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Ectodermal appendages in mammals, such as teeth, mammary glands, sweat glands and hair follicles, are generated during embryogenesis through a series of mesenchymal-epithelial interactions. Canonical Wnt signaling and its inhibitors are implicated in the early steps of ectodermal appendage development and patterning. To study the activation dynamics of the Wnt target and inhibitor Dickkopf4 (Dkk4) in ectodermal appendages, we used CRSIPR/Cas9 to generate a Dkk4-Cre knock-in mouse (Mus musculus) line, where the Cre recombinase cDNA replaces the expression of endogenous Dkk4. Using Cre reporters, the Dkk4-Cre activity was evident at the prospective sites of ectodermal appendages, overlapping with the Dkk4 mRNA expression. Unexpectedly, a predominantly mesenchymal cell population in the embryo posterior also showed Dkk4-Cre activity. Lineage-tracing suggested that these cells are likely derived from a few Dkk4-Cre-expressing cells in the epiblast at early gastrulation. Finally, our analyses of Dkk4-Cre-expressing cells in developing hair follicle epithelial placodes revealed intra- and inter-placodal cellular heterogeneity, supporting emerging data on the positional and transcriptional cellular variability in placodes. Collectively, we propose the new Dkk4-Cre knock-in mouse line as a suitable model to study Wnt and DKK4 inhibitor dynamics in early mouse development and ectodermal appendage morphogenesis., (© 2023 The Authors. genesis published by Wiley Periodicals LLC.)

The Forkhead box (Fox) gene family is an evolutionarily ancient gene family named after the Drosophila melanogaster forkhead gene (fkh). Fox genes are highly conserved transcription factors critical for embryogenesis and carcinogenesis. In the current study, we report a whole-genome survey of Fox genes and their expression patterns in the leech Helobdella austienesis. Phylogenetic analysis suggests that some Fox genes of leeches are correlated with other Lophotrochozoa and vertebrate Fox genes. Here we have performed semiquantitative reverse transcription polymerase chain reaction and whole-mount in situ hybridization of Fox genes throughout the embryonic development of H. austinensis. We found that each one of the leech Fox genes (FoxA1, FoxA3, FoxC, FoxL2, FoxO1, and FoxO2) is expressed in a specific set of cells or tissue type. From Stages 9-11, Hau-FoxA1 was expressed in the foregut of the anterior region, and Hau-FoxL2 was expressed in mesodermal muscle fiber. Hau-FoxA3 was temporally expressed in the ventral neuroectoderm. At Stage 11, Hau-FoxC was expressed in the foregut. Hau-FoxO genes have a ubiquitous expression. Our results provide more insight on the evolutionary linkage and role of the Fox gene function in Bilateria., (© 2018 Wiley Periodicals, Inc.)

The neural crest (NC) multipotent progenitor cells form at the neural plate border and migrate to diverse locations in the embryo to differentiate into many cell types. NC is specified by several embryonic pathways, however the role of noncanonical Wnt signaling in this process remains poorly defined. Daam1 is a formin family protein that is present in embryonic ectoderm at the time of NC formation and can mediate noncanonical Wnt signaling. Our interference experiments indicated that Daam1 is required for NC gene activation. To further study the function of Daam1 in NC development we used a transgenic reporter Xenopus line, in which GFP transcription is driven by sox10 upstream regulatory sequences. The activation of the sox10:GFP reporter in a subset of NC cells was suppressed after Daam1 depletion and in embryos expressing N-Daam1, a dominant interfering construct. Moreover, N-Daam1 blocked reporter activation in neuralized ectodermal explants in response to Wnt11, but not Wnt8 or Wnt3a, confirming that the downstream pathways are different. In complementary experiments, a constitutively active Daam1 fragment expanded the NC territory, but this gain-of-function activity was eliminated in a construct with a point mutation in the FH2 domain that is critical for actin polymerization. These observations suggest a new role of Daam1 and actin remodeling in NC specification., (© 2018 Wiley Periodicals, Inc.)

The lens originates from a simple cuboidal epithelium, which, on its basal side, contacts the optic vesicle, whilst facing the extraembryonic environment on its apical side. As this epithelium changes into the pseudostratified lens placode, its cells elongate and become narrower at their apical ends. This is due to the formation of an apical actin network, whose appearance is restricted to cells of the placodal region, as a result of region-specific signaling mechanisms that remain largely unknown. Here, we investigated the role of the polarity protein PAR3 and the phosphorylation state of its Threonine 833 (T833) aPKC-binding site in the recruitment of aPKC and in the establishment of actin network in the chick lens placode. Overexpression of wild type PAR3 recruited aPKC and punctate actin clusters to the basolateral membranes of the placodal cells. Recruitment of aPKC depended on the charge of the residue that replaced the T833 residue. In contrast, induction of the ectopic actin spots was independent on the charge of this residue., (© 2017 Wiley Periodicals, Inc.)

The decision by embryonic ectoderm to give rise to epidermal versus neural derivatives is the result of signaling events during blastula and gastrula stages. However, there also is evidence in Xenopus that cleavage stage blastomeres contain maternally derived molecules that bias them toward a neural fate. We used a blastomere explant culture assay to test whether maternally deposited transcription factors bias 16-cell blastomere precursors of epidermal or neural ectoderm to express early zygotic neural genes in the absence of gastrulation interactions or exogenously supplied signaling factors. We found that Foxd4l1, Zic2, Gmnn, and Sox11 each induced explants made from ventral, epidermis-producing blastomeres to express early neural genes, and that at least some of the Foxd4l1 and Zic2 activities are required at cleavage stages. Similarly, providing extra Foxd4l1 or Zic2 to explants made from dorsal, neural plate-producing blastomeres significantly increased the expression of early neural genes, whereas knocking down either significantly reduced them. These results show that maternally delivered transcription factors bias cleavage stage blastomeres to a neural fate. We demonstrate that mouse and human homologs of Foxd4l1 have similar functional domains compared to the frog protein, as well as conserved transcriptional activities when expressed in Xenopus embryos and blastomere explants. genesis 54:334-349, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

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, fibroblast growth factor, vascular endothelial growth factor, and Wnt5. Each is necessary for explicit tasks in skeleton production., (Copyright © 2014 Wiley Periodicals, Inc.)

