission using a unique organ known as the rastrum. In other photosymbiotic species, the mode is constrained by the distribution pattern of photosymbionts in the host colony. Species of different genera have similar modes of transmission wherein they harbor cyanobacterial cells at similar sites within colonies. Accordingly, host species of distinct lineages likely acquired similar modes of transmission convergently. Why obligate photosymbiosis was established exclusively in these didemnid ascidians remains uncertain. Ascidian photosymbiosis is discussed from the viewpoint of evolution and diversification of vertical transmission. © 2014 Wiley Periodicals, Inc.

Description: The Cre-loxP recombination system has been used to promote DNA recombination both in vitro and in vivo . For in vivo delivery, Cre expression is commonly achieved through the use of tissue/cell type-specific promoters, viral infection, or drug inducible transcription and protein translocation to promote targeted DNA excision. The development of cell permeable (or penetrating) peptide tagged proteins has facilitated the delivery of Cre recombinase protein into cells in culture, organotypic slide culture, or in living animals. In this report, we generated bacterially expressed, his-tagged Cre protein with either a cardiac targeting peptide (CTP) or an antennapedia peptide (ANTP) at the C-terminus and demonstrated efficient uptake and recombination in both cell culture and mice. To facilitate delivery to cardiac and skeletal muscle, we mixed proteins with pluronic F-127 hydrogel and delivered Cre protein into reporter Rosa26mTmG mouse skeletal muscle or Rosa26LacZ cardiac muscle via ultrasound guided injection. Activation of reporter gene expression indicated that these Cre proteins were enzymatically active. Recombination events were detected only in the vicinity of injection areas. In conclusion, we have developed a method to deliver enzymatically active Cre protein locally to skeletal muscle and cardiac muscle that may be adapted for use with other proteins. © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Nipbl ( Scc2 ) and Mau2 ( Scc4 ) encode evolutionary conserved proteins that play a vital role for loading the cohesin complex onto chromosomes, thereby ensuring accurate chromosome segregation during cell division. While mutations in human NIPBL are known to cause the developmental disorder Cornelia de Lange syndrome, the functions of Nipbl and Mau2 in mammalian development are poorly defined. Here we generated conditional alleles for both genes in mice and show that neural crest cell-specific inactivation of Nipbl or Mau2 strongly affects craniofacial development. Surprisingly, the early phase of neural crest cell proliferation and migration is only moderately affected in these mutants. Moreover, we found that Mau2 single homozygous mutants exhibited a more severe craniofacial phenotype when compared to that of Nipbl ; Mau2 double homozygous mutants. This raises the possibility that the Mau2/Nipbl protein interaction is not only required for cohesin loading, but may also be required to restrict the level of Nipbl involved in regulating gene expression. Together, the data suggest that proliferating neural crest cells tolerate a substantial reduction of cohesin loading proteins and we propose that the successive decrease of cohesin loading proteins in neural crest cells may alter developmental gene regulation in a highly dynamic manner. © 2014 Wiley Periodicals, Inc.

Description: The introduction of a transgene into the genome through homologous recombination or sequence-specific enzymatic modification is a key technique for producing transgenic mice. The Rosa26 gene has been widely used to produce transgenic mice because the gene is transcriptionally active in various cell types and, at many developmental stages, is permissive for constitutive expression of integrated transgenes, and is dispensable for normal development. However, permissive loci other than Rosa26 are needed to generate mice that harbor multiple transgenes for complex studies. Here, we identified the Cd6 locus on mouse chromosome 19 as a transgene integration site in a transgenic mouse strain showing widespread reporter expression. Using this locus, we generated a knock-in mouse line that harbors a CAG promoter-driven reporter transgene, and found that the homozygous transgenic mice are viable and fertile, although transgene insertion disrupted Cd6 gene expression. The transgene on the Cd6 locus expressed reporter genes extensively throughout embryos, neonates, and adults. Combined with the Cre/loxP binary system, blood and lymphatic endothelial cell-specific reporter expression from the transgenic locus was achieved. These results suggest that Cd6 is valuable as an alternative site for targeted transgenesis. © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT A central unresolved question in the molecular cascade that drives establishment of left-right (LR) asymmetry in vertebrates are the mechanisms deployed to relay information between the midline site of symmetry-breaking and the tissues which will execute a program of asymmetric morphogenesis ( Figure 1 ). The cells located between these two distant locations must provide the medium for signal relay. Of these, the gut endoderm is an attractive candidate tissue for signal transmission since it comprises the epithelium that lies between the node, where asymmetry originates, and the lateral plate, where asymmetry can first be detected. Here, focusing on the mouse as a model, we review our current understanding and entertain open questions concerning the relay of LR information from its origin. © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT The organization of echinoderm Hox clusters is of interest due to the role that Hox genes play in deuterostome development and body plan organization, and the unique gene order of the Hox complex in the sea urchin Strongylocentrotus purpuratus , which has been linked to the unique development of the axial region. Here, we report that the Hox and ParaHox clusters of Acanthaster planci , a corallivorous starfish found in the Pacific and Indian oceans, generally resemble the chordate and hemichordate clusters. The A. planci Hox cluster shares with sea urchins the loss of one of the medial Hox genes, even-skipped ( Evx) at the anterior of the cluster, as well as organization of the posterior Hox genes. © 2014 Wiley Periodicals, Inc.

