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We have previously reported that RING1 and YY1 binding protein (RYBP) is important for central nervous system development in mice and that Rybp null mutant (Rybp -/- ) mouse embryonic stem (ES) cells form more progenitors and less terminally differentiated neural cells than the wild type cells in vitro. Accelerated progenitor formation coincided with a high level of Pax6 expression in the Rybp -/- neural cultures. Since Pax6 is a retinoic acid (RA) inducible gene, we have analyzed whether altered RA signaling contributes to the accelerated progenitor formation and impaired differentiation ability of the Rybp -/- cells. Results suggested that elevated Pax6 expression was driven by the increased activity of the RA signaling pathway in the Rybp -/- neural cultures. RYBP was able to repress Pax6 through its P1 promoter. The repression was further attenuated when RING1, a core member of ncPRC1s was also present. According to this, RYBP and PAX6 were rarely localized in the same wild type cells during in vitro neural differentiation. These results suggest polycomb dependent regulation of Pax6 by RYBP during in vitro neural differentiation. Our results thus provide novel insights on the dynamic regulation of Pax6 and RA signaling by RYBP during mouse neural development., (© 2022. The Author(s).)

The organization and development of the nervous system are traditionally used for phylogenetic analysis and may be useful for clarification of evolution and phylogeny of some poor studied groups. One of these groups is brachiopods: most data on their nervous system organization were obtained in 19 th century. In this research, antibody staining and confocal laser scanning microscopy were used to study the nervous system of early ontogenetic stages of the brachiopod Novocrania anomala. Although N. anomala adults are thought to lack a supraenteric ganglion, a large supraenteric ganglion exists in N. anomala juveniles with either a trocholophe or a schizolophe. During ontogenesis, the supraenteric ganglion in the juvenile changes its shape: the commissure between the two lobes of the ganglion extends. This commissure possibly gives rise to the main brachial nerve in adults. The supraenteric ganglion gives rise to the cross (transversal) nerves that extend to the accessory brachial nerve, which gives rise to the tentacular nerves. In juveniles with a trocholophe, the accessory brachial nerve gives rise to the frontal and intertentacular nerves of tentacles that form a single row. When the trocholophe transforms into the schizolophe, the second row of tentacles appears and the innervation of the tentacles changes. The intertentacular nerves disappear and the second accessory nerve forms and gives rise to the laterofrontal tentacular nerves of the inner and outer tentacles and to the abfrontal nerves of the inner tentacles. The so-called subenteric ganglion, which was described as a ganglion in N. anomala adults, is represented by a large circumvisceral nerve in N. anomala juveniles.The results suggest that 'phoronid-like' non-specialized tentacles may be regarded as the ancestral type of tentacles for brachiopods and probably for all lophophorates. The presence of intertentacular nerves is the ancestral feature of all lophophorates. The transformation of the juvenile supraenteric ganglion into the main brachial nerve of N. anomala adults suggests that research is needed on the development and organization of the supraenteric ganglion and the main brachial nerve in other brachiopods, whose adults have a prominent supraenteric ganglion.

Immature multipotent embryonic peripheral glial cells, the Schwann cell precursors (SCPs), differentiate into melanocytes, parasympathetic neurons, chromaffin cells, and dental mesenchymal populations. Here, genetic lineage tracing revealed that, during murine embryonic development, some SCPs detach from nerve fibers to become mesenchymal cells, which differentiate further into chondrocytes and mature osteocytes. This occurred only during embryonic development, producing numerous craniofacial and trunk skeletal elements, without contributing to development of the appendicular skeleton. Formation of chondrocytes from SCPs also occurred in zebrafish, indicating evolutionary conservation. Our findings reveal multipotency of SCPs, providing a developmental link between the nervous system and skeleton., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

In amniotes, the development of the primitive streak and its accompanying 'organizer' define the first stages of gastrulation. Although these structures have been characterized in detail in model organisms, the human primitive streak and organizer remain a mystery. When stimulated with BMP4, micropatterned colonies of human embryonic stem cells self-organize to generate early embryonic germ layers 1 . Here we show that, in the same type of colonies, Wnt signalling is sufficient to induce a primitive streak, and stimulation with Wnt and Activin is sufficient to induce an organizer, as characterized by embryo-like sharp boundary formation, markers of epithelial-to-mesenchymal transition and expression of the organizer-specific transcription factor GSC. Moreover, when grafted into chick embryos, human stem cell colonies treated with Wnt and Activin induce and contribute autonomously to a secondary axis while inducing a neural fate in the host. This fulfils the most stringent functional criteria for an organizer, and its discovery represents a milestone in human embryology.

Hensen's node is the "organizer" of the avian and mammalian early embryo. It has many functions, including neural induction and patterning of the ectoderm and mesoderm. Some of the signals responsible for these activities are known but these do not explain the full complexity of organizer activity. Here we undertake a functional screen to discover new secreted factors expressed by the node at this time of development. Using a Signal Sequence Trap in yeast, we identify several candidates. Here we focus on Calreticulin. We show that in addition to its known functions in intracellular Calcium regulation and protein folding, Calreticulin is secreted, it can bind to BMP4 and act as a BMP antagonist in vivo and in vitro. Calreticulin is not sufficient to account for all organizer functions but may contribute to the complexity of its activity., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

We provide novel insights into the function(s) of β-carotene-15,15'-oxygenase (CMOI) during embryogenesis. By performing in vivo and in vitro experiments, we showed that CMOI influences not only lecithin:retinol acyltransferase but also acyl CoA:retinol acyltransferase reaction in the developing tissues at mid-gestation. In addition, LC/MS lipidomics analysis of the CMOI-/- embryos showed reduced levels of four phosphatidylcholine and three phosphatidylethanolamine acyl chain species, and of eight triacylglycerol species with four or more unsaturations and fifty-two or more carbons in the acyl chains. Cholesteryl esters of arachidonate, palmitate, linoleate, and DHA were also reduced to less than 30% of control. Analysis of the fatty acyl CoA species ruled out a loss in fatty acyl CoA synthetase capability. Comparison of acyl species suggested significantly decreased 18:2, 18:3, 20:1, 20:4, or 22:6 acyl chains within the above lipids in CMOI-null embryos. Furthermore, LCAT, ACAT1 and DGAT2 mRNA levels were also downregulated in CMOI-/- embryos. These data strongly support the notion that, in addition to cleaving β-carotene to generate retinoids, CMOI serves an additional function(s) in retinoid and lipid metabolism and point to its role in the formation of specific lipids, possibly for use in nervous system tissue., (© 2013.)

