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Tissue, cellular and sub-cellular localization of the Vangl2 protein during embryonic development: effect of the Lp mutation.

Loop-tail (Lp) mice show a very severe neural tube defect, craniorachischisis, which is caused by mis-sense mutations in the Vangl2 gene. The membrane protein Vangl2 belongs to a highly conserved group of proteins that regulate planar polarity in certain epithelia, and that are also important for convergent extension movements during gastrulation and neurulation. A specific anti-Vangl2 antiserum was produced and used to examine the tissue, cell type, and sub-cellular localization of Vangl2 during embryogenesis. Vangl2 protein is expressed at high levels in the neural tube and shows a dynamic expression profile during neurulation. After neural tube closure, robust Vangl2 staining is detected in several neural and neurosensory tissues, including cerebral cortex, dorsal root ganglia, olfactory epithelium, retina, mechanosensory hair cells of the cochlea, and optic nerve. Vangl2 is also expressed during organogenesis in a number of tubular epithelia, including the bronchial tree, intestinal crypt/villus axis, and renal tubular segments derived from ureteric bud and from metanephric mesenchyme. Examination of Vangl2 localization in the neural tubes and cochleas of the normal and Lp/Lp embryos shows disruption of normal membrane localization of Vangl2 in independent alleles at Lp (Lp, Lp(m1Jus)) as well as overall decrease in the expression level.

Animals↗

Induction and the Turing-Child field in development.

The central problem in biological development is the understanding of epigenesis. The dominant theory of development in the last 80 years that also purports to explain epigenesis is induction theory. It suggests that development is driven by sequential inductions where each "induction" (in one sense of the word induction) is effected by the action of an inducing part of the embryo on a responding part of the embryo. The theory stems from Spemann and Mangold (W.Roux' Arch.f.Entw.d.Organis.u.mikrosk.Anat.100 (1924) 599) who transplanted a tissue from the dorsal blastopore lip of Triturus into the ventral ectoderm of another gastrula and thus initiated and "induced" (in another sense of the word induction) gastrulation and embryogenesis in the ventral side of the host that became a double embryo (siamese twins). We explain this induction, i.e. the formation of the double embryo, according to the Child theory and the Turing-Gierer-Meinhardt theory when it is also assumed that cAMP and ATP are the Turing activator and inhibitor, respectively. Spemann and Mangold (W.Roux' Arch.f.Entw.d.Organis.u.mikrosk.Anat.100 (1924) 599) also suggested that the ingressing mesoderm induces the overlying ectoderm to form the neural plate and neural tube. This 'neural induction', the 'primary embryonic induction', became the cornerstone of induction theory, i.e. of the sequential induction concept referred to above. But we argue that the metabolic gradients that precede and accompany neurulation, as obtained by Child, also for Triturus, arise through a Turing self-organization if it is assumed that cAMP and ATP are the Turing morphogens, and these gradients are the cause and primary event of neurulation. Thus there is no need to invoke the 'neural induction'. It is argued that fundamental events such as gastrulation and also organ formation are caused by the Turing-Child field and not by sequential induction. Similar principles, such as bud formation caused by a radial metabolic pattern that transforms to a longitudinal pattern, govern the formation, for example, of the mouth and the gut. The formation and localization of bottle cells is explained according to the Child-Turing field and modern biochemistry. The chemical metabolic pre-pattern precedes, and causes, morphogenesis and differentiation as envisaged by Turing. The Spemann and Mangold (W.Roux' Arch.f.Entw.d.Organis.u.mikrosk.Anat.100 (1924) 599) transplantation experiment when performed on a sea urchin duplicates not only the phenotype but also the metabolic (reduction) pattern. These experimental results, by Horstadius, predicted by Child, follow from the Turing-Gierer-Meinhardt theory if it is assumed that cAMP and ATP are the Turing morphogens. If the transplantation is performed not onto the whole sea urchin but onto only a part of it, that manifests only a part of the metabolic pattern, then from the part a phenotypic whole underlain by a normal and a whole metabolic pattern can be rescued. These experimental results of Horstadius follow from Turing theory if cAMP and ATP are the Turing morphogens. Understanding how to transform a part into a whole can be valuable in regenerative medicine. Unspecific induction of a secondary amphibian embryo is similar to the induction of posterior structures at the anterior pole of an insect, and the "double abdomen" (and Kalthoff's experimental results) of the midge Smittia resulting from UV irradiation of the anterior pole, can be explained by Meinhardt theory of unspecific induction if ATP is the Turing morphogen. When not working on regeneration, Child investigated intact living organisms and his observation method was not disruptive to normal development, whereas workers in induction theory work with pieces and in general disrupt normal development. We conclude that the Turing-Child field causes all development and explains epigenesis. Sequential induction does not explain epigenesis and does not exist in normal development. But induction in the sense of a transplantation leading to double embryo or rescuing a whole phenotype from a part is of interest.