Description: Hox and ParaHox genes are involved in patterning the anterior-posterior body axis in metazoans during embryo development. Body plan evolution and diversification are affected by variations in the number and sequence of Hox and ParaHox genes, as well as by their expression patterns. For this reason Hox and ParaHox gene investigation in the phylum Mollusca is of great interest, as this is one of the most important taxa of protostomes, characterized by a high morphological diversity. The comparison of the works reviewed here indicates that species of molluscs, belonging to different classes, share a similar composition of Hox and ParaHox genes. Therefore evidence suggests that the wide morphological diversity of this taxon could be ascribed to differences in Hox gene interactions and expressions and changes in the Hox downstream genes rather than to Hox cluster composition. © 2014 Wiley Periodicals, Inc.

Description: Understanding the role of conserved non-coding elements (CNEs) throughout the genome is taking advantage of the improved efficiency of genome-sequencing techniques and bioinformatics tools. Tunicates diverged before the vertebrate whole genome duplications and therefore, represent an optimal model system to study the evolution of complex regulatory networks. Here, we review the current knowledge on the characterization of CNEs during embryonic development, focusing on the evolutionary similarity and divergence between tunicates and other chordates. Many vertebrate specific CNEs that regulate developmental processes were identified based on high level of sequence conservation, but only few of them have been recognized in tunicates or other invertebrates because of genomic sequences divergence. We discuss recent studies demonstrating that a combination of different methodologies, based not only on high sequence identity, can collectively be used to identify CNEs with regulatory activity in phylogenetically distant species. Here, a low sequence constraints approach was successfully used to search orthologous chordate gene regions for cross-species conserved regulatory elements that control developmental genes. © 2014 Wiley Periodicals, Inc.

Description: Historically, mutations have had a significant impact on the study of developmental processes and phenotypic evolution. Lesions in DNA are created by artificial methods or detected by natural genetic variation. Random mutations are then ascribed to genetic change by direct sequencing or positional cloning. Tunicate species of the ascidian genus Ciona represent nearly fully realized model systems in which gene function can be investigated in depth. Additionally, tunicates are valuable organisms for the study of naturally occurring mutations due to the capability to exploit genetic variation down to the molecular level. Here, we summarize the available information about how mutations are studied in ascidians with examples of insights that have resulted from these applications. We also describe notions and methodologies that might be useful for the implementation of easy and tight procedures for mutations studies in Ciona . This article is protected by copyright. All rights reserved.

Description: Biocuration, the field of biology concerned with organizing, representing, checking and making biological information accessible to both humans and computers, has become an essential part of biological and biomedical research. However, curation increasingly lags behind data generation in funding, development and recognition. In this work, I describe the biocuration efforts accomplished by the community of laboratories working on Tunicata, as well as challenges that face. I argue that biocuration is essential for the future of scientific research, and that the experience gathered by tunicate community could prove extremely useful to other biologists' communities. This article is protected by copyright. All rights reserved.