The bovine trophectoderm (TE) undergoes a dramatic morphogenetic transition prior to uterine endometrial attachment. Many studies have documented trophoblast-specific gene expression profiles at various pre-attachment stages, yet genetic interactions within the transitioning TE gene regulatory network are not well characterized. During bovine embryogenesis, transcription factors OCT4 and CDX2 are co-expressed during early trophoblast elongation. In this study, the bovine trophectoderm-derived CT-1 cell line was utilized as a genetic model to examine the roles of CDX2 and OCT4 within the bovine trophoblast gene regulatory network. An RT-PCR screen for TE-lineage transcription factors identified expression of CDX2, ERRB, ID2, SOX15, ELF5, HAND1, and ASCL2. CT-1 cells also express a nuclear-localized, 360 amino acid OCT4 ortholog of the pluripotency-specific human OCT4A. To delineate the roles of CDX2 and OCT4 within the CT-1 gene network, CDX2 and OCT4 levels were manipulated via overexpression and siRNA-mediated knockdown. An increase in CDX2 negatively regulated OCT4 expression, but increased expression of IFNT, HAND1, ASCL2, SOX15, and ELF5. A reduction of CDX2 levels exhibited a reciprocal effect, resulting in decreased expression of IFNT, HAND1, ASCL2, and SOX15. Both overexpression and knockdown of CDX2 increased ETS2 transcription. In contrast to CDX2, manipulation of OCT4 levels only revealed a positive autoregulatory mechanism and upregulation of ASCL2. Together, these results suggest that CDX2 is a core regulator of multiple trophoblast genes within CT-1 cells., (© 2013 Wiley Periodicals, Inc.)

BMP signaling is pivotal for normal limb bud development in vertebrate embryos and genetic analysis of receptors and ligands in the mouse revealed their requirement in both mesenchymal and ectodermal limb bud compartments. In this study, we genetically assessed the potential essential functions of SMAD4, a mediator of canonical BMP/TGFß signal transduction, in the mouse limb bud ectoderm. Msx2-Cre was used to conditionally inactivate Smad4 in the ectoderm of fore- and hindlimb buds. In hindlimb buds, the Smad4 inactivation disrupts the establishment and signaling by the apical ectodermal ridge (AER) from early limb bud stages onwards, which results in severe hypoplasia and/or aplasia of zeugo- and autopodal skeletal elements. In contrast, the developmentally later inactivation of Smad4 in forelimb buds does not alter AER formation and signaling, but prolongs epithelial-mesenchymal feedback signaling in advanced limb buds. The late termination of SHH and AER-FGF signaling delays distal progression of digit ray formation and inhibits interdigit apoptosis. In summary, our genetic analysis reveals the temporally and functionally distinct dual requirement of ectodermal Smad4 during initiation and termination of AER signaling., (Copyright © 2013 Wiley Periodicals, Inc.)

The commitment of regions of the embryo to form particular tissues or organs is a central concept in development, but the mechanisms controlling this process remain elusive. The well-studied model of lens induction is ideal for dissecting key phases of the commitment process. We find in Xenopus tropicalis, at the time of specification of the lens, i.e., when presumptive lens ectoderm (PLE) can be isolated, cultured, and will differentiate into a lens that the PLE is not yet irreversibly committed, or determined, to form a lens. When transplanted into the posterior of a host embryo lens development is prevented at this stage, while ~ 3 h later, using the same assay, determination is complete. Interestingly, we find that specified lens ectoderm, when cultured, acquires the ability to become determined without further tissue interactions. Furthermore, we show that specified PLE has a different gene expression pattern than determined PLE, and that determined PLE can maintain expression of essential regulatory genes (e.g., foxe3, mafB) in an ectopic environment, while specified PLE cannot. These observations set the stage for a detailed mechanistic study of the genes and signals controlling tissue commitment., (Copyright © 2012 Wiley Periodicals, Inc.)

In vertebrates, different Wnt-signaling pathways are required in a temporally coordinated manner to promote cardiogenesis. In Drosophila, wingless holds an essential role in heart development. Among the known Drosophila Wnts is DWnt4, the function of which has been studied in various developmental processes except for heart development. We re-evaluated the expression pattern of DWnt4 during embryogenesis and show that transcripts are not restricted to the dorsal ectoderm but are also present in the cardiogenic mesoderm. Moreover, we detect DWnt4 mRNA transcripts in myocardial cells by stage 16. The heart phenotype in DWnt4 mutant embryos is characterized by various degrees of disrupted expression of different cardiac markers. Overexpression of Dwnt4 also affects heart marker expression, which can be partially rescued by simultaneous inhibition of PKC. Our data reveal a role for DWnt4 in cardiogenesis; however, integration of DWnt4 with other known signaling pathways that function in heart development still awaits further investigation., (Copyright © 2012 Wiley Periodicals, Inc.)