Adenosine Triphosphate↗

Retinoid receptors and vitamin A deficiency: differential patterns of transcription during early avian development and the rapid induction of RARs by retinoic acid.

The functional links of specific retinoid receptors to early developmental events in the avian embryo are not known. Before such studies are undertaken, knowledge is required of the spatiotemporal expression patterns of the receptor genes and their regulation by endogenous retinoic acid levels during the early stages of development. Here, we report the expression patterns of mRNAs for RARalpha, RARalpha2, RARbeta2, RARgamma, RARgamma2, RXRalpha, and RARgamma from neurulation to HH10 in the normal and vitamin A-deficient (VAD) quail embryo. The transcripts for all retinoid receptors are detectable at HH5, except for RXRgamma, which is detected at the beginning of HH6. At the 4/5 somite stage of HH8, when retinoid signaling is initiated in the avian embryo, mRNAs of all receptors are present, with very strong and ubiquitous expression patterns for RARalpha, RARalpha2, RARgamma, RARgamma2, and RXRalpha, a persistent expression of RARgamma in the neural tissues, a strong expression of RARbeta2 in lateral plate mesoderm and somites, and an anterior expression of RXRgamma. All retinoid receptors are expressed in the heart primordia. In the VAD quail embryo, the general pattern of retinoid receptor transcript localization is similar to that of the normal, except that the expression of RARalpha2 and RARbeta2 is severely diminished. Administration of retinol or retinoic acid to VAD embryos at or before the 4/5 somite stage rescues the expression of RARalpha2 and RARbeta2 within approximately 45 min and restores normal development. RARbeta2 expression requires the expression of RARalpha2. After neurulation, the expression of all retinoid receptors in the VAD quail embryo becomes independent of vitamin A status and is similar to that of the normal. The mRNA levels and sites of expression of the key enzyme for retinoic acid biosynthesis, Raldh-2, are not affected by vitamin A status; the expression pattern is restricted and does not correspond to that of retinoid receptors at all sites. The general patterns and intensity of retinoid receptor gene expression during early quail development are comparable to those of the mammalian and thus validate the application of results from retinoid-regulated avian development studies to those of the mammalian.

Aldehyde Oxidoreductases↗

Cordon-bleu is a conserved gene involved in neural tube formation.

The axial midline is an important source of patterning and morphogenesis cues in the vertebrate embryo. The midline derives from a small group of cells in the gastrulating embryo, known as "the organizer" in recognition of its ability to organize an entire body plan. The mammalian organizer, the node, gives rise to axial midline structures: the notochord, dorsal foregut, and part of the floor plate of the neural tube. Only some of the genes that direct midline development are known. In this study, we present the complete coding sequence for a novel gene, cordon-bleu (cobl), expressed specifically in the node and its derivatives until organogenesis stages. The deduced sequence does not resemble any gene of known function. However, cobl is widely conserved: apparent orthologs and paralogs are found in many vertebrate species, with several sequence domains of high conservation but unknown function. We find that chicken cordon-bleu is similarly expressed in the node and its derivatives, suggesting functional conservation. We also report the sequence and nonoverlapping expression of a related mouse gene, Coblr1. Finally, we show that cobl interacts with the neurulation gene Vangl2 to facilitate midbrain neural tube closure, demonstrating roles for both cobl and Vangl2 in midbrain neurulation.

Alleles↗

Spinal cord arteriovenous fistulas involving the conus medullaris: presentation, management, and embryologic considerations.