Description: Transcription factors Pax3 and Zic1 are among the earliest genes activated at the neural plate border. In Xenopus , they are necessary and sufficient to promote the formation of multiple neural plate border cell types, including the neural crest, cranial placodes and hatching gland. Pax3 is especially critical for the formation of the hatching gland, a group of cells that produce proteolytic enzymes essential to digest the egg vitelline envelope and jelly coat in order to release the tadpole into the environment. In a screen designed to identify downstream targets of Pax3 we isolated a member of the astacin family of metalloproteases, related to Xenopus hatching enzyme (Xhe), that we named Xhe2 . Xhe2 is exclusively expressed in hatching gland cells as they first emerge at the lateral edge of the anterior neural plate, and persists in this tissue up to the tadpole stage. Knockdown experiments show that Xhe2 expression depends entirely on Pax3 function. Gain-of-function studies demonstrate that Pax3 can induce premature hatching through the upregulation of several proteolytic enzymes including Xhe2. Interestingly, Xhe2 overexpression is sufficient to induce early hatching, indicating that Xhe2 is one of the key components of the degradation mechanism responsible for breaking down the vitelline membrane. This article is protected by copyright. All rights reserved.

Description: The discovery and application of the CRISPR/Cas9 genome editing method has greatly enhanced the ease with which transgenic manipulation can occur. We applied this technology to the mollusc, Crepidula fornicata , and have successfully created transgenic embryos expressing mCherry fused to endogenous β-catenin. Specific integration of the fluorescent reporter was achieved by homologous recombination with a β -catenin -specific donor DNA containing the mCherry coding sequence. This fluorescent gene knock-in strategy permits in vivo observations of β-catenin expression during embryonic development and represents the first demonstration of CRISPR/Cas9-mediated transgenesis in the Lophotrochozoa superphylum. The CRISPR/Cas9 method is a powerful and economical tool for genome modification and presents an option for analysis of gene expression in not only major model systems, but also in those more diverse species that may not have been amenable to the classic methods of transgenesis. This approach will allow one to generate transgenic lines of snails for future studies. This article is protected by copyright. All rights reserved.

Description: Botryllus schlosseri is a colonial ascidian with characteristics that make it an attractive model for studying immunology, stem cell biology, evolutionary biology, and regeneration. Transcriptome sequencing and the recent completion of a draft genome sequence for B. schlosseri have revealed a large number of genes, both with and without vertebrate homologs, but analyzing the spatial and temporal expression of these genes in situ has remained a challenge. Here we report a robust protocol for in situ hybridization that enables the simultaneous detection of multiple transcripts in whole adult B. schlosseri using Tyramide Signal Amplification in conjunction with digoxigenin- and dinitrophenol-labeled RNA probes. Using this protocol we have identified a number of genes that can serve as markers for developing and mature structures in B. schlosseri , permitting analysis of phenotypes induced in loss-of-function experiments. © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Summary: Glial cells missing (Gcm) is the primary regulator of glial cell fate in Drosophila . Gcm belongs to a small family of transcriptional regulators involved in fundamental developmental processes found in diverse animal phyla including vertebrates. Gcm proteins contain the highly conserved DNA-binding GCM domain, which recognizes an octamer DNA sequence. To date, studies in Drosophila have primarily relied on gcm alleles caused by P-element induced DNA deletions at the gcm locus, as well as a null allele caused by a single base pair substitution in the GCM domain that completely abolishes DNA binding. Here I characterize two hypomorphic missense alleles of gcm with intermediate glial cells missing phenotypes. In embryos homozygous for either of these gcm alleles the number of glial cells in the central nervous cystem (CNS) is reduced approximately in half. Both alleles have single amino acid changes in the GCM domain. These results suggest that Gcm protein activities in these mutant alleles have been attenuated such that they are operating at threshold levels, and trigger glial cell differentiation neural precursors in the CNS in a stochastic fashion. These hypomorphic alleles provide additional genetic resources for understanding Gcm functions and structure in Drosophila and other species. © 2014 Wiley Periodicals, Inc.