Gastrulation in vertebrates is a conserved process, which involves transition from cellular pluripotency to early precursors of ectoderm, mesoderm, and endoderm. Pluripotency control during this stage is far from being understood. Recent genetic and transcriptomic studies in zebrafish suggest that the core pluripotency transcription factors (TFs) Pou5f1 and TFs of the SoxB1 group are critically involved in large-scale temporal coordination of gene expression during gastrulation. A significant number of evolutionary conserved target genes of Pou5f1 in zebrafish are also involved in stem-cell circuit in mammalian ES cell cultures. Here, I will review the roles of Pou5f1 in development and discuss the evolutionary conservation of Pou5f1 functions and their relation to pluripotency control., (Copyright © 2011 Wiley Periodicals, Inc.)

Epithelial invagination in many model systems is driven by apical cell constriction, mediated by actin and myosin II contraction regulated by GTPase activity. Here we investigate apical constriction during chick lens placode invagination. Inhibition of actin polymerization and myosin II activity by cytochalasin D or blebbistatin prevents lens invagination. To further verify if lens placode invaginate through apical constriction, we analyzed the role of Rho-ROCK pathway. Rho GTPases expression at the apical portion of the lens placode occurs with the same dynamics as that of the cytoskeleton. Overexpression of the pan-Rho inhibitor C3 exotoxin abolished invagination and had a strong effect on apical myosin II enrichment and a mild effect on apical actin localization. In contrast, pharmacological inhibition of ROCK activity interfered significantly with apical enrichment of both actin and myosin. These results suggest that apical constriction in lens invagination involves ROCK but apical concentration of actin and myosin are regulated through different pathways upstream of ROCK. genesis 49:368-379, 2011., (2011 Wiley-Liss, Inc.)

Neurogenic placodes are transient, thickened patches of embryonic vertebrate head ectoderm that give rise to the paired peripheral sense organs and most neurons in cranial sensory ganglia. We present the first analysis of gene expression during neurogenic placode development in a basal actinopterygian (ray-finned fish), the North American paddlefish (Polyodon spathula). Pax3 expression in the profundal placode confirms its homology with the ophthalmic trigeminal placode of amniotes. We report the conservation of expression of Pax2 and Pax8 in the otic and/or epibranchial placodes, Phox2b in epibranchial placode-derived neurons, Sox3 during epibranchial and lateral line placode development, and NeuroD in developing cranial sensory ganglia. We identify Sox3 as a novel marker for developing fields of electrosensory ampullary organs and for ampullary organs themselves. Sox3 is also the first molecular marker for actinopterygian ampullary organs. This is consistent with, though does not prove, a lateral line placode origin for actinopterygian ampullary organs., (Copyright © 2011 Wiley-Liss, Inc.)

cDNA derived from trophectoderm (TE) and embryonic disc (ED) of a single day 12 porcine embryo was subjected to next-generation sequencing using the Illumina platform. The short sequencing reads from triplicate sequencing runs were aligned to a custom database designed to represent the known porcine transcriptome. As expected, genes involved in epithelial cell function and steroid biosynthesis were more abundant in cells from the TE; genes involved in maintenance of pluripotency and chromatin remodeling were more highly expressed in cells from the ED. Quantitative real-time PCR was used to confirm the validity of the approach. We conclude that gene expression profiles of even extremely small samples (

During the metamorphosis of tunicate ascidians, the swimming larva uses its three anterior papillae to detect the substrate for settlement, reabsorbs its chordate-like tail, and becomes a sessile oozooid. In view of the crucial role played by the anterior structures and their nerve relations, we applied electron microscopy and immunocytochemistry to study the larva of the colonial ascidian Botryllus schlosseri, following differentiation of the anterior epidermis during late embryogenesis, the larval stage, and the onset of metamorphosis. Rudiments of the papillae appear in the early tail-bud stage as ectodermic protrusions, the apexes of which differentiate into central and peripheral bipolar neurons. Axons fasciculate into two nerves direct to the brain. Distally, the long, rod-like dendritic terminations extend during the larval stage, becoming exposed to sea water. After the larva selects and adheres to the substrate, these neurons retract and regress. Adjacent to the papillae, other scattered neurons insinuate dendrites into the tunic and form the net of rostral trunk epidermal neurons (RTENs) which fasciculate together with the papillary neurons. Our data indicate that the papillae are simple and coniform, the papillary neurons are mechanoreceptors, and the RTENs are chemoreceptors. The interpapillary epidermal area, by means of an apocrine secretion, provides sticky material for temporary adhesion of the larva to the substrate., (2009 Wiley-Liss, Inc.)

Lens formation in mouse is critically dependent on proper development of the retinal neuroectoderm that is located close beneath the head surface ectoderm. Signaling from the prospective retina triggers lens-specific gene expression in the surface-ectoderm. Supression of canonical Wnt/beta-catenin signaling in the surface ectoderm is one of the prerequisites for lens development because, as we show here, ectopic Wnt activation in the retina and lens abrogates lens formation. Wnt inhibiton is mediated by signals coming from the retina but its exact mechanism is unknown. We show that Pax6 directly controls expression of several Wnt inhibitors such as Sfrp1, Sfrp2, and Dkk1 in the presumptive lens. In accordance, absence of Pax6 function leads to aberrant canonical Wnt activity in the presumptive lens that subsequently impairs lens development. Thus Pax6 is required for down-regulation of canonical Wnt signaling in the presumptive lens ectoderm., ((c) 2009 Wiley-Liss, Inc.)

The largest of the cranial ganglia, the trigeminal ganglion, relays cutaneous sensations of the head to the central nervous system. Its sensory neurons have a dual origin from both ectodermal placodes and neural crest. Here, we show that the birth of neurons derived from the chick ophthalmic trigeminal placode begins prior to their ingression (HH11), as early as HH8, and considerably earlier than previously suspected (HH16). Furthermore, cells exiting the cell cycle shortly thereafter express the ophthalmic trigeminal placode marker Pax3 (HH9). At HH11, these postmitotic Pax3+ placode cells begin to express the pan-neuronal marker neurofilament while still in the ectoderm. Analysis of the ectodermal origin and distribution of these early postmitotic neurons reveals that the ophthalmic placode extends further rostrally than anticipated, contributing to neurons that reside in and make a significant contribution to the ophthalmic trigeminal nerve. These data redefine the timing and extent of neuron formation from the ophthalmic trigeminal placode.