The nuclear casein kinase and cyclin-dependent kinase substrate 1 (NUCKS) is a highly phosphorylated nuclear protein that is overexpressed in many types of cancer. The flexibility of NUCKS and its extensive posttranslational modifications indicate that it is multifunctional, and its expression in most cell types suggests a housekeeping function. However, spatiotemporal expression of the Nucks protein during rodent development has not been reported. Thus, we investigated the expression of both the Nucks mRNA and protein during rat and mouse development by immunohistochemistry, in situ hybridization, Western immunoblotting, and reverse-transcription PCR analysis. We also used BLAST analysis against expressed sequence tag databases to determine whether a NUCKS homologue is expressed in invertebrate organisms. We found that Nucks expression increased during the initial stages of embryonic development, and then gradually decreased until birth in all tissues except the nervous tissue and muscle fibers. Interestingly, the expression of Nucks was very strong in migrating neural crest cells at E13.5 and ectoderm-derived tissues. In most tissues analyzed, the levels of Nucks correlated with the levels of Bax and activated caspase-3, which are indicative of apoptosis. Moreover, Nucks was upregulated very early during neuronal apoptosis in vitro. Expression analysis revealed that no transcript with close homology to the Nucks gene was present in invertebrates. The expression of Nucks in both proliferating and quiescent cells and its correlation with Bax levels and apoptosis strongly suggest that Nucks plays complex roles in cell homeostasis. Furthermore, the lack of homology in invertebrate organisms indicates a specific role for Nucks in vertebrate embryogenesis., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: Tissue microenvironment plays key roles in regulating the progression of aggressive tumors. Tumors are uncommon in the early embryo, suggesting that embryonic tissue microenvironments are nonpermissive for tumors. Yet, the effects of embryonic tissue microenvironments on tumor cells have not been extensively studied. We have, therefore, tested the behavior of human glioblastoma multiforme (GBM) cells transplanted into a central neural tissue microenvironment in the chicken embryo., Results: GBM cells were cultured as spheres to enrich for GBM stem cells (GSCs) and transduced with GFP for identification. Within the proliferative embryonic neural tissue, GSC-enriched GBM cells exhibited reduced proliferation and survival, altered gene expression, and formed no tumors, in marked contrast to their aggressive behavior in vitro and tumor formation in other tissue microenvironments including the chorioallantoic membrane of the chicken embryo and the brain of adult severe combined immunodeficiency (SCID) mice. Surviving cells in the spinal neural tube exhibited tumor-atypical expression profiles of neuron-, glia-, stem cell-, and tumor-related genes., Conclusions: Embryonic neural tissue provides a poor environment for GBM cell survival and tumor formation, and redirects differentiation toward a more benign phenotype. Understanding the anti-tumorigenic effects of this embryonic tissue microenvironment could provide opportunities to develop novel therapies for GBM treatment., (Copyright © 2013 Wiley Periodicals, Inc.)

Wnt signaling is an evolutionarily ancient pathway used to regulate many events during metazoan development. Genetic results from Caenorhabditis elegans more than a dozen years ago suggested that Wnt signaling in this nematode worm might be different than in vertebrates and Drosophila: the worm had a small number of Wnts, too many β-catenins, and some Wnt pathway components functioned in an opposite manner than in other species. Work over the ensuing years has clarified that C. elegans does possess a canonical Wnt/β-catenin signaling pathway similar to that in other metazoans, but that the majority of Wnt signaling in this species may proceed via a variant Wnt/β-catenin signaling pathway that uses some new components (mitogen-activated protein kinase signaling enzymes), and in which some conserved pathway components (β-catenin, T-cell factor [TCF]) are used in new and interesting ways. This review summarizes our current understanding of the canonical and novel TCF/β-catenin-dependent signaling pathways in C. elegans.

This study of spontaneous abortions and fetal deaths in utero used immunostains to evaluate the structure of developing cutaneous nerves. Melan-A immunostains were also used to screen 25 cases of grossly normal fetal skin for occult fetal nevi. Discrete portions of epidermis were generally supplied by branches emanating from regularly spaced deep cutaneous nerves, producing a wedge shape, interpreted as neurocutaneous units (NCU). Deeper nerves embraced broader portions of epidermis. Some nerves ran parallel to epidermis, especially near the superficial vascular plexus at the junction of superficial and deep dermis. Nerve sheath stem cells in each NCU may supply the melanocytes needed by the corresponding portion of epidermis. Transformed nerve sheath stem cells may lead to formation of occult prenatal nevi, whose histology and histogenesis may best be understood in terms of NCUs. In particular, the size and shape of a nevus may be largely determined by its NCU of origin. Six fetal nevi were detected, and 3 occult lumbosacral Mongolian spots; all in deep dermis, no later than the middle of the second trimester, mainly with a pattern of singly dispersed deep dermal melanocytes. These findings suggest that congenital (prenatal) nevi begin as intradermal nevi. In addition to explaining congenital nevi, these findings have implications for the histogenesis of acquired (postnatal) nevi and dysplastic nevi.

The Hippo pathway is a conserved pathway that interconnects with several other pathways to regulate organ growth, tissue homoeostasis and regeneration, and stem cell self-renewal. This pathway is unique in its capacity to orchestrate multiple processes, from sensing to execution, necessary for organ expansion. Activation of the Hippo pathway core kinase cassette leads to cytoplasmic sequestration of the nuclear effectors YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif), consequently disabling their transcriptional co-activation function. Components upstream of the core kinase cassette have not been well understood, especially in vertebrates, but are gradually being elucidated and include cell polarity and cell adhesion proteins.

Despite the key role of γ-aminobutyric acid (GABA) neurons in the modulation of cerebral cortical output, little is known about their development in the human cortex. We analyzed several GABAergic parameters in standardized regions of the cerebral cortex and white matter in a total of 38 human fetuses and infants from 19 gestational weeks to 2.7 postnatal years using immunocytochemistry, Western blotting, tissue autoradiography, and computer-based cellular quantitation. At least 20% of GABAergic neurons in the white matter migrated toward the cortex over late gestation. After term, migration declined and ended within 6 postnatal months. In parallel, the GABAergic neuronal density increased in the cortex over late gestation, also with a peak at term. From midgestation to infancy, the pattern of GABAA receptor binding changed from uniformly low across all cortical layers to high levels concentrated in the middle laminae; glutamic acid decarboxylase (GAD65 and GAD67) levels differentially increased. Thus, the second half of gestation is a period of rapid development of the cortical GABAergic system that continues into early infancy. This period corresponds to the peak window of vulnerability to perinatal hypoxia-ischemia in which GABAergic neurons are potentially developmentally susceptible, including in the preterm infant.