Description: The colonial tunicate Botryllus schlosseri is a widespread filter-feeding ascidian that lives in shallow waters and is easily reared in aquaria. Its peculiar blastogenetic cycle, characterized by the presence of three blastogenetic generations (filtering adults, buds and budlets) and by recurrent generation changes, has resulted in over 60 years of studies aimed at understanding how sexual and asexual reproduction are coordinated and regulated in the colony. The possibility of using different methodological approaches, from classical genetics to cell transplantation, contributed to the development of this species as a valuable model organism for the study of a variety of biological processes. Here, we review the main studies detailing rearing, staging methods, reproduction and colony growth of this species, emphasizing the asymmetry in sexual and asexual reproduction potential, sexual reproduction in the field and the laboratory, and self- and cross-fertilization. These data, opportunely matched with recent tanscriptomic and genomic outcomes, can give a valuable help to the elucidation of some important steps in chordate evolution. © 2014 Wiley Periodicals, Inc.

Description: Embryonic and post-embryonic development in ascidians have been studied for over a century, but it is only in the last 10 years that the complex molecular network involved in coordinating post-larval development and metamorphosis has started to emerge. In most ascidians, the transition from the larval to the sessile juvenile/adult, or metamorphosis, requires a combination of environmental and endogenous signals and is characterized by coordinated global morphogenetic changes that are initiated by the adhesion of the larvae. Cloney was the first to describe cellular events of ascidians' metamorphosis in 1978 and only recently elements of the molecular regulation of this crucial developmental step have been revealed. This review aims to present a thorough view of this crucial developmental step by combining recent molecular data to the already established cellular events. © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Tunicates are invertebrate members of the chordate phylum, and are considered to be the sister group of vertebrates. Tunicates are composed of ascidians, thaliaceans and appendicularians. With the advent of inexpensive high throughput sequencing, the number of sequenced tunicate genomes is expected to rise sharply within the coming years. To facilitate comparative genomics within the tunicates, and between tunicates and vertebrates, standardized rules for the nomenclature of tunicate genetic elements need to be established. Here we propose a set of nomenclature rules, consensual within the community, for predicted genes, pseudogenes, transcripts, operons, transcriptional cis -regulatory regions, transposable elements, and transgenic constructs. In addition, the document proposes guidelines for naming transgenic and mutant lines. The latest version of these guidelines can be downloaded from the Tunicate Portal ( http://www.tunicate-portal.org/ ). To better identify the latest version, the file name for the guidelines should follow the following syntax: Genetic_Guidelines_Tunicate_[year]_[month]_[day] (example, Genetic_Guidelines_Tunicate_2014_05_01). © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT We describe a novel zebrafish line that fluorescently tags a previously unknown protein, CT74a, allowing us to follow its endogenous expression in real time and at subcellular resolution in live embryos. Our results showed that CT74a-Citrine fusion protein is expressed in the developing pharyngeal arches, hindbrain, and fin buds in a pattern highly reminiscent of transcription factors belonging to anterior Hox gene families, including expression in a subset of neuronal nuclei. Consistent with this, splinkerette-PCR revealed that CT74a-Citrine’s genomic integration is within the HoxB region, and 3’ RACE demonstrated that its downstream coding sequence has no recognizable homology. Thus, CT74a is a previously unknown protein located within the HoxB cluster adjacent to Hoxb4a and is expressed in a Hoxb4a-like pattern. © 2014 Wiley Periodicals, Inc.

Description: Congenital heart disease (CHD) is the most common birth defect. However, the majority of CHD cases have unknown etiology. Here we report the identification of ASXL2 and ASXL1, two homologous chromatin factors, as novel regulators of heart development. Asxl2 -/- fetuses have reduced body weight and display congenital heart malformations including thickened compact myocardium in the left ventricle, membranous ventricular septal defect and atrioventricular valval stenosis. Although most Asxl2 -/- animals survive to term, the neonates have patent ductus arteriosus and consequent lung hemorrhage and die soon after birth. Asxl1 -/- fetuses have reduced body weight and display cleft palate, anophthalmia as well as ventricular septal defects and a failure in lung maturation. From these results, we conclude that normal heart development requires both ASXL proteins. In particular, ASXL2 plays an important role in heart morphogenesis and the transition from fetal to postnatal circulation. © 2014 Wiley Periodicals, Inc.