SOX genes encode transcription factors acting in various developmental processes in bilaterian animals, such as stem cell maintenance and the control of specification and differentiation of cell types in a variety of contexts, notably in the developing nervous system. To gain insights into the early evolution of this important family of developmental regulators, we investigated the expression of one subgroup B, two subgroup E, one subgroup F and two divergent SOX genes in the cydippid larva and in the adult of the ctenophore Pleurobrachia pileus. Transcripts of the two unclassified SOX (PpiSOX2/12) were detected in the female germ line and in various populations of putative somatic stem cells/undifferentiated progenitors. The remaining genes had spatially restricted expression patterns in ciliated epithelial cells, notably within neuro-sensory territories. These data are compatible with an ancient involvement of SOX proteins in controlling aspects of stem cell maintenance, cellular differentiation and specification, notably within neuro-sensory epithelia. In addition, the results highlight the complexity of the ctenophore anatomy and suggest that the SOX played an important role in the elaboration of the unique ctenophore body plan during evolution, through multiple gene co-option., (Copyright 2008 Wiley-Liss, Inc.)

Nodal factors play crucial roles during embryogenesis of chordates. They have been implicated in a number of developmental processes, including mesoderm and endoderm formation and patterning of the embryo along the anterior-posterior and left-right axes. We have analyzed the function of the Nodal signaling pathway during the embryogenesis of the sea urchin, a non-chordate organism. We found that Nodal signaling plays a central role in axis specification in the sea urchin, but surprisingly, its first main role appears to be in ectoderm patterning and not in specification of the endoderm and mesoderm germ layers as in vertebrates. Starting at the early blastula stage, sea urchin nodal is expressed in the presumptive oral ectoderm where it controls the formation of the oral-aboral axis. A second conserved role for nodal signaling during vertebrate evolution is its involvement in the establishment of left-right asymmetries. Sea urchin larvae exhibit profound left-right asymmetry with the formation of the adult rudiment occurring only on the left side. We found that a nodal/lefty/pitx2 gene cassette regulates left-right asymmetry in the sea urchin but that intriguingly, the expression of these genes is reversed compared to vertebrates. We have shown that Nodal signals emitted from the right ectoderm of the larva regulate the asymmetrical morphogenesis of the coelomic pouches by inhibiting rudiment formation on the right side of the larva. This result shows that the mechanisms responsible for patterning the left-right axis are conserved in echinoderms and that this role for nodal is conserved among the deuterostomes. We will discuss the implications regarding the reference axes of the sea urchin and the ancestral function of the nodal gene in the last section of this review.

Putative stem cells have recently been isolated from several extra-embryonic tissues, including Wharton's Jelly and umbilical cord blood. Relevant studies have focused on primary cultures established from freshly isolated tissues. In this report, we examine the plasticity of 472 cells, a cryopreserved human amniocyte cell line originally isolated in 1974. Under conditions conducive for proliferation, the amniocytes displayed fibroblast-like morphologies and expressed Oct4 and Rex1, genes associated with pluripotency. Perhaps indicative of inherent plasticity, 472 cells simultaneously expressed ectodermal beta-III-tubulin and mesodermal fibronectin. When cultured under conditions that promote neural differentiation, the cells adopted neuronal morphologies and expressed neuronal genes, including Gap-43, NF-M, tau, and synaptophysin. Exposure to culture conditions that encourage osteogenic differentiation resulted in increased expression of alkaline phosphatase (ALP) and the deposition of mineralized matrix, established markers of bone cell differentiation. In sum, this population of human amniocytes appears to be multipotent, capable of in vitro differentiation to ectodermal and mesodermal cell types. Retention of this plasticity through decades of cryopreservation suggests that amniocytes might be candidates for future cell-based therapies.

Rat primary microglia (MG) acquired a multipotent property to give rise to neuroectodermal cells through two-step culture in 10 and 70% serum-supplemented media for 5 days. Such multipotent MG, called promicroglioblasts (ProMGBs), formed cell aggregates, which generated cells with neuroectodermal phenotypes shortly after their transfer into serum-free medium. As revealed by immunohistochemistry, there were a few MG expressing NG2 chondroitin sulfate proteoglycan (NG2) in the neonatal rat brain. Primary culture from the neonatal brain contained NG2+ MG, which appeared to be the source of NG2+ ProMGB aggregates. The aggregates were MG marker+/NG2+/GFAP+/NCAM+/S-100beta- and had alkaline phosphatase activity. The marked accumulation of NG2+ MG was observed close to stab wounds made in the mature rat brain. The accumulated NG2+ MG in the wound gradually decreased in number, but the cells persisted up to 150 days postlesioning. In addition, GFAP immunoreactivity increased markedly around the wound. The NG2+ MG in the wounds separated with trypsin-EDTA formed NG2+ aggregates in 70% serum-supplemented medium and then transformed into cells with neuroectodermal phenotypes in serum-free medium. Although it is difficult to separate viable neurons from mature brains, cells from stab wounds generated process-bearing beta-tubulin III+ cells in vitro easily. These data suggest that NG2+ MG in normal developing or pathologic brains are involved in the genesis or regeneration of the brain., (Copyright 2006 Wiley-Liss, Inc.)