The morphogenesis of vertebrate body parts remains an open question. It is not clear whether the existence of different structures, such as a head, can be addressed by fundamental laws of tissue movement and deformation, or whether they are only a sequence of stop-and-go genetic instructions. I have filmed by time-lapse microscopy the formation of the presumptive head territory in chicken embryos. I show that the early lateral evagination of the eye cups and of the mesencephalic plate is a consequence of a sudden change in boundary conditions of the initial cell flow occurring in these embryos. Due to tissue flow, and collision of the two halves of the embryo, the tissue sheet movement is first dipolar, and next quadrupolar. In vivo air puff tonometry reveals a simple visco-elastic behaviour of the living material. The jump from a dipolar to a quadrupolar flow changes the topology of the early morphogenetic field which is observed towards a complex vortex winding with a trail (the eye cups and brain folds). The hydrodynamical model accounts for the discontinuity of the vector field at the moment of collision of the left and right halves of the embryo, at a quantitative level. This suggests a possible mechanism for the morphogenesis of the head of amniotes, as compared to cephalochordates and anamniotes.

The central nervous system contains a wide variety of neuronal subclasses generated by neural progenitors. The achievement of a unique neural fate is the consequence of a sequence of early and increasingly restricted regulatory events, which culminates in the expression of a specific genetic combinatorial code that confers individual characteristics to the differentiated cell. How the earlier regulatory events influence post-mitotic cell fate decisions is beginning to be understood in the Drosophila NB 5-6 lineage. However, it remains unknown to what extent these events operate in other lineages. To better understand this issue, we have used a very highly specific marker that identifies a small subset of abdominal cells expressing the Drosophila neuropeptide Capa: the ABCA neurons. Our data support the birth of the ABCA neurons from NB 5-3 in a cas temporal window in the abdominal segments A2-A4. Moreover, we show that the ABCA neuron has an ABCA-sibling cell which dies by apoptosis. Surprisingly, both cells are also generated in the abdominal segments A5-A7, although they undergo apoptosis before expressing Capa. In addition, we have performed a targeted genetic screen to identify players involved in ABCA specification. We have found that the ABCA fate requires zfh2, grain, Grunge and hedgehog genes. Finally, we show that the NB 5-3 generates other subtype of Capa-expressing cells (SECAs) in the third suboesophageal segment, which are born during a pdm/cas temporal window, and have different genetic requirements for their specification., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Although mosquito genome projects have uncovered orthologues of many known developmental regulatory genes, extremely little is known about mosquito development. In this study, the role of semaphorin-1a (sema1a) was investigated during vector mosquito embryonic ventral nerve cord development. Expression of sema1a and the plexin A (plexA) receptor are detected in the embryonic ventral nerve cords of Aedes aegypti (dengue vector) and Anopheles gambiae (malaria vector), suggesting that Sema1a signaling may regulate mosquito nervous system development. Analysis of sema1a function was investigated through siRNA-mediated knockdown in A. aegypti embryos. Knockdown of sema1a during A. aegypti development results in a number of nerve cord phenotypes, including thinning, breakage, and occasional fusion of the longitudinal connectives, thin or absent commissures, and general distortion of the nerve cord. Although analysis of Drosophila melanogaster sema1a loss-of-function mutants uncovered many similar phenotypes, aspects of the longitudinal phenotypes differed between D. melanogaster and A. aegypti. The results of this investigation suggest that Sema1a is required for development of the insect ventral nerve cord, but that the developmental roles of this guidance molecule have diverged in dipteran insects.

The Ly6 superfamily, present in most metazoan genomes, codes for different cell-surface proteins and secreted ligands containing an extracellular motif called a Ly6 domain or three-finger domain. We report the identification of 36 novel genes coding for proteins of this family in Drosophila. One of these fly Ly6 proteins, coded by the gene boudin (bou), is essential for tracheal morphogenesis in the fly embryo and contributes to the maintenance of the paracellular barrier and the organisation of the septate junctions in this tissue. Bou, a glycosylphosphatidylinositol anchored membrane protein, is also required for septate junction organisation in epithelial tissues and in the chordotonal organ glial cells, but not in the central nervous system. Our study reveals interesting parallelisms between the Ly6 proteins of flies and vertebrates, such as the CD59 antigen. Similarly to this human protein, Bou travels from cell to cell associated with extracellular particles and, consistently, we show that it is required in a non-cell-autonomous fashion. Our work opens the way for future studies addressing the function of Ly6 proteins using Drosophila as a model system.

Amphibians have been used for over a century as experimental animals. In the field of developmental biology in particular, much knowledge has been accumulated from studies on amphibians, mainly because they are easy to observe and handle. Xenopus laevis is one of the most intensely investigated amphibians in developmental biology at the molecular level. Thus, Xenopus is highly suitable for studies on the mechanisms of organ differentiation from not only a single fertilized egg, as in normal development, but also from undifferentiated cells, as in the case of in vitro organogenesis. Based on the established in vitro organogenesis methods, we have identified many genes that are indispensable for normal development in various organs. These experimental systems are useful for investigations of embryonic development and for advancing regenerative medicine. Developmental Dynamics 238:1309-1320, 2009. (c) 2009 Wiley-Liss, Inc.

Mind bomb1 (Mib1)-mediated endocytosis of the Notch ligand DeltaD is essential for activation of Notch in a neighboring cell. Although most DeltaD is localized in cytoplasmic puncta in zebrafish neural tissue, it is on the plasma membrane in mib1 mutants because Mib1-mediated endocytosis determines the normal subcellular localization of DeltaD. Knockdown of Notch increases cell surface DeltaA and DeltaD, but not DeltaC, suggesting that, like Mib1, Notch regulates endocytosis of specific ligands. Transplant experiments show that the interaction with Notch, both in the same cell (in cis) and in neighboring cells (in trans), regulates DeltaD endocytosis. Whereas DeltaD endocytosis following interaction in trans activates Notch in a neighboring cell, endocytosis of DeltaD and Notch following an interaction in cis is likely to inhibit Notch signaling by making both unavailable at the cell surface. The transplantation experiments reveal a heterogeneous population of progenitors: in some, cis interactions are more important; in others, trans interactions are more important; and in others, both cis and trans interactions are likely to contribute to DeltaD endocytosis. We suggest that this heterogeneity represents the process by which effective lateral inhibition leads to diversification of progenitors into cells that become specialized to deliver or receive Delta signals, where trans and cis interactions with Notch play differential roles in DeltaD endocytosis.