Description: The Drosophila Trithorax-like (Trl) gene encodes a GAGA factor which regulates a number of developmentally important genes. In this study, we identify a new function for Drosophila GAGA factor in male germ cell development. Trl mutants carrying strong hypomorphic alleles display loss of primordial germ cells during their migration in embryogenesis and severe disruption in mitochondria structure during early spermatogenesis. The mutation resulted in small testes formation, a deficit of germ cells, abnormal mitochondrial morphogenesis, spermatocyte death through autophagy and partial or complete male sterility. Pleiotropic mutation effects can be explained by the misexpression of GAGA factor target genes, the products of which are required for germ cell progression into mature sperm. © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Notch signaling is important in angiogenesis during embryonic development. However, the embryonic lethal phenotypes of knock-out and transgenic mice have precluded studies of the role of Notch post-natally. To develop a mouse model that would bypass the embryonic lethal phenotype and investigate the possible role of Notch signaling in adult vessel growth, we developed transgenic mice with Cre-conditional expression of the constitutively active intracellular domain of Notch1 (IC-Notch1). Double transgenic IC-Notch1/Tie2-Cre embryos with endothelial specific IC-Notch1 expression died at embryonic day 9.5. They displayed collapsed and leaky blood vessels and defects in angiogenesis development. A tetracycline-inducible system was used to express Cre recombinase postnatally in endothelial cells. In adult mice IC-Notch1 expression inhibited bFGF-induced neovascularization and female mice lacked mature ovarian follicles, which may reflect the block in bFGF-induced angiogenesis required for follicle growth. Our results demonstrate that Notch signaling is important for both embryonic and adult angiogenesis and indicate that the Notch signaling pathway may be a useful target for angiogenic therapies. © 2014 Wiley Periodicals, Inc.

Description: Mixl1 is the only member of the Mix/Bix homeobox gene family identified in mammals. During mouse embryogenesis, Mixl1 is first expressed at embryonic day (E)5.5 in cells of the visceral endoderm (VE). At the time of gastrulation, Mixl1 expression is detected in the vicinity of the primitive streak. Mixl1 is expressed in cells located within the primitive streak, in nascent mesoderm cells exiting the primitive streak, and in posterior VE overlying the primitive streak. Genetic ablation of Mixl1 in mice has revealed its crucial role in mesoderm and endoderm cell specification and tissue morphogenesis during early embryonic development. However, the early lethality of the constitutive Mixl1 -/- mutant precludes the study of its role at later stages of embryogenesis and in adult mice. To circumvent this limitation, we have generated a conditional Mixl1 allele ( Mixl1 cKO ) that permits temporal as well as spatial control of gene ablation. Animals homozygous for the Mixl1 cKO conditional allele were viable and fertile. Mixl1 KO/KO embryos generated by crossing of Mixl1 cKO/cKO mice with Sox2-Cre or EIIa-Cre transgenic mice were embryonic lethal at early somite stages. By contrast to wild-type embryos, Mixl1 KO/KO embryos contained no detectable Mixl1, validating the Mixl1 cKO as a protein null after Cre-mediated excision. Mixl1 KO/KO embryos resembled the previously reported Mixl1 -/- mutant phenotype. Therefore, the Mixl1 cKO allele provides a tool for investigating the temporal and tissue-specific requirements for Mixl1 in the mouse. © 2014 Wiley Periodicals, Inc.

Description: Grafting and transplantation experiments in embryology require proper distinction between host and donor tissues. For the avian model this has traditionally been achieved by using two closely related species (e.g., chick and quail) followed by species-specific antibody staining. Here, we show that an in situ hybridization probe against the HINTW gene is a robust and reliable marker for female-derived chicken cells. At all pre-circulation stages tested, all cells in female embryos, independently confirmed by PCR analysis, were strongly positive for HINTW , whereas all male embryos were negative. This probe is broadly applicable in intra-specific chick/chick chimera studies, and as a proof of principle, we utilized this probe to detect female cells in three experimental settings: 1) to mark female donor cells in a node transplantation assay; 2) to distinguish female cells in male/female twins generated by the Cornish pasty culture; and 3) to detect female half of the embryo in artificially generated bilateral gynandromorphs. A rapid, PCR based pre-screening step increases the efficiency of obtaining desired donor/host sex combination from 25% to 100%. For most avian chimera studies, this female-specific in situ probe is a low cost alternative to the commonly-used QCPN antibody and to ubiquitous-GFP chicken strains which are not widely available to the research community. © 2014 Wiley Periodicals, Inc.