Radial glial cell origins and functions have been studied extensively in the brain; however, questions remain relating to their origin and fate in the spinal cord. In the present study, radial glia are investigated in vivo using the neuroepithelial markers nestin and vimentin and the gliogenic markers GLAST, BLBP, 3CB2, and glial fibrillary acidic protein (GFAP). This has revealed heterogeneity among nestin/vimentin-positive precursor cells and suggests a lineage progression from neuroepithelial cell through to astrocyte in the developing spinal cord. A population of self-renewing radial cells, distinct from an earlier pseudo-stratified neuroepithelium, that resemble radial glial cells in morphology but do not express GLAST, BLBP, or 3CB2, is revealed. These radial cells arise directly from the spinal cord neuroepithelium and are probably the progenitors of neurons and the earliest appearing radial glial cells. GLAST/BLBP-positive radial glia first appear in the ventral cord at E14, and these cells gradually transform through one or more intermediate stages into differentiated astrocytes. Few if any neurons appear to be derived from radial glial cells, which are instead the major sources of astrocytes in the spinal cord. Evidence for the nonradial glial cell origins of some white matter astrocytes is also presented.

Members of the Dlx gene family play essential roles in the development of the zebrafish and mouse inner ear, but little is known regarding Dlx genes and avian inner ear development. We have examined the inner ear expression patterns of Dlx1, Dlx2, Dlx3, Dlx5, and Dlx6 during the first 7 days of chicken embryonic development. Dlx1 and Dlx2 expression was seen only in nonneuronal cells of the cochleovestibular ganglion and nerves from stage 21 to stage 32. Dlx3 marks the otic placode beginning at stage 9 and becomes limited to epithelium adjacent to the hindbrain as invagination of the placode begins. Dlx3 expression then resolves to the dorsal otocyst and gradually becomes limited to the endolymphatic sac by stage 30. Dlx5 and Dlx6 expression in the developing inner ear is first seen at stages 12 and 13, respectively, in the rim of the otic pit, before spreading throughout the dorsal otocyst. As morphogenesis proceeds, Dlx5 and Dlx6 expression is seen throughout the forming semicircular canals and endolymphatic structures. During later stages, both genes are seen to mark the distal surface of the forming canals and display expression complementary to that of BMP4 in the vestibular sensory regions. Dlx5 expression is also seen in the lagena macula and the cochlear and vestibular nerves by stage 30. These findings suggest important roles for Dlx genes in the vestibular and neural development of the avian inner ear., ((c) 2005 Wiley-Liss, Inc.)

Krüppel-like factor 4 (Klf4) belongs to the family of transcription factors that are thought to be involved in the regulation of epithelial and germ cell differentiation, based on their expression in postproliferative cells of the skin, gut, and testes. Gene ablation experiments suggest that Klf4 plays a role in keratinocyte differentiation, since mice lacking Klf4 fail to establish proper barrier function and, as a consequence, die postnatally due to dehydration. Recent studies have shown that Klf4 is also expressed in postnatal male mice, in postmeiotic sperm cells undergoing terminal differentiation into sperm cells. However, prior to the current study, the expression pattern of Klf4 during early and mid-embryogenesis had not been examined. Here we demonstrate that Klf4 transcripts can be detected from embryonic day 4.5 (E4.5) on in the developing conceptus, and that Klf4 expression before E10 is restricted to extraembryonic tissues. The embryo proper displays a highly dynamic and changing Klf4 signal from E10 of murine development on. In addition to being expressed in a stripe of mesenchymal cells extending from the forelimb bud rostrally over the branchial arches to the developing eye, Klf4 is also expressed in the mesenchyme surrounding the nasal pit at day E11.5. In addition, Klf4 has been detected in the apical ectodermal ridge and adjacent mesenchymal cells in the limb buds, and in mesenchymal cells of the developing body wall in trunk areas. These findings suggest that Klf4 plays an important role in regulating cellular proliferation, which underlies the morphogenetic changes that shape the developing embryo., (Copyright 2003 Wiley-Liss, Inc.)

The Bex1/Rex3 gene was recently identified as an X-linked gene that is differentially expressed between parthenogenetic and normal fertilized, preimplantation stage mouse embryos. The Bex1/Rex3 gene appears to be expressed preferentially from the maternal X chromosome in blastocysts, but from either X chromosome in later stage embryonic tissues and adult tissues. To investigate whether differential expression of the Bex1/Rex3 gene between normal and parthenogenetic blastocyst stage embryos reflects genomic imprinting at the Bex1/Rex3 locus itself, or instead is the result of preferential inactivation of the paternal X chromosome or differences in timing of cellular differentiation, we examined in detail the expression pattern of the Bex1/Rex3 mRNA in normal preimplantation stage embryos, and compared its expression between androgenetic, gynogenetic, and normal fertilized embryos. Expression data reveal that the Bex1/Rex3 gene is initially transcribed at the 2-cell stage, transiently induced at the 8-cell stage, and then increases in expression again at the blastocyst stage. Very little expression is observed in isolated inner cell masses, indicating selective expression in the trophectoderm. Comparisons of Bex1/Rex3 mRNA expression between male and female androgenetic and control embryos and gynogenetic embros failed to reveal any significant difference in expression between the different classes of embryos at the 8-cell stage, or the expanding blastocyst stage (121 hr post-hCG). At the late blastocyst stage (141 hr post-hCG), expression was significantly lower in XY control embryos as compared with XX controls. Bex1/Rex3 mRNA expression did not differ between XX and XY androgenones at the blastocyst stage or between gynogenones and XX control embryos. Thus, the Bex1/Rex3 gene does not appear to be regulated directly by genomic imprinting during the preimplantation period, just as it is not regulated by imprinting at later stages. Apparent differences in gene expression may arise through the effects of trophectoderm-specific expression coupled with differences in timing of trophectoderm differentiation between the different classes of embryos and effects of preferential paternal X chromosome inactivation (XCI)., (Copyright 2002 Wiley-Liss, Inc.)