Background: The PKD family of serine/threonine kinases comprises a single member in Drosophila (dPKD), two isoforms in C. elegans (DKF-1 and 2) and three members, PKD1, PKD2 and PKD3 in mammals. PKD1 and PKD2 have been the focus of most studies up to date, which implicate these enzymes in very diverse cellular functions, including Golgi organization and plasma membrane directed transport, immune responses, apoptosis and cell proliferation. Concerning PKD3, a role in the formation of vesicular transport carriers at the trans-Golgi network (TGN) and in basal glucose transport has been inferred from in vitro studies. So far, however, the physiological functions of the kinase during development remain unknown., Results: We have examined the expression pattern of PKD3 during the development of mouse embryos by immunohistochemistry. Using a PKD3 specific antibody we demonstrate that the kinase is differentially expressed during organogenesis. In the developing heart a strong PKD3 expression is constantly detected from E10 to E16.5. From E12.5 on PKD3 is increasingly expressed in neuronal as well as in the supporting connective tissue and in skeletal muscles., Conclusion: The data presented support an important role for PKD3 during development of these tissues.

Adult urodeles (salamanders) are unique in their ability to regenerate complex organs perfectly. The recently developed Accessory Limb Model (ALM) in the axolotl provides an opportunity to identify and characterize the essential signaling events that control the early steps in limb regeneration. The ALM demonstrates that limb regeneration progresses in a stepwise fashion that is dependent on signals from the wound epidermis, nerves and dermal fibroblasts from opposite sides of the limb. When all the signals are present, a limb is formed de novo. The ALM thus provides an opportunity to identify and characterize the signaling pathways that control blastema morphogenesis and limb regeneration. Our previous study provided data on cell contribution, cell migration and nerve dependency indicating that an ectopic blastema is equivalent to an amputation-induced blastema. In the present study, we have determined that formation of both ectopic blastemas and amputation-induced blastemas is regulated by the same molecular mechanisms, and that both types of blastema cells exhibit the same functions in controlling growth and pattern formation. We have identified and validated five marker genes for the early stages of wound healing, dedifferentiation and blastema formation, and have discovered that the expression of each of these markers is the same for both ectopic and amputation-induced blastemas. In addition, ectopic blastema cells interact coordinately with amputation-induced blastema cells to form a regenerated limb. Therefore, the ALM is appropriate for identifying the signaling pathways regulating the early events of tetrapod limb regeneration.

LRRC4, leucine-rich repeat C4 protein, has been identified in human (GenBank accession No. AF196976), mouse (GenBank accession No. DQ177325), rat (GenBank accession No. DQ119102) and bovine (GenBank accession No. DQ164537) with identical domains. In terms of their similarity, the genes encoding LRRC4 in these four mammalian species are orthogs and therefore correspond to the same gene entity. Based on previous research, and using in situ hybridization, we found that LRRC4 had the strongest expression in hippocampal CA1 and CA2, the granule cells of the dentate gyrus region, the mediodoral thalamic nucleus, and cerebella Purkinje cell layers. Using a P19 cell model, we also found that LRRC4 participates in the differentiation of neuron and glia cells. In addition, extracellular proteins containing both an LRR cassette and immunoglobulin domains have been shown to participate in axon guidance. Our data from neurite outgrowth assays indicated that LRRC4 promoted neurite extension of hippocampal neurons, and induced differentiation of glioblastoma U251 cells into astrocyte-like cells, confirmed by morphology observation and glial fibrillary acidic protein expression.

Background: Previous studies demonstrated that the spinal cord within a fetal myelomeningocele (MMC) lesion suffers progressive destruction during gestation. This study aims at elucidating this pathophysiologic feature on a cellular and ultrastructural level in a model of genetically determined MMC., Methods: Curly tail/loop tail mouse fetuses at various gestational stages and neonates were analyzed electron-microscopically to document time-point and nature of neural tissue development and pathologic alterations within the MMC., Results: At embryonic day (E) 8.5 and E9.5, round cells displaying multiple microvilli covered the entire region of interest, and some specimens showed initial stages of neurulation. At E10.5, neurulation was terminated in normal animals, whereas the neural placode remained unfolded in MMC fetuses and became distinguishable from adjacent epidermal layers. At E15.5, an apparently normal differentiation was found. Until this time-point, there was no tissue damage or inflammation. Thereafter, increasingly severe tissue alterations were identified with ongoing gestation leading to almost complete loss of neural tissue at birth., Conclusion: We show here in fetal mice with MMC that, apart from absent neurulation, growth and development of the otherwise perfectly intact exposed spinal cord appear normal in early gestation, whereas later, the unprotected neural tissue is progressively destroyed.

Nerves and blood vessels often follow parallel trajectories as they course through the body to their distal targets. Proteins that regulate the process of axon guidance have likewise been shown to play a crucial role in blood vessel migration. With the recent description of the endothelial tip cell as an analog of the axonal growth cone, the nerve-vessel analogy seems complete. Notwithstanding these considerable similarities, one crucial difference remains between neural and vascular guidance. While a navigating axon is but a single cell, a sprouting vessel is composed of multiple cells that must be co-ordinately regulated. Recent studies of the Dll4-Notch1 signaling pathway have provided valuable insight into how the vasculature accomplishes this crucial task.

The gene GTF2IRD1 is localized within the critical region on chromosome 7 that is deleted in Williams syndrome patients. Genotype-phenotype comparisons of patients carrying variable deletions within this region have implicated GTF2IRD1 and a closely related homolog, GTF2I, as prime candidates for the causation of the principal symptoms of Williams syndrome. We have generated mice with an nls-LacZ knockin mutation of the Gtf2ird1 allele to study its functional role and examine its expression profile. In adults, expression is most prominent in neurons of the central and peripheral nervous system, the retina of the eye, the olfactory epithelium, the spiral ganglion of the cochlea, brown fat adipocytes and to a lesser degree myocytes of the heart and smooth muscle. During development, a dynamic pattern of expression is found predominantly in musculoskeletal tissues, the pituitary, craniofacial tissues, the eyes and tooth buds. Expression of Gtf2ird1 in these tissues correlates with the manifestation of some of the clinical features of Williams syndrome.