BACKGROUND: Spinal cord arteriovenous fistulas (SCAVF) are uncommon congenital lesions that usually involve the most caudal aspects of the cord. We present three cases of SCAVF that illustrate the clinical manifestations and possible management options. The characteristic involvement of the conus medullaris and an associated tethered spinal cord in one of our patient suggests that a disorder of secondary neurulation may be involved in the formation of these arteriovenous shunt lesions. METHODS: Review of records and radiologic studies in three consecutive patients with SCAVF's treated at this institution. RESULTS: All three patients had SCAVF involving the lower lumbar spinal cord segments or the conus. One of the conus lesions was associated with tethering of the spinal cord. One small lesion (Type A) was treated surgically, whereas the two larger lesions (Type B) were treated using interventional neuroradiologic techniques. CONCLUSIONS: Both surgical and endovascular method have a role in management of these unusual spinal cord vascular malformations. The association with tethered cord suggests that the propensity for SCAVM to occur in the most caudal portions of the spinal cord may result from failure of secondary neurulation to properly develop the unique and complex vascular anatomy of the region.

Adult↗

Spinal dysraphism: MR imaging rationale.

Spinal cord development occurs through the three consecutive periods of gastrulation (weeks 2-3), primary neurulation (weeks 3-4), and secondary neurulation (weeks 5-6). Spinal cord malformations derive from defects in these early embryonic stages, and are collectively called spinal dysraphisms. Spinal dysraphisms may be categorized clinically into open and closed, based on whether the abnormal nervous tissue is exposed to the environment or covered by skin. Open spinal dysraphisms include myelomeningocele and other rare abnormalities such as myelocele, hemimyelomeningocele, and hemimyelocele, and are always associated with a Chiari II malformation. Closed spinal dysraphisms are further divided into two subsets based on whether a subcutaneous mass is present in the low back. Closed spinal dysraphisms with mass comprise lipomyelocele, lipomyelomeningocele, meningocele, and myelocystocele. Closed spinal dysraphisms without mass comprise simple dysraphic states (tight filum terminale, filar and intradural lipomas, persistent terminal ventricle, and dermal sinuses) and complex dysraphic states. The latter category involves abnormal notochordal development, either in the form of failed midline integration (ranging from complete dorsal enteric fistula to neurenteric cysts and diastematomyelia) or of segmental agenesis (caudal agenesis and spinal segmental dysgenesis). Magnetic resonance imaging is the imaging modality of choice for evaluation of this complex group of disorders.

Adolescent↗

Moderate alcohol exposure compromises neural tube midline development in prenatal brain.

We previously reported that fetal alcohol treatment compromised the development of the midline raphe and the serotonin neurons contained in it. In this study, we report that the timely development of midline neural tissue during neural tube formation is sensitive to alcohol exposure. Pregnant dams were treated from embryonic day 7 (E7, prior to neurulation) or E8.5 (at neurulation) with the following diets: (a) alcohol (ALC), given as either a 20% or 25% ethanol-derived calorie (EDC) liquid diet, or (b) isocaloric liquid diet pair-fed (PF), or (c) standard rat chow (Chow). Fetal brains from each group were examined on E13, E15, or E18. Neural tube development was compromised as a result of alcohol exposure in the following ways: (1) approximately 60% of embryos at E13 and 20% at E15 showed perforation of the floor plate in the diencephalic vesicle, (2) although completely closed at E13, 70-80% of embryos failed to complete the formation of neural tissue at the roof as the alcohol exposure continued to E15, and (3) 60-80% of embryos show delayed 'occlusion' of the ventral canal by newly formed nestin-positive neuroepithelial cells and S100beta-positive glia in the brainstem of E15. The compromised (incomplete) neural tube midline (cNTM) occurred near the ventricles at E13 and E15, but was later completed at E18. In all cases, the cNTM was accompanied by an enlarged ventricle, and dose-dependent brain weight reduction. The midline of the neural tube at the roof and floor plates is known to mediate timely trophic induction for neural differentiation. Prenatal midline deficits also have the potential to affect the development of midline neurons such as raphe, septal nuclei, and the timely crossing of commissural fibers. The results of the liquid diet alcohol exposure paradigm suggest it is more a model for Alcohol-Related Neurodevelopmental Disorder (ARND) featuring neuropsychiatric disorders than for full-blown fetal alcohol syndrome (FAS) with noticeable facial dysmorphogenesis and gross brain retardation.