The origin of vascular pericytes (PCs) and smooth muscle cells (vSMCs) in the brain has hitherto remained an open question. In the present study, we used the quail-chick chimerization technique to elucidate the lineage of cranial PCs/vSMCs. We transplanted complete halves of brain anlagen, or dorsal (presumptive neural crest [NC]) or ventral cranial neural tube. Additional experiments included transplantations of neuroectoderm into limb mesenchyme, and of head mesoderm or limb mesenchyme into paraxial head mesoderm. After interspecific transplantation of quail brain rudiment, graft-derived vSMCs were found in the vessel walls of the grafted brain. Notably, transplanted ventral neural tube also gave rise to vSMCs. After grafting of quail head mesoderm, quail endothelial cells were found in the host brain, but no vSMCs of donor origin. Grafting of quail whole or ventral neural tube into the limb bud led to endowment of graft and host vessels with graft-derived vSMCs. Quail limb bud mesenchyme contributed to vSMCs in the ectopic neural graft, but, when transplanted into paraxial head mesenchyme, it did not form intraneural vSMCs. After orthotopic transplantation of cranial NC, graft-derived vSMCs were not only found in meninges and brain of the operated side, but also on the contralateral side. Our results show that 1) avian cranial neuroectoderm is able to differentiate into vSMCs of the brain; 2) this potential is not restricted to the prospective NC; and 3) neither cranial mesoderm nor cranially transplanted limb bud mesoderm can give rise to brain vSMC., (Copyright 2002 Wiley-Liss, Inc.)

In vertebrates, there are six or more copies of genes related to the Drosophila pattern formation homeodomain gene Distal-less. Among this family, Dlx3 and Dlx5 share extensive sequence homology and have similar, but distinctive, expression patterns, suggesting that these two factors may have substantially redundant developmental functions. Here we show that at the earliest phases of embryogenesis in Xenopus, there are significant differences between Dlx3 and Dlx5 expression and that this correlates with different functions in the restriction of neural crest and neural plate boundaries, respectively., (Copyright 2001 Wiley-Liss, Inc.)

The developmental control genes of the Pax family are essential for brain development. Several Pax genes are also involved in chromosomal translocations causing malignancies in humans, and Pax5 expression is deregulated in medulloblastomas. We have investigated whether Pax5 can induce tumors in the developing mouse brain. Primary mouse embryonic neuroectodermal cells were retrovirally transduced with mouse Pax5 and transplanted into the brain of syngeneic host mice. No tumors developed in 36 transplants after one year, and there were no alterations in the differentiation pattern of the neural transplants. We then generated transgenic mice expressing human Pax5 under control of the Engrailed-2 promoter, which is expressed in the cerebellar external granule cell layer and in medulloblastomas. Sustained expression was achieved in the cerebellum of transgenic animals throughout lifetime. Expression levels were similar to those observed in human medulloblastomas. Again, cerebellar morphogenesis was undisturbed, and no tumors arose. These results strongly argue against a dominant transforming activity of PAX5 in NEC and in cerebellar granule cell precursors of mice, and underline the restricted tissue-specificity of PAX5 related oncogenesis., (Copyright 2001 Wiley-Liss, Inc.)

The mixed lineage leukemia gene (MLL) was originally identified through its involvement in reciprocal translocations in leukemias. MLL codes for a large multidomain protein and bears homology to the Drosophila developmental control gene trithorax in two small domains in the amino terminal region, the central zinc finger domain and the carboxy SET domain. Like the Drosophila trx, MLL has also been shown to be a positive regulator of Hox gene expression. We have targeted Mll (the murine homologue of MLL) in exon 5 causing expression of three truncated in-frame Mll transcripts. These transcripts retain all or some of the AT hook motifs and the DMT domain. This mutant allele causes early in vivo preimplantation lethality of homozygous embryos prior to the 2-cell stage. Embryos cultured in vitro progress to the 2-cell stage, but further development is arrested. The heterozygotes exhibit mild skeletal defects as well as defects in some neuroectodermal derivatives.

The mucus secreting cement gland is the anterior-most ectodermal organ of the Xenopus embryo. The homeobox genes Pltx1 and Pitx2c are expressed in the cement gland primordium. Misexpression of both genes induced ectopic cement gland tissue in whole embryos and transcription of the marker genes Xag1 and Xag2 in animal cap explant cultures. Antisense morpholino oligonucleotides against Pitx1 and Pitx2c inhibited ectopic cement gland formation induced by otx2. Gene knock downs generated by morpholino oligonucleotides were specific and could be rescued by coinjection of Pitx mRNAs. These data demonstrate for the first time the requirement of specific genes for cement gland formation by loss-of-function experiments. genesis 30:144--148, 2001., (Copyright 2001 Wiley-Liss, Inc.)

We have cloned and sequenced cDNAs encoding two isoforms of XMAP215, a high molecular weight microtubule-associated protein identified in Xenopus eggs. XMAP215 is approximately 80% identical in amino acid sequence to the product of ch-TOG, a cDNA that is over expressed in certain human tumors [Charrasse et al., 1995: Eur J Biochem 234:406-413]. Northern and Western blots demonstrated that XMAP215 is expressed throughout development, from oogenesis to tadpole. We identified two XMAP215 transcripts differing only in the presence of a 108-bp sequence encoding a 36 amino acid insert. RT-PCR revealed that the transcripts encoding these two isoforms are expressed at distinct times during development: a transcript containing the insert (encoding XMAP215(M)) is expressed during oogenesis and is present through gastrulation. The second transcript (encoding XMAP215(Z)) lacks the 108-bp insert and is expressed from gastrulation onward. In situ hybridization demonstrated that XMAP215 transcripts are localized to the ectoderm of early embryos and in the developing nervous system during later development. These results suggest that XMAP215 plays important roles in at least two phases of development: (1) regulating the assembly of MTs during the rapid cell divisions after fertilization, and (2) regulating MT assembly during the development of the nervous system., (Copyright 2000 Wiley-Liss, Inc.)