Biomaterials prepared from polyacrylamide, ethyl acrylate (EA), and hydroxyethyl acrylate (HEA) in various blend ratios, methyl acrylate and chitosan, were tested in vitro as culture substrates and compared for their ability to be colonized by the cells migrating from embryonic brain explants. Neural explants were isolated from proliferative areas of the medial ganglionic eminence and the cortical ventricular zone of embryonic rat brains and cultured in vitro on the different biomaterials. Chitosan, poly(methyl acrylate), and the 50% wt copolymer of EA and HEA were the most suitable substrates to promote cell attachment and differentiation of the neural cells among those tested. Immunofluorescence microscopy analysis showed that progenitor cells had undergone differentiation and that the resulting glial and neuronal cells expressed their intrinsic morphological characteristics in culture.

Stem cell persistence into adulthood requires self-renewal from early developmental stages. In the developing mouse brain, only apical progenitors located at the ventricle are self-renewing, whereas basal progenitors gradually deplete. However, nothing is known about the mechanisms regulating the fundamental difference between these progenitors. Here we show that the conditional deletion of the small Rho-GTPase cdc42 at different stages of neurogenesis in mouse telencephalon results in an immediate increase in basal mitoses. Whereas cdc42-deficient progenitors have normal cell cycle length, orientation of cell division and basement membrane contact, the apical location of the Par complex and adherens junctions are gradually lost, leading to an increasing failure of apically directed interkinetic nuclear migration. These cells then undergo mitoses at basal positions and acquire the fate of basal progenitors. Thus, cdc42 has a crucial role at the apical pole of progenitors, thereby regulating the position of mitoses and cell fate.

The relative contributions of different FGF ligands and spliceforms to mesodermal and neural patterning in Xenopus have not been determined, and alternative splicing, though common, is a relatively unexplored area in development. We present evidence that FGF8 performs a dual role in X. laevis and X. tropicalis early development. There are two FGF8 spliceforms, FGF8a and FGF8b, which have very different activities. FGF8b is a potent mesoderm inducer, while FGF8a has little effect on the development of mesoderm. When mammalian FGF8 spliceforms are analyzed in X. laevis, the contrast in activity is conserved. Using a loss-of-function approach, we demonstrate that FGF8 is necessary for proper gastrulation and formation of mesoderm and that FGF8b is the predominant FGF8 spliceform involved in early mesoderm development in Xenopus. Furthermore, FGF8 signaling is necessary for proper posterior neural formation; loss of either FGF8a or a reduction in both FGF8a and FGF8b causes a reduction in the hindbrain and spinal cord domains.

A new robotic microscope system, called the Frogatron 3000, was developed to collect time-lapse images from arbitrary viewing angles over the surface of live embryos. Embryos are mounted at the center of a horizontal, fluid-filled, cylindrical glass chamber around which a camera with special optics traverses. To hold them at the center of the chamber and revolve them about a vertical axis, the embryos are placed on the end of a small vertical glass tube that is rotated under computer control. To demonstrate operation of the system, it was used to capture time-lapse images of developing axolotl (amphibian) embryos from 63 viewing angles during the process of neurulation and the in-plane kinematics of the epithelia visible at the center of each view was calculated. The motions of points on the surface of the embryo were determined by digital tracking of their natural surface texture, and a least-squares algorithm was developed to calculate the deformation-rate tensor from the motions of these surface points. Principal strain rates and directions were extracted from this tensor using decomposition and eigenvector techniques. The highest observed principal true strain rate was 28 +/- 5% per hour, along the midline of the neural plate during developmental stage 14, while the greatest contractile true strain rate was--35 +/- 5% per hour, normal to the embryo midline during stage 15.

The expressions of mRNAs for pituitary adenylate cyclase-activating polypeptide (PACAP), vasoactive intestinal peptide (VIP), and their receptors (PAC1, VPAC1 and VPAC2) were examined in the five steps of the in vitro neuronal culture model of embryonic stem (ES) cell differentiation. mRNAs for PACAP, VIP, PAC1 receptor, and VPAC2 receptor were moderately expressed in neural stem cell-enriched cultures, while VPAC1 receptor mRNA was most prominently expressed in embryoid bodies (EBs). The expression of PAC1 receptor mRNA was further upregulated after terminal differentiation into neurons. In contrast, the expressions of PAC1 receptor and PACAP mRNAs were markedly decreased after glial differentiation. These results suggest that this in vitro neuronal culture system will be a useful model for future studies on the functional role of the PACAPergic system during different stages of neuronal development.

Versican, an extracellular matrix proteoglycan, has been implicated in limb development and is expressed in precartilage mesenchymal condensations. However, studies have lacked precise spatial and temporal investigation of versican localization during skeletogenesis and its relationship to patterning of muscle and nerve during mammalian limb development. The transgenic mouse line hdf (heart defect), which bears a lacZ reporter construct disrupting Cspg2 encoding versican, allowed ready detection of hdf transgene expression through histochemical analysis. Hdf transgene expression in whole mount heterozygous embryos and localization of versican relative to cartilage, muscle, and nerve tissues in paraffin-embedded limb sections of wild-type embryos from 10.5-14 days postcoitum were evaluated by lacZ histochemistry, immunohistochemistry, and in situ hybridization. Versican was localized within precartilage condensations and nascent cartilages with expression diminishing during maturation of the cartilage model at later time points. Interestingly, versican remained highly expressed in developing synovial joint interzones, suggesting potential function for versican in joint morphogenesis. Isolated myoblasts, incipient skeletal muscle masses, and neurites were not present in areas of strong versican expression within the developing limb. Versican-expressing tissues may reserve space for the future limb skeleton and developing joints and may aid in patterning of muscle and nerve by deterring muscle migration and innervation into these regions.