Description: Neurod1 is a transcription factor involved in several developmental programs of the gastrointestinal tract, pancreas, neurosensory and central nervous system. In the brain, Neurod1 was shown to be essential for neurogenesis as well as migration, maturation and survival of newborn neurons during development and adulthood. Interestingly, Neurod1 is also expressed in a subset of fully mature neurons where its function remains unclear. To study the role of Neurod1, systems are required that allow the temporal and spatial genetic manipulation of Neurod1-expressing cells. To this aim we have generated four Neurod1-CreER T2 mouse lines in which CreER T2 expression, although at different levels, is restricted within areas of physiological Neurod1 expression and Neurod1 positive cells. In particular, the different levels of CreER T2 expression in different mouse lines offers the opportunity to select the one that is more suited for a given experimental approach. Hence, our Neurod1-CreER T2 lines provide valuable new tools for the manipulation of newborn neurons during development and adulthood as well as for studying the subpopulation of mature neurons that retain Neurod1 expression throughout life. In this context, we here report that Neurod1 is not only expressed in immature newborn neurons of the adult hippocampus, as already described, but also in fully mature granule cells of the dentate gyrus . © 2014 Wiley Periodicals, Inc.

Description: During development, vertebrates form a progression of up to three different kidneys that are comprised of functional units termed nephrons. Nephron composition is highly conserved across species, and an increasing appreciation of the similarities between zebrafish and mammalian nephron cell types has positioned the zebrafish as a relevant genetic system for nephrogenesis studies. A key component of the nephron blood filter is a specialized epithelial cell known as the podocyte. Podocyte research is of the utmost importance as a vast majority of renal diseases initiate with the dysfunction or loss of podocytes, resulting in a condition known as proteinuria that causes nephron degeneration and eventually leads to kidney failure. Understanding how podocytes develop during organogenesis may elucidate new ways to promote nephron health by stimulating podocyte replacement in kidney disease patients. In this review, we discuss how the zebrafish model can be used to study kidney development, and how zebrafish research has provided new insights into podocyte lineage specification and differentiation. Further, we discuss the recent discovery of podocyte regeneration in adult zebrafish, and explore how continued basic research using zebrafish can provide important knowledge about podocyte genesis in embryonic and adult environments. © 2014 Wiley Periodicals, Inc.

Description: Genes required for an organism to develop to maturity (for which no other gene can compensate) are considered essential. The continuing functional annotation of the mouse genome has enabled the identification of many essential genes required for specific developmental processes including cardiac development. Patterns are now emerging regarding the functional nature of genes required at specific points throughout gestation. Essential genes required for development beyond cardiac progenitor cell migration and induction include a small and functionally homogenous group encoding transcription factors, ligands and receptors. Actions of core cardiogenic transcription factors from the Gata, Nkx, Mef, Hand and Tbx families trigger a marked expansion in the functional diversity of essential genes from mid-gestation onwards. As the embryo grows in size and complexity, genes required to maintain a functional heartbeat and to provide muscular strength and regulate blood flow are well represented. These essential genes regulate further specialisation and polarisation of cell types along with proliferative, migratory, adhesive, contractile and structural processes. The identification of patterns regarding the functional nature of essential genes across numerous developmental systems may aid prediction of further essential genes and those important to development and/or progression of disease. © 2014 Wiley Periodicals, Inc.

Description: Transforming growth factor beta2 (TGFβ2) is a multifunctional protein which is expressed in several embryonic and adult organs. TGFB2 mutations can cause Loeys Dietz syndrome, and its dysregulation is involved in cardiovascular, skeletal, ocular and neuromuscular diseases, osteoarthritis, tissue fibrosis, and various forms of cancer. TGFβ2 is involved in cell growth, apoptosis, cell migration, cell differentiation, cell-matrix remodeling, epithelial-mesenchymal transition, and wound healing in a highly context-dependent and tissue-specific manner. Tgfb2 -/- mice die perinatally from congenital heart disease, precluding functional studies in adults. Here, we have generated mice harboring Tgfb2 β geo (knockout-first lacZ -tagged insertion) gene-trap allele and Tgfb2 flox conditional allele . Tgfb2 β geo/ β geo or Tgfb2 β geo/- mice died at perinatal stage from the same congenital heart defects as Tgfb2 -/- mice. β-galactosidase staining successfully detected Tgfb2 expression in the heterozygous Tgfb2 βgeo fetal tissue sections. Tgfb2 flox mice were produced by crossing the Tgfb2+/ β geo mice with the FLPeR mice. Tgfb2flox/- mice were viable. Tgfb2 conditional knockout (Tgfb2cko/-) fetuses were generated by crossing of Tgfb2flox/- mice with Tgfb2+/-;EIIaCre mice. Systemic Tgfb2cko/- embryos developed cardiac defects which resembled the Tgfb2 β geo/ β geo, Tgfb2 β geo/-, and Tgfb2 -/- fetuses. In conclusion, Tgfb2 β geo and Tgfb2 flox mice are novel mouse strains which will be useful for investigating the tissue specific expression and function of TGFβ2 in embryonic development, adult organs, and disease pathogenesis and cancer. © 2014 Wiley Periodicals, Inc.