SPARC (Secreted Protein, Acidic, Rich in Cysteine) was detected by immunohistochemistry in the sensorial layer of the bilayered embryonic epidermis of Xenopus laevis during neurulation, when a subset of the sensorial cells are selected to differentiate into ciliated cell precursors. After the ciliated cells had intercalated into the outer layer and had undergone ciliogenesis, intense SPARC immunostaining was associated with the cilia and remained associated with the cilia throughout their persistence on the epidermis. Circumferential SPARC immunostaining was also detected at the interface between surface epithelial cells. Animal cap explants indicated that the embryonic activation of SPARC expression in the dorsal ectoderm does not require signaling from factors secreted by the underlying mesoderm. Immunoelectron microscopy revealed that SPARC is intimately associated with the 9 + 2 microtubule arrays of cilia. Our data indicate that SPARC plays a role in the development and function of the surface ciliated epidermis of Xenopus embryos. We propose that the counter-adhesive activity of SPARC facilitates the intercalation of ciliary cell precursors to the surface epithelial layer, where its Ca(2+)-binding abilities promote cell-cell adhesion. Based on its association with ciliary microtubule arrays, we also propose that intracellular SPARC may play a role in regulating ciliary beat frequency and polarity., (Copyright 2000 Wiley-Liss, Inc.)

On day 12-13 of gestation, the preimplantation pig conceptus undergoes a dramatic morphologic change from an approximately 1-cm sphere to a nearly 1-m long thread. This transformation, referred to as elongation, occurs in just 12-24 h. Elongation is primarily the result of trophectodermal cell shape changes, as there is relatively little mitosis during this stage of development. Thus far, descriptions of elongation have been limited to histologic and immunofluorescent studies of cell morphology and gross biochemical evaluations. We hypothesized that the changes in trophectoderm morphology likely involves significant changes in gene expression. Therefore, we used RNA arbitrarily primed-PCR (RAP-PCR) to characterize potential differential gene expression by trophectodermal cells during pig conceptus elongation. We found that the porcine heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 was shown to be differentially expressed by trophectodermal cells during elongation. We suggest that regulated alternative splicing may contribute to the morphogenetic process of elongation. genesis 26:9-14, 2000. Published 2000 Wiley-Liss, Inc.

Dactylaplasia, or Dac, was recently mapped to the distal portion of mouse chromosome 19 and shown to be inherited as an autosomal semi-dominant trait characterized by missing central digital rays. The most common locus for human split hand split foot malformation, also typically characterized by missing central digital rays, is 10q25, a region of synteny to the Dac locus. The Dac mouse appears to be an ideal genotypic and phenotypic model for this human malformation syndrome. Several genes lie in this region of synteny, however, only Fibroblast Growth Factor 8, or Fgf-8, has been implicated to have a role in limb development. We demonstrate that the developmental mechanism underlying loss of central rays in Dac limbs is dramatic cell death of the apical ectodermal ridge, or AER. This cell death pattern is apparent in E10.5-11.5 Dac limb buds stained with the supravital dye Nile Blue Sulfate. We demonstrate that Fgf8 expression in wild type limbs colocalizes spatially and temporally with AER cell death in Dac limbs. Furthermore, in our mapping panel, there is an absence of recombinants between Fgf-8 and the Dac locus in 133 backcross progeny with a median linkage estimate of approximately 0.5 cM. Thus, our results demonstrate that cell death of the AER in Dac limbs silences the role of the AER as key regulator of limb outgrowth, and that Fgf-8 is a strong candidate for the cause of the Dac phenotype.

Although gap junctions are not known to be important in mediating cell-cell interactions amongst migratory cells, our studies showed that the connexin 43 (Cx43) gap junction gene is widely expressed in mouse neural crest cell lineages. Using in situ hybridization analysis, Cx43 expression was detected in presumptive neural crest cells emerging from the neural folds of the early postimplantation embryo. Neural crest expression of the Cx43 gap junction gene was also indicated by the analysis of transgenic mice containing a lacZ reporter construct driven by the Cx43 promoter. In neural tube explant cultures of these transgenic mice, lacZ expression was observed in the emerging neural crest outgrowths. Whole mount X-gal staining of these transgenic embryos at various stages of development showed lacZ expression in neural crest cells distributed along the entire craniocaudal axis, with expression found in both cranial and trunk neural crest cells contributing to a wide range of embryonic tissues. This included presumptive cardiac neural crest cells localized in the heart. In light of the widespread expression of Cx43 in neural crest cell lineages, dye injection studies, were carried out to determine if neural crest cells are functionally coupled via gap junctions. Such studies revealed extensive dye coupling among presumptive neural crest cells, thus demonstrating that these migratory cells are indeed gap junctional communication competent. In total, these observations suggest that gap junctions may play a role in mouse neural crest development. This possibility is particularly intriguing given the recent finding that the Cx43 knockout mice die of defects associated with the outflow tract [Reaume et al., 1995], a region of the heart in which neural crest cells are required for normal development.