Animals↗

Failure of neural tube closure in the loop-tail (Lp) mutant mouse: analysis of the embryonic mechanism.

Loop-tail (Lp) is unique among mouse mutants in failing to initiate neural tube closure at the cervical/hindbrain boundary (so-called 'Closure 1'), at the 5-7 somite stage. Lp/Lp embryos go on to develop a malformation that closely resembles cranio-rachischisis, the most severe neural tube defect found in humans. We investigated several possible embryological mechanisms that may underlie this failure of neural tube closure in Lp. The genotypes of Lp/Lp, Lp/+ and +/+ embryos from mixed litters were identified using the polymerase chain reaction to amplify a polymorphic microsatellite sequence that is very closely linked to Lp. At post-neurulation stages of development, Lp/Lp embryos have a shortened body axis, which could suggest a defect of axial elongation as the primary anomaly in Lp. However, we found that axial elongation is normal in Lp homozygotes prior to the stage of defective Closure 1, indicating that the shortened body axis of later embryos is a secondary effect of the neurulation anomaly, or an independent effect of the Lp mutation. Some workers have reported cell proliferation rates to be abnormal in later stage Lp/Lp embryos. We observed variations in [3H]thymidine labelling index, and mitotic index, between embryonic tissues, and between embryos at different somite stages. However, Lp/Lp, Lp/+ and +/+ embryos had closely similar cell proliferation parameters, arguing against a mechanism based on faulty embryonic growth. Thirdly, we tested the hypothesis that the defect in loop-tail results from an inability of the neural folds to become apposed, specifically at the site of Closure 1. By tying a silk suture around the embryonic axis, at the future site of Closure 1, we were able to effect convergence of the neural folds at this site. Neural fold closure failed to progress along the body axis in sutured Lp/Lp embryos, however, in contrast to operated Lp/+ and +/+ embryos which exhibited normal progression of neural tube closure. The embryonic defect in loop-tail appears, therefore, to involve either a general inability of the spinal neural folds to become apposed along the spinal region, or a defect in the process of neural fold fusion.

Animals↗

Cloning of cDNAs encoding retinoic acid receptors RAR gamma 1, RAR gamma 2, and a new splicing variant, RAR gamma 3, from Aambystoma mexicanum and characterization of their expression during early development.

To analyze retinoic acid (RA) receptor (RAR) expression during early development in the urodele embryo, we have isolated cDNAs for four members of the axolotl (Ambystoma mexicanum) RAR family, namely RAR alpha (NR1B1), aRAR gamma 1 (NR1B3a), aRAR gamma 2 (NR1B3b), and a new splicing variant of aRAR gamma 2, aRAR gamma 3 (NR1B3c), which contains an insertion of five hydrophobic amino acids in the C-terminal region of the DNA binding domain. The temporal expression pattern of the RAR gamma isoforms was established by RT-PCR using total RNA from embryos of different stages. The expression of aRAR gamma 2 coincides with neurulation and is enhanced in the extremities of the embryo's anteroposterior axis. The aRAR gamma 3 is specifically expressed during gastrulation and early neurulation, whereas aRAR gamma 1 is expressed later during organogenesis. Global aRAR gamma 2 mRNA levels, as well as their spatio-temporal expression pattern in the neurula, were not affected by treatment with RA. These results show that several RARs are expressed in the axolotl embryo during early development, and reveal the existence of a new RAR gamma variant.

Alternative Splicing↗

Altered expression of mitochondrial 16S ribosomal RNA in p53-deficient mouse embryos revealed by differential display.