Frodo is a novel conserved regulator of Wnt signaling that has been identified by its association with Dishevelled, an intracellular component of Wnt signal transduction. To understand further how Frodo functions, we have analyzed its role in neural development using specific morpholino antisense oligonucleotides. We show that Frodo and the closely related Dapper synergistically regulate head development and morphogenesis. Both genes were cell-autonomously required for neural tissue formation, as defined by the pan-neural markers sox2 and nrp1. By contrast, beta-catenin was not required for pan-neural marker expression, but was involved in the control of the anteroposterior patterning. In the mesoderm, Frodo and Dapper were essential for the expression of the organizer genes chordin, cerberus and Xnr3, but they were not necessary for the expression of siamois and goosecoid, established targets of beta-catenin signaling. Embryos depleted of either gene showed a decreased transcriptional response to TCF3-VP16, a beta-catenin-independent transcriptional activator. Whereas the C terminus of Frodo binds Dishevelled, we demonstrate that the conserved N-terminal domain associates with TCF3. Based on these observations, we propose that Frodo and Dapper link Dsh and TCF to regulate Wnt target genes in a pathway parallel to that of beta-catenin.

Objective: Our objective was to investigate the relation between the embryological development and neural tissue maturation at the site where the neural plate failed to form a neural tube., Material and Methods: Samples from 15 aborted human fetuses with neural tube defects (NTD). All of the fetuses were between 20 and 25 gestational weeks old. Indicators of neural tissue maturation, formation of basal lamina, expression of integrins and neuron specific class III beta tubulin (tuj1) were investigated. To detect the adverse effects of the environment, if any, p53 and bcl-2 activity at both sites of the open and closed neural plate were investigated as well., Results: No difference was found in the expression of maturation-related molecules at the site of the neural plate that remained open compared with the site where the neural tube is normally formed. While high p53 activity was noted in neural tissue at the site of the neural tube defect, no such activity was detected in the neural tissue where the neural tube is normally formed., Conclusion: Our results suggested that maturation and differentiation of neural tissue continued regardless of the failure of neural tube closure. Therefore, the neurological deficits that are encountered in NTD patients should be related to secondary damage such as amnion fluid toxicity, uterus contractions, labor, etc. It seems valuable to save the neural plate before the negative effects of the environment renders the neural tissue functionless.

Different experimental systems are used to study developmental processes in mammals. In this study, three experimental models were analysed and correlated: (1) cultivation of rat embryos in vitro; (2) cultivation in vitro and then transplantation in vivo; (3) direct transplantation in vivo. When embryos were cultivated in vitro and then transplanted in vivo, after the initial in vitro restriction, developmental potential was recovered. The in vitro restriction depended on medium used and duration of culture. Pre-cultivation in serum-free medium for 7 days restricted developmental potential for nervous tissue, and for 14 days restricted developmental potential for skeletal muscles, adipose tissue and glandular epithelia. Transferrin addition improved in vitro differentiation of neuroblasts, cartilage and columnar epithelium. In the combined in vitro and in vivo method, transferrin preserved developmental potential in comparable extent to the addition of the serum. Even in serum-free conditions in vitro, the subsequent in vivo wide expression of developmental potential was possible. Therefore, the combination of in vitro and in vivo methods turned to be advantageous than the isolated approaches (in vitro or in vivo only), and enabled testing in more detail the influence of a single substance on developmental course and potential.

The poor regenerative capacity of the spiral ganglion neurons of the mammalian inner ear has initiated research on how to assist the functional recovery of the injured auditory system. A possible treatment is to use a biological implant with a potential to establish central or peripheral synaptic contacts to develop into a functional auditory unit. The feasibility of this approach was tested by xenograft implantation of dorsal root ganglion (DRG) neurons from embryonic days 13 to 14, mouse expressing either LacZ or enhanced green fluorescent protein (EGFP) into the scala tympani of the adult rat inner ear. Transplanted DRG neurons survived in the scala tympani for a postoperative period ranging from 3 to 10 weeks, as verified by histochemical detection of LacZ, EGFP fluorescence and immunohistochemical labeling of the neuronal markers neurofilament and Thy 1.2. DRG neurons were found close to structures near the sensory epithelium (the organ of Corti) as well as adjacent to the spiral ganglion neurons with their peripheral dendrites. These results illustrate not only the survival of xenografted DRG neurons in the adult inner ear but also the feasibility of a neuronal transplantation strategy in the degenerated auditory system, thereby creating possibilities to replace spiral ganglion neurons.

Objective: We examined the effects of maternal corticosteroid administration on water content in regional tissue in ovine fetuses at 60%, 80%, and 90% of gestation., Methods: After catheters were placed in the fetuses, the ewes were given four 6-mg doses of dexamethasone or placebo injections 12 hours apart over 48 hours. Water content of fetal tissue was determined 18 hours after the last injection was given to the ewes. Tissue water was determined by wet-to-dry weight ratio in brain (cerebral cortex, caudate nucleus, cerebellum, midbrain, and medulla) and non-neural tissues (kidney, liver, muscle, and skin) at each gestational age., Results: Water content (P <.05) in brain regions was lower in fetuses from dexamethasone-treated than placebo-treated ewes at 60% but not 80% or 90% of gestation and in non-neural tissues at each gestational age., Conclusions: Maternal treatment with a corticosteroid regimen similar to that used in the clinical setting was associated with small decreases in brain water content early but not later in gestation. This corticosteroid treatment regimen was also associated with decreased regional non-neural tissue water content at 60%, 80%, and 90% of gestation.

Thirty years ago, patients with spinal cord injury (SCI) and their families were told "nothing can be done" to improve function. Since the SCI patient population is reaching normal life expectancy through better health care, it has become an obviously worthwhile enterprise to devote considerable research effort to SCI. Targets for intervention in SCI toward improved function have been identified using basic research approaches and can be simplified into a list: (1) reduction of edema and free-radical production, (2) rescue of neural tissue at risk of dying in secondary processes such as abnormally high extracellular glutamate concentrations, (3) control of inflammation, (4) rescue of neuronal/glial populations at risk of continued apoptosis, (5) repair of demyelination and conduction deficits, (6) promotion of neurite growth through improved extracellular environment, (7) cell replacement therapies, (8) efforts to bridge the gap with transplantation approaches, (9) efforts to retrain and relearn motor tasks, (10) restoration of lost function by electrical stimulation, and (11) relief of chronic pain syndromes. Currently, over 70 clinical trials are in progress worldwide. Consequently, in this millennium, unlike in the last, no SCI patient will have to hear "nothing can be done."