The changes in protein synthesis that occur during differentiation of the primitive germ layers were examined by high-resolution, two-dimensional gel electrophoresis of proteins synthesized in 6.5 and 7.5 days postcoitum (d.p.c.) mouse embryos. For 6.5 d.p.c. embryos, protein synthesis patterns were compared between whole extraembryonic and embryonic regions and between embryonic visceral endoderm and embryonic ectoderm. For 7.5 d.p.c. embryos, comparisons were made between extraembryonic and embryonic regions and between isolated embryonic endoderm, mesoderm, and ectoderm. Each of the isolated 7.5 d.p.c. germ layers was divided into anterior and posterior fragments in order to evaluate possible regional differences in gene expression along the anterior-posterior axis. Comparisons of protein synthesis patterns revealed the greatest difference between isolated endoderm and ectoderm, indicating that by as early as 6.5 d.p.c. patterns of gene expression differ significantly between these tissues. The greatest similarities were found between ectoderm and whole embryonic regions and between endoderm and whole extraembryonic regions, which most likely reflects the overall cellular compositions of the embryonic and extraembryonic regions. Based on their patterns of synthesis, four groups of proteins were identified that were preferentially synthesized in either endoderm or ectoderm. These provide useful markers for studying differentiation in these tissues. One other protein, migrating at the position expected for vimentin, was synthesized at an elevated rate in isolated mesoderm. We also observed differences in rates of synthesis of alpha-tubulin and tropomyosin-5 indicative of potential differences in cytoskeletal composition among the germ layers beyond those previously described. The difference in overall protein synthesis patterns between anterior and posterior regions was greatest in the embryonic endoderm, indicating that differentiation along the anterior-posterior axis may be initiated sooner or may proceed more rapidly in the endoderm than in the other germ layers. These data provide the first quantitative evaluation of the degree to which differentiation of the three primitive germ layers affects protein synthesis patterns and reveal potentially useful markers of endoderm and ectoderm differentiation.

We report results from a study of Hox-7 expression during mouse embryonic and fetal development and compare the localization of Hox-7 transcripts with those of the retinoic acid receptors. Transcripts were detected by in situ hybridization. Hox-7 expression occurs in (1) cephalic neural crest and its derivatives, (2) sites of ectomesodermal interaction, (3) extraembryonic tissues, and (4) endocardial cells. Hox-7 does not seem to be involved in defining rostrocaudal boundaries, but instead appears to be expressed along the proximodistal axes at these sites. We further investigated the active sites of morphogenesis, which involve an ectomesodermal interaction (e.g., limb buds, visceral arches), including genital tubercle and tail ridge. These are regions highly positive for Hox-7 transcripts, and many are known to be sites for the expression of gamma-retinoic acid receptors (RARs) and cellular retinoic acid binding proteins. Most regions that express Hox-7 are subregions of gamma-RAR expression. In the developing limb bud, expression of Hox-7 takes place in the interdigital region, where it overlaps areas of beta-RAR expression.

The effects of cytosine arabinoside, 6-aminonicotinamide, and 6-mercaptopurine riboside on the incorporation of [14C] glucose moieties and [32P] phosphate into acid-soluble material and lipids, RNA, DNA, and protein were measured in the dissected mesoderm and ectoderm of mouse limb buds at the 42-45 (day 11) somite stage. Due to the different proliferative capacities of the two tissues the incorporation of the precursors into mesodermal cells was considerably higher the than into ectodermal ones. Cytosine arabinoside inhibited the incorporation of the precursor moieties only into DNA, but very early after its application. This effect was more obvious in mesoderm than ectoderm. 6-Aminonicotinamide interfered only with glucose metabolism, whereas the incorporation of phosphate was not affected. 14C radioactivity in the various cell components was similarly reduced in mesoderm and ectoderm. 6-mercaptopurine riboside caused an increased incorporation of precursor material in all fractions studied in the mesoderm as well as in the ectoderm during the first 12 hours. This was succeeded by a dramatic decrease of incorporated 14C and 32P radioactivity. Differences of response in the tissues could not be detected with this drug. It is suggested that the malformations of the extrmities caused by these antimetabolites may be predominantly attributed to changes in the cell function rather than to gross effects on cell metabolism.

The plant lectin concanavalin A (con A) causes malformations of rabbit embryos when 160 micrograms (in 40 microliter) are injected into the exocoelom on gestational days 12-15 but does not cause malformations on days 10-11. The purpose of this study was to investigate the mechanism for increased susceptibility of day 12-15 embryos to con A teratogenicity. Light microscopy of day 11 embryos 15-20 hr after treatment with con A revealed no observable difference from controls. Day 13 embryos at similar times exhibited limb buds with large areas that were denuded of ectoderm. Concurrent addition of alpha-methyl-D-mannoside (alpha MM), a specific inhibitor of con A, to the injection solution of day 13 embryos resulted in limb buds that appeared normal. The regions of con A binding to day 11 and day 13 embryos were visualized through epifluorescent microscopy of untreated embryos stained with fluorescein-labelled con A. Day 11 embryos exhibited moderate fluorescence on the surface of limb buds and the pericardial region. Day 13 embryos exhibited strong fluorescence of limb bud surfaces; the pericardial region remained moderately fluorescent. Addition of alpha MM to the incubation medium resulted in no fluorescence above background. Visualization of con A receptors was accomplished by ultrastructural analysis of forelimb buds stained with ferritin-labelled con A. Ferritin label was observed only on the surfaces of the ectoderm and was sparse over all regions of day 11 limb buds. In contrast, ferritin label was moderately heavy in all regions of the day 13 limb buds. No labelling occurred when the ferritin-labelled con A was preincubated with alpha MM. These observations indicate that the number of exposed con A receptors on limb buds of teratogenically sensitive embryos (day 13) is increased, compared with the number of exposed receptors on limb buds of younger, insensitive (day 11) embryos. The increased number of exposed con A receptors on limb buds during the teratogenically sensitive period provides not only increased binding of the lectin to sensitive embryos but also a potential mechanism for the anomalous attachment of distal regions of the limb buds to the body wall.