Inactivation of the tumor suppressor p53 is associated with neural tube defects and altered teratogenicity in early embryos. To gain insight into the function of p53 during early embryogenesis, RNA profiles of wild-type p53(+/+) and p53(-/-) null mutant mouse embryos were compared at the head-fold stage (day 8 post coitum) using HPLC-based mRNA differential display. The results of this screen revealed a deficiency of mitochondrial 16S ribosomal RNA in p53(-/-) embryos. RT-PCR showed abnormalities in 16S rRNA levels relative to some representative nuclear (COIV, beta-actin) and mitochondrial (COIII) transcripts in p53(-/-) embryos, and that 16S rRNA expression increased with development of p53(+/+) embryos during neurulation. Embryos that lack p53 also displayed weakened cytochrome c oxidase staining and reduced ATP content. During neurulation, the mouse embryo switches from an anaerobic (glycolytic) to an aerobic (oxidative) metabolism. The preliminary results of the present study suggest that p53 may be involved, directly or indirectly, in this transition.

Adenosine Diphosphate↗

The forkhead gene FH1 is involved in evolutionary modification of the ascidian tadpole larva.

The forkhead gene FH1 encodes a HNF-3beta protein required for gastrulation and development of chordate features in the ascidian tadpole larva. Although most ascidian species develop via a tadpole larva, the conventional larva has regressed into an anural (tailless) larva in some species. Molgula oculata (the tailed species) exhibits a tadpole larva with chordate features (a dorsal neural sensory organ or otolith, a notochord, striated muscle cells, and a tail), whereas its sister species Molgula occulta (the tailless species) has evolved an anural larva, which has lost these features. Here we examine the role of FH1 in modifying the larval body plan in the tailless species. We also examine FH1 function in tailless speciesxtailed species hybrids, in which the otolith, notochord, and tail are restored. The FH1 gene is expressed primarily in the presumptive endoderm and notochord cells during gastrulation, neurulation, and larval axis formation in both species and hybrids. In the tailless species, FH1 expression is down-regulated after neurulation in concert with arrested otolith, notochord, and tail development. The FH1 expression pattern characteristic of the tailed species is restored in hybrid embryos prior to the development of chordate larval features. Antisense oligodeoxynucleotides (ODNs) shown previously to disrupt FH1 function were used to compare the developmental roles of this gene in both species and hybrids. As described previously, antisense FH1 ODNs inhibited endoderm invagination during gastrulation, notochord extension, and larval tail formation in the tailed species. Antisense FH1 ODNs also affected gastrulation in the tailless species, although the effects were less severe than in the tailed species, and an anural larva was formed. In hybrid embryos, antisense FH1 ODNs blocked restoration of the otolith, notochord, and tail, reverting the larva back to the anural state. The results suggest that changes in FH1 expression are involved in re-organizing the tadpole larva during the evolution of anural development.

Amino Acid Sequence↗

Morphological diversity of dying cells during regression of the human tail.

During normal human development a number of transient structures form and subsequently regress completely. One of the most prominent structures that regress during development is the human tail. We report here a histological and ultrastructural study of cell death in the cranial and caudal (tail) parts of the neural tube in 4 to 6-week-old human embryos. Initially, the human tail is composed of tail bud mesenchyme which differentiates into caudal somites, secondary neural tube, notochord and tail gut. Later on, these structures gradually regress by cell death. During the investigated period, we observed two morphologically distinct types of dying cells. The well-described apoptotic type of cell death was observed only in the cranial neural tube that forms during primary neurulation. The other type of cell death characterized by necrotic morphology was observed in the tail mesenchyme and in the caudal neural tube that forms during secondary neurulation. This morphological diversity suggests that besides differences in origin and fate there are different mechanisms of developmental cell death between two parts of the human neural tube. We can speculate that the apoptotic type of cell death is associated with the precise control of cell numbers and that the other morphologically distinct type of cell death is responsible for the massive removal of transitory structures.

Abortion, Induced↗

Mammalian neural crest and neural crest derivatives.