The X11-like (X11L) protein was originally isolated as a protein bound to the cytoplasmic domain of the beta-amyloid precursor protein (APP), which is associated with Alzheimer's disease. In mammals, X11L is believed to play an important role in the regulation of APP metabolism. Here we isolated and characterized the Drosophila X11L (dX11L) protein, also may be referred to this protein as Drosophila Mint (dMint), Lin 10 (dLin10) or X11 (dX11), is thought to be expressed in neuronal tissues from late embryonic through to the adult stages of the fly. The phosphotyrosine interaction domain of dX11L interacts with the cytoplasmic domain of the Drosophila amyloid precursor protein-like (APPL) similar to the way human X11L (hX11L) interacts with APP. Overexpression of dX11L on post-mitotic neurons had a lethal effect on flies and, when it was localized to the eye imaginal disc, disruption of compound eye morphology due to enhanced apoptosis of neuronal cells was observed. Overexpression of hX11L and the PDZ domain of dX11L resulted in identical eye phenotypes. The PDZ domain is highly conserved between Drosophila and human, and appears to be responsible for this phenotype. Our findings suggest that the X11L family may be involved with the regulation of apoptosis during neural cell development and that aberrant X11L function could be contribute in this way to the neuronal degeneration observed in Alzheimer's disease.

Dishevelled (Dsh) is required for the specification of cell fate and polarity by secreted Wnt proteins. Frodo, a novel conserved Dsh-binding protein, synergized with Xenopus Dsh (XDsh) in secondary axis induction in Xenopus laevis embryos. A dominant inhibitory construct and antisense oligonucleotide-mediated depletion of Frodo inhibited axial development in response to XDsh and XWnt8, and suppressed transcriptional activation of a reporter construct. At later embryonic stages, both dominant negative Frodo and antisense oligonucleotides interfered with the expression of regional neural markers and caused eye deficiencies, indicating that Frodo is required for normal eye and neural tissue development. Full-length Frodo RNA suppressed these loss-of-function phenotypes, attesting to their specificity. These findings establish a function for Frodo as an essential positive regulator of Wnt signalling.

Drosophila neuroectodermal embryonic cells were transplanted into the occipital brain region of adult rats. The first series of experiments used a transgenic strain expressing lacZ to detect the presence of Drosophila cells. The second series used a strain carrying a is lethal (ts403) in the X chromosome; this mutation strongly inhibits the synthesis of heat shock proteins (hsps) and their transport into the nuclei. Immunostaining reveals a strong induction of hsp70 in the xenografts in the first series of experiments, in which no glial scar was detectable. By contrast, where the ts mutation was xenotransplanted, the condition of xenografts was worse, and a glial scar was readily evident between the xenograft and host tissue.

Lack of type XV collagen in mice results in mild skeletal myopathy and increases vulnerability to exercise-induced skeletal muscle and cardiac injury [Proc. Natl. Acad. Sci. USA 98 (2001), 1194]. The expression of type XV collagen was studied during murine fetal development from 10.5 to 18.5 dpc using immunofluorescence. The first sign of type expression was seen in the capillaries of many tissues at 10.5 dpc, some of them showing developmental transitions in the expression. Interestingly, capillaries forming the blood-brain barrier and those of the sinusoidal type were essentially lacking in this collagen. Early expression was also detected in the skeletal muscle and peripheral nerves, while expression in the heart, kidney and lung appeared to be developmentally regulated. In addition, distinct staining was found in the perichondrium of the cartilage. Collectively, the dynamics of its expression during development, its localization in the basement membrane--fibrillar matrix interface and the consequences of its absence in mice suggest a structural role in providing stability at least in skeletal muscle and capillaries. The early prominent expression of type XV collagen in newly forming blood vessels could also indicate a possible role in angiogenic processes.

Aim: To analyze neural tissue differentiation in a unique, chemically-defined in vitro culture model of gastrulating rat embryo proper by use of transmission electron microscopy (TEM), proliferating cell nuclear antigen (PCNA) expression, and in vivo transplantation after a 2-week culture in serum-free or serum-supplemented media. Influence of protein-free medium unfavorable for differentiation of neural tissue in vitro was compared with favorable serum-free media enriched with transferrin or albumin. Differentiation of mesodermal derivatives in transplants was also investigated., Methods: We cultivated 9.5-day-old Fischer rat embryos on the gas-liquid interface in the protein-free Eagle's Minimum Essential Medium (MEM), in MEM with either iron-saturated holotransferrin (50 microg/mL) or iron-free apotransferrin (50 microg/mL), and in medium saturated with either bovine serum albumin (BSA) (4 mg/mL or 400 microg/mL) or rat serum (50%). After the two-week culture period, light microscopy, TEM, and immunohistochemical method for detection of PCNA were done. Some explants were transplanted under the kidney capsule of adult male rats to be cultured in vivo for additional two weeks. Chi-square test or Fisher exact test were used to compare the proportion of tissues developed., Results: Proportion of differentiated neural tissue was similar in explants cultivated in apotransferrin- and holotransferrin-supplemented media (13/33 and 9/20, respectively), but higher than in explants cultivated in protein-free medium (1/13). Neurons and glia cells produced a neuropil structure. Myelinization occurred only in serum-supplemented medium. PCNA expression was detected in a small number of neural tissue cells, even in serum-free cultivated embryos. Differentiation of brain-like tissue, cerebrospinal, and vegetative ganglionic cells occurred in all groups of transplants. However, in the transplants derived from protein-free medium, the proportion of neural tissue, cartilage, bone, skeletal and smooth muscle was significantly lower than in transferrin-supplemented media (p<0.01). Albumin seemed to promote differentiation of all tissues except vegetative ganglionic cells., Conclusion: Nerve tissue differentiated to a rather high degree in a two-week in vitro postimplantation embryo culture. Transferrin or albumin, as the only proteins used for serum-free precultivation, significantly improved subsequent differentiation of nerve tissue and mesodermal derivatives in transplants in vivo.

Little axonal regeneration occurs after spinal cord injury in adult mammals. Regrowth of mature CNS axons can be induced, however, by altering the intrinsic capacity of the neurons for growth or by providing a permissive environment at the injury site. Fetal spinal cord transplants and neurotrophins were used to influence axonal regeneration in the adult rat after complete spinal cord transection at a midthoracic level. Transplants were placed into the lesion cavity either immediately after transection (acute injury) or after a 2-4 week delay (delayed or chronic transplants), and either vehicle or neurotrophic factors were administered exogenously via an implanted minipump. Host axons grew into the transplant in all groups. Surprisingly, regeneration from supraspinal pathways and recovery of motor function were dramatically increased when transplants and neurotrophins were delayed until 2-4 weeks after transection rather than applied acutely. Axonal growth back into the spinal cord below the lesion and transplants was seen only in the presence of neurotrophic factors. Furthermore, the restoration of anatomical connections across the injury site was associated with recovery of function with animals exhibiting plantar foot placement and weight-supported stepping. These findings suggest that the opportunity for intervention after spinal cord injury may be greater than originally envisioned and that CNS neurons with long-standing injuries can reinitiate growth, leading to improvement in motor function.