Description: Fast and slow TnI are co-expressed in E11.5 embryos, and fast TnI is present from the very beginning of myogenesis. A novel green fluorescent protein (GFP) reporter mouse lines (FastTnI/GFP lines) that carry the primary and secondary enhancer elements of the mouse fast troponin I (fast TnI), in which reporter expression correlates precisely with distribution of the endogenous f TnI protein was generated. Using the FastTnI/GFP mouse model, we characterized the early myogenic events in mice, analyzing the migration of GFP-positive myoblasts, and the formation of primary and secondary myotubes in transgenic embryos. Interestingly, we found that the two contractile fast and slow isoforms of TnI are expressed during the migration of myoblasts from the somites to the limbs and body wall, suggesting that both participate in these events. Since no sarcomeres are present in myoblasts, we speculate that the function of fast TnI in early myogenesis is, like Myosin and Tropomyosin, to participate in cell movement during the initial myogenic stages. © 2014 Wiley Periodicals, Inc.

Description: Matrix metalloproteinases (MMPs) are a large and complex family of zinc-dependent endoproteinases widely recognized for their roles in remodeling the extracellular matrix (ECM) during embryonic development, wound healing, and tissue homeostasis. Their mis-regulation is central to many pathologies, and they have therefore been the focus of biomedical research for decades. These proteases have also recently emerged as mediators of neural development and synaptic plasticity in vertebrates, however understanding of the mechanistic basis of these roles, and the molecular identities of the MMPs involved remains far from complete. We have identified a zebrafish orthologue of mmp25 (a.k.a. leukolysin; MT6-MMP), a membrane-type, furin-activated MMP associated with leukocytes and invasive carcinomas, but which we find is expressed by a subset of the sensory neurons during normal embryonic development. We detect high levels of Mmp25β expression in the trigeminal, craniofacial, and posterior lateral line ganglia in the hindbrain, and in Rohon-Beard cells in the dorsal neural tube during the first 48 hours of embryonic development. Knockdown of Mmp25β expression with morpholino oligonucleotides results in larvae that are uncoordinated and insensitive to touch, and which exhibit defects in the development of sensory neural structures. Using in vivo zymography, we observe that Mmp25β morphant embryos show reduced type IV collagen degradation in regions of the head traversed by elongating axons emanating from the trigeminal ganglion, suggesting that Mmp25β may play a pivotal role in mediating ECM remodeling in the vicinity of these elongating axons. © 2014 Wiley Periodicals, Inc.

Description: The Down syndrome cell adhesion molecule ( DSCAM ) is an Ig containing cell adhesion molecule with remarkable structural conservation throughout metazoans. In insects, DSCAM has 38,000 potential isoforms that convey axon guidance, fasciculation and dendrite morphogenesis during neurodevelopment. In vertebrates, DSCAM is expressed throughout the nervous system and seems to also mediate proper axonal guidance and synaptogenesis without the isoform diversity found in insects. Differences in DSCAM function among several vertebrate species complicate the understanding of an evolutionarily conserved role during embryogenesis. We take advantage of the frog developmental model Xenopus tropicalis to study DSCAM function in early development by expression analysis and morpholino-mediated knockdown. Our results indicate that DSCAM is expressed early in development and restricted to the head and nervous system. Knockdown of protein expression results in early morphogenetic phenotypes characterized by failed gastrulation and improper posterior neural tube closure. Our results reveal a specific, fundamental role of DSCAM in early morphogenetic movements, presumably through its well-known role in homophilic cell adhesion. © 2014 Wiley Periodicals, Inc.