In the mammalian embryonic trunk, neural crest cells emigrate from the closed neural tube in a cranio-caudal sequences and appear to have similar migration pathways and derivatives to those of avian embryos. In the cranial region, however, there are mammalian-specific features, which are related to the mammalian-specific pattern of cranial neurulation. Midbrain and rostral hindbrain neural crest cells emigrate from widely open neural folds; caudal hindbrain crest emigrates in a caudo-rostral sequence, following the sequence of neural tube closure in this region. The forebrain is also a source of neural crest cells at early stages of neurulation; both forebrain and midbrain crest cells contribute to the frontonasal mesenchyme, although their relative contributions have not been analysed. Few studies have provided direct information about mammalian neural crest cell derivatives. Studies on the effects of retinoid excess on craniofacial development provide indirect evidence that mammalian cranial neural crest, like that of avian embryos, includes two populations whose differentiated phenotype and morphological tissue structure are determined prior to emigration. Retinoid-induced shortening of the preotic hindbrain leads to abnormal migration pathways of the neural crest cells that normally migrate into the mandibular arch to form Meckel's cartilage, so that an ectopic Meckel's cartilage-like structure forms in the maxillary region of the face. Slow descent of the heart in retinoid-exposed embryos enables the "wrong" crest cell population to populate the wall of the truncus arteriosus. These observations correlate well with observations of retinoid-induced craniofacial and heart abnormalities in human infants.

Animals↗

Inducible gene expression in transgenic Xenopus embryos.

The amphibian Xenopus laevis has been successfully used for many years as a model system for studying vertebrate development. Because of technical limitations, however, molecular investigations have mainly concentrated on early stages. We have developed a straightforward method for stage-specific induction of gene expression in transgenic Xenopus embryos [1] [2]. This method is based on the Xenopus heat shock protein 70 (Xhsp70 [3]) promoter driving the expression of desired gene products. We found that ubiquitous expression of the transgene is induced upon relatively mild heat treatment. Green fluorescent protein (GFP) was used as a marker to monitor successful induction of gene expression in transgenic embryos. We used this method to study the stage specificity of Wnt signalling function. Transient ectopic Wnt-8 expression during early neurulation was sufficient to repress anterior head development and this capacity was restricted to early stages of neurulation. By transient over-expression at different stages of development, we show that frizzled-7 disrupted morphogenesis sequentially from anterior to posterior along the dorsal axis as development proceeds. These results demonstrate that this method for inducible gene expression in transgenic Xenopus embryos will be a very powerful tool for temporal analysis of gene function and for studying molecular mechanisms of vertebrate organogenesis.

Animals↗

Expression of the bHLH transcription factor Tcf12 (ME1) gene is linked to the expansion of precursor cell populations during neurogenesis.

In this study, we focused on the potential function of the murine gene Tcf12 (also known as ME1 or HEB) encoding the bHLH E-protein ME1 during brain development. An exencephaly phenotype of low penetrance has consistently been observed in both Tcf12 null mice and Tcf12(dm) homozygous mice. Thus, to address the possible underlying mechanism of the Tcf12 gene during the early steps of brain development, we performed a detailed analysis of its spatio-temporal expression pattern at distinct steps of gastrulation and neurogenesis. We found that Tcf12 transcripts are detected in the embryonic ectoderm prior to neural induction during gastrulation. During neurulation, Tcf12 transcripts are evident at high levels in the proliferating neuroepithelium of the neural folds and the cephalic mesenchyme. Thus, Tcf12 gene expression coincides with the massive proliferation occurring in the forming neuroepithelium and cephalic mesenchyme during neural tube formation, which is consistent with the exencephaly phenotype of Tcf12 null mice. In the developing cortex and spinal cord, Tcf12 expression is restricted to the proliferative ventricular zones, indicating that Tcf12 expression is down regulated when these neuronal cells undergo their final differentiation. Interestingly, we found that the postnatal Tcf12 expression parallels the ongoing adult neurogenesis in the mitotically active subventricular zone. Thus, the timing and location of Tcf12 expression combined with this severe neurulation defect support our hypothesis that the Tcf12 gene may be involved in the control of proliferating neural stem cells and progenitor cells and that it may be critical to sustain their undifferentiated state during embryonic and adult neurogenesis.

Animals↗

Genetic control of caudal development.