To study the pathogenesis of X-linked recessive myotubular myopathy (XLMTM), we used a nerve-muscle coculture system which allows the reconstitution of functional motor units in vitro after coupling of human skeletal muscle cells with embryonic rat spinal cord explants. We used three skeletal muscle cell lines derived from subjects with known mutations in the MTM1 gene (two from embryonic tissues, associated with mutations predicted to give a severe phenotype, and one from a neonate still alive at 3 years 6 months and exhibiting a mild phenotype). We compared these three XLMTM muscle cell cultures with control cultures giving special attention to behaviour of living cocultures (formation of the myofibres, contractile activity, survival), expression of muscular markers (desmin, dystrophin, alpha-actinin, troponin-T, myosin heavy chain isoforms), and nerve-muscle interactions (expression and aggregation of the nicotinic acetylcholine receptors). We were unable to reproduce any 'myotubular' phenotype since XLMTM muscle cells behaved like normal cells with regard to all the investigated parameters. Our results suggest that XLMTM muscle might be intrinsically normal and emphasize the possible involvement of the myotubularin-deficient motor neurons in the development of the disease.

Background: Embryonic xenogeneic neural tissue is an alternative for transplantation in Parkinson's disease, but immune responses limit the application. The aims of this study were to enhance the in vitro viability rates by donor tissue pretreatment; to compare the efficacy of cyclosporine A (CsA) and tacrolimus (FK) in inhibiting xenograft rejection in rats; to evaluate additional inductive therapy with prednisolone (PRE) or mycophenolate mofetil (MMF)., Methods: Tirilazad (a lipid peroxidase inhibitor) or FK and acYVAD-cmk (a caspase inhibitor), were added to embryonic porcine ventral mesencephalic tissue and viability was assessed in vitro. Tirilazad-treated tissue was grafted to the striatum of rats that were either left untreated or immunosuppressed with FK (1 mg/kg) or CsA (15 mg/kg) alone or in combination with a 2-week PRE (20 mg/kg) or MMF (40 mg/kg) induction course. Xenograft survival and host responses were determined using immunohistochemistry., Results: Pretreatment with tirilazad enhanced tissue survival in vitro. After transplantation into untreated controls, there was no graft survival at twelve weeks. Neural cell counts were significantly improved in immunosuppressed recipients, but there were no differences between the treatment groups. Additional inductive treatment reduced the infiltration with CD4+ and CD8+ cells, and macrophage infiltration was reduced compared with animals given CsA or FK alone., Conclusion: Pretreatment of the donor tissue with free-radical scavengers reduces cell loss caused by tissue trauma. Porcine neural tissue xenografts survive significantly better in animals immunosuppressed with either FK or CsA. Additional inductive treatment with PRE or MMF reduced the infiltration of host cells into the xenografts.

The effect of implanted embryonic nervous tissue on restoration of axonal connections in the cerebral cortex after mechanical injury was studied on albino rats using fluorescent lipophilic probe DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate) and confocal laser scanning microscope. Implantation of embryonic tissue to damaged area promotes the growth of axons through the transplant to adjacent tissue. The damaged area is impenetrable for axons growing without implantation.

A review of the mechanisms underlying cytogenesis and of the nervous tissues, the role of microenvironment, cell interactions in differentiation, and plastic rearrangements in the mammalian brain after transplantation of the embryonic nervous tissue. A common concept has been proposed, which unites biological processes involved in neurotransplantation and may serve as a theoretical base for the formation of ideas on pathways of differentiation of the nervous tissue, stimulation of compensatory processes and regeneration in the developing and pathological brain.

Polycomb group (PcG) proteins form multimeric protein complexes which are involved in the heritable stable repression of genes. Previously, we identified two distinct human PcG protein complexes. The EED-EZH protein complex contains the EED and EZH2 PcG proteins, and the HPC-HPH PcG complex contains the HPC, HPH, BMI1, and RING1 PcG proteins. Here we show that YY1, a homolog of the Drosophila PcG protein pleiohomeotic (Pho), interacts specificially with the human PcG protein EED but not with proteins of the HPC-HPH PcG complex. Since YY1 and Pho are DNA-binding proteins, the interaction between YY1 and EED provides a direct link between the chromatin-associated EED-EZH PcG complex and the DNA of target genes. To study the functional significance of the interaction, we expressed the Xenopus homologs of EED and YY1 in Xenopus embryos. Both Xeed and XYY1 induce an ectopic neural axis but do not induce mesodermal tissues. In contrast, members of the HPC-HPH PcG complex do not induce neural tissue. The exclusive, direct neuralizing activity of both the Xeed and XYY1 proteins underlines the significance of the interaction between the two proteins. Our data also indicate a role for chromatin-associated proteins, such as PcG proteins, in Xenopus neural induction.

Background: Glutamate decarboxylase (GAD) is the biosynthetic enzyme for the neurotransmitter gamma-aminobutyric acid (GABA). Mouse embryos lacking the 67-kDa isoform of GAD (encoded by the Gad1 gene) develop a complete cleft of the secondary palate. This phenotype suggests that this gene may be involved in the normal development of tissues outside of the CNS. Although Gad1 expression in adult non-CNS tissues has been noted previously, no systematic analysis of its embryonic expression outside of the nervous system has been performed. The objective of this study was to define additional structures outside of the central nervous system that express Gad1, indicating those structures that may require its function for normal development., Results: Our analysis detected the localized expression of Gad1 transcripts in several developing tissues in the mouse embryo from E9.0-E14.5. Tissues expressing Gad1 included the tail bud mesenchyme, the pharyngeal pouches and arches, the ectodermal placodes of the developing vibrissae, and the apical ectodermal ridge (AER), mesenchyme and ectoderm of the limb buds., Conclusions: Some of the sites of Gad1 expression are tissues that emit signals required for patterning and differentiation (AER, vibrissal placodes). Other sites correspond to proliferating stem cell populations that give rise to multiple differentiated tissues (tail bud mesenchyme, pharyngeal endoderm and mesenchyme). The dynamic expression of Gad1 in such tissues suggests a wider role for GABA signaling in development than was previously appreciated.