Description: LHX9 is a LIM-homeodomain transcription factor essential for the development of gonads, spinal cord interneurons, and thalamic neurons to name a few. We recently reported the expression of LHX9 in retinal amacrine cells during development. In this study, we generated an Lhx9 - GFPCreER T2 (GCE) knock-in mouse line by knocking-in a GCE cassette at the Lhx9 locus, thus inactivating endogenous Lhx9 . Lhx9 GCE/+ mice were viable, fertile, and displayed no overt phenotypical characteristics. Lhx9 GCE/GCE mice were all phenotypically female, smaller in size, viable, but infertile. The specificity and efficacy of the Lhx9-GCE mouse line was verified by crossing it to a Rosa26 - tdTomato reporter mouse line, which reveals the Cre recombinase activities in retinal amacrine cells, developing limbs, testis, hippocampal neurons, thalamic neurons, and cerebellar neurons. Taken together, the Lhx9-GCE mouse line could serve as a beneficial tool for lineage tracing and gene manipulation experiments. © 2014 Wiley Periodicals, Inc.

Description: This year marks the 125 th anniversary of the beginning of regeneration research in the ascidian Ciona intestinalis . A brief note was published in 1891 reporting the regeneration of the Ciona neural complex and siphons. This launched an active period of Ciona regeneration research culminating in the demonstration of partial body regeneration: the ability of proximal body parts to regenerate distal ones, but not vice versa . In a process resembling regeneration, wounds in the siphon tube were discovered to result in the formation of an ectopic siphon. Ciona regeneration research then lapsed into a period of relative inactivity following the purported demonstration of the inheritance of acquired characters using siphon regeneration as a model. Around the turn of the present century, Ciona regeneration research experienced a new blossoming. The current studies established the morphological and physiological integrity of the regeneration process and its resemblance to ontogeny. They also determined some of the cell types responsible for tissue and organ replacement and their sources in the body. Finally, they showed that regenerative capacity is reduced with age. Many other aspects of regeneration now can be studied at the mechanistic level because of the extensive molecular tools available in Ciona . © 2014 Wiley Periodicals, Inc.

Description: As a dual function protein, β-catenin affects both cell adhesion and mediates canonical Wnt/β-catenin cell signaling. β-Catenin is prominently expressed in somatic Sertoli cells in the testis and postmeiotic germ cells, suggesting an additional role in spermatogenesis. It was reported previously that Cre/loxP-mediated conditional inactivation of the β-catenin gene ( Ctnnb1 ) in male gonads using a protamine promoter-driven Cre transgene ( Prm-cre ) resulted in partial infertility, reduced sperm count, and abnormal spermatogenesis. In this report we demonstrated that the conditional deletion of Ctnnb1 using a germ cell specific Cre transgene ( Stra8-icre ) had no effect on male fertility. We have shown that the Stra8-icre transgene was highly efficient in generating deletion in early pre-meiotic and post-meiotic cells. No differences in anatomical or histological presentation were found in the mutant testis, the production of viable sperm was similar, and no abnormalities in DNA sperm content were detected. We concluded that β-catenin is fully dispensable in germ cells for spermatogenesis. The conflicting results from the earlier study may have been due to off-target expression of Prm-cre in testicular somatic cells. In future studies, the analysis of conditional mutants using several Cre-transgenes should be encouraged to reduce potential errors. © 2014 Wiley Periodicals, Inc.

Description: Asymmetries in the nervous system have been observed throughout the animal kingdom. Deviations of brain asymmetries are associated with a variety of neurodevelopmental disorders; however, there has been limited progress in determining how normal asymmetry is established in vertebrates. In the C. elegans chemosensory system, two pairs of morphologically symmetrical neurons exhibit molecular and functional asymmetries. This review focuses on the development of antisymmetry of the pair of AWC olfactory neurons, from transcriptional regulation of general cell identity, establishment of asymmetry through neural network formation and calcium signaling, to the maintenance of asymmetry throughout the life of the animal. Many of the factors that are involved in AWC development have homologs in vertebrates, which may potentially function in the development of vertebrate brain asymmetry. © 2014 Wiley Periodicals, Inc.