Several lines of evidence suggest that caudal development involves a distinct programme. This is illustrated by the fact that a specific pattern of malformations affects the caudal end of the human embryo. In addition, neurulation, the process leading to the formation of the neural tube, proceeds through different morphogenetic movements caudally. In mammals, as in birds, the caudal neural tube arises from cavitation and not from folding of the neural plate as in more rostral levels. However, recent fate mapping studies have suggested that the two modes of neurulation represent a continuous programme, possibly involving similar cellular or molecular mechanisms. Finally, analyses of mutant mice have shown that T-box transcription factors and components of the Wnt signalling pathway control cellular migration and the promotion of mesoderm formation in the caudal embryo. In humans, mutation in the HLXB9 transcription factor causes an autosomal dominant form of sacral agenesis. Thus, the combination of classical embryological and molecular genetics approaches has provided critical reference points for the delineation of the developmental programme of the caudal embryo.

Animals↗

Expression of transforming growth factor-beta 2 and beta 3 mRNAs and proteins in the developing chicken embryo.

Specific cDNA probes and antibodies for chicken transforming growth factor (TGF)-beta 2 and beta 3 were used to study expression of TGF-beta 2 and beta 3 mRNAs and proteins in the developing chicken embryo. Expression of the mRNAs for both TGF-beta isoforms was detected by day 1.5 of incubation (Hamburger and Hamilton stage 10) by RNA Northern blot analysis and increased with developmental age. Expression of TGF-beta 2 and beta 3 mRNAs was detected in every embryonic tissue examined, with the level of expression of both isoforms being high in heart, brain and muscle and low in kidney and liver. Coordinate unidirectional upregulation of expression of TGF-beta 2 and beta 3 mRNAs occurred in most embryonic tissues with development except the heart, where the steady-state level of expression of TGF-beta 2 mRNA decreased with age, while that of TGF-beta 3 mRNA increased. In situ hybridization analysis detected TGF-beta 2 and beta 3 mRNAs as early as the definitive primitive streak stage (stage 4). During neurulation (stage 10), TGF-beta 2 and beta 3 mRNAs were detected in cells of all three germ layers; TGF-beta 3 mRNA was detected in neurectoderm as well. Following neurulation, TGF-beta 3 mRNA was detected in the neural tube, notochord, ectoderm, endoderm, sclerotome and dermomyotome at stage 16; expression of TGF-beta 2 mRNA was not as prominent as TGF-beta 3 mRNA in these structures. By stage 29, both TGF-beta 2 and beta 3 mRNAs were localized in several tissues including heart, lung, gizzard and feathers. Immunohistochemical staining analysis detected immunoreactive TGF-beta 2 and beta 3 proteins in all three germ layers of stage 4 embryos. Staining for TGF-beta 2 and beta 3 proteins was detected in several cell types and tissues in the early developing embryo frequently in the same locations as TGF-beta 2 and beta 3 mRNAs, with staining for TGF-beta 2 being less intense than TGF-beta 3. However, in some cases, localization of TGF-beta 2 and beta 3 proteins was different from that of the mRNAs, indicating that a complex mechanism of transcription, translation and secretion exits during chicken embryogenesis. These and other results suggest that TGF-beta 2 and beta 3 may play important roles and act through both autocrine and paracrine mechanisms in the development of many tissues in the chicken.

Animals↗

MARCKS deficiency in mice leads to abnormal brain development and perinatal death.

The MARCKS protein is a widely distributed cellular substrate for protein kinase C. It is a myristoylprotein that binds calmodulin and actin in a manner reversible by protein kinase C-dependent phosphorylation. It is also highly expressed in nervous tissue, particularly during development. To evaluate a possible developmental role for MARCKS, we disrupted its gene in mice by using the techniques of homologous recombination. Pups homozygous for the disrupted allele lacked detectable MARCKS mRNA and protein. All MARCKS-deficient pups died before or within a few hours of birth. Twenty-five percent had exencephaly and 19% had omphalocele (normal frequencies, < 1%), indicating high frequencies of midline defects, particularly in cranial neurulation. Nonexencephalic MARCKS-deficient pups had agenesis of the corpus callosum and other forebrain commissures, as well as failure of fusion of the cerebral hemispheres. All MARCKS-deficient pups also displayed characteristic lamination abnormalities of the cortex and retina. These studies suggest that MARCKS plays a vital role in the normal developmental processes of neurulation, hemisphere fusion, forebrain commissure formation, and formation of cortical and retinal laminations. We conclude that MARCKS is necessary for normal mouse brain development and postnatal survival.

Animals↗