Immunolocalization of Periostin-like factor and Periostin during embryogenesis.

Periostin-like factor (PLF) and Periostin are alternatively spliced mRNAs. Our findings are the first to show similarities and differences between PLF and Periostin location using isoform-specific antibodies. The differences in when and where they are present during mouse embryogenesis suggest that they may have different functions. Using immunostaining techniques, we observed that PLF was highly expressed at 12.5 days postconception (dpc) in the intermediate and outer zones of most brain regions, spinal cord, cranial and spinal nerves, and chondrocytes in developing bone and in the heart wall. By 16.5 dpc, PLF was also present in ameloblasts and odontoblasts in developing teeth, and by 19.5 dpc, PLF was present at low levels only in vagal nerve bundles, discrete white matter bundles in the brain, and chondrocytes of developing ribs. Periostin, on the other hand, was absent at 12.5 dpc from dorsal spinal cord and from cranial and spinal nerves. By 16.5 dpc, Periostin was present in many spinal nerves, but absent thereafter, and at 19.5 dpc, Periostin was present in chondrocytes in developing bone but not in neural tissues. The different spatial and temporal location of PLF and Periostin in cartilage and bone cells suggests different roles for these proteins in endochondral bone formation. The early expression of PLF in brain differentiation zones and in developing axon bundles and nerves suggests that it may facilitate axon growth.     

Targeted disruption of the neurochondrin/norbin gene results in embryonic lethality

Neurochondrin/norbin is a cytoplasmic protein involved in dendrite outgrowth. The expression of the gene has been restricted to neural, bone, and chondral tissues. To identify the functions of the gene in vivo, we have generated mice with a disrupted mutation in the neurochondrin/norbin gene. Histological analysis of heterozygous mutant mice indicates the possibility of specific functions of neurochondrin/norbin in chondrocyte differentiation. We defined the expression patterns of neurochondrin/norbin-lacZ fusion protein in the central nervous system. In the developing olfactory bulb, beta-galactosidase activity was detected in the mantle layer at 12.5 dpc and the strongest activity was detected in the presumptive mitral or tufted cell layer at 15.5 dpc. beta-Galactosidase activity was also detected in the lateral choroid plexus. In homozygous (-/-) mutant mice, the disruption of the neurochondrin/norbin gene leads to early embryonic death between 3.5 and 6.5 dpc. This result indicates that neurochondrin/norbin gene function is essential for the early embryogenesis.     

小鼠ERb 基因片段的克隆及其在胚胎发育中的表达

为探讨ERb在小鼠胚胎发育过程中的表达, 首先设计ERb引物并扩增ERb基因片段, 构建ERb/pGEM-3Z重组质粒进行克隆, 分别用EcoRⅠ和HindⅢ进行酶切得到线性化DNA片段, 以Sp6和T7聚合酶合成地高辛标记的(dig )正、反义RNA探针。然后通过胚胎整体原位杂交技术分析ERb 在小鼠胚胎中的表达。运用该探针检测到ERb 基因在10.5 dpc胚胎的脑、脊神经管、生殖脊、心包、肢芽及颌弓部位表达, 在13.5 dpc胚胎的端脑、中脑、延髓、脊髓、肢芽中表达。推测ERb基因可能在小鼠胚胎性别分化过程中起调节作用; 可能在小鼠胚胎神经管的早期区域化过程中起作用并在3个原始脑泡进一步分化及脊髓的分化过程中起作用; 可能在小鼠胚胎肢芽中的骨与软骨的形成与分化中起调控作用; 可能在小鼠胚胎心脏发育过程中起作用, 可能在小鼠胚胎颌弓的表面分化过程中起调控作用。     

Aortic carboxypeptidase-like protein is expressed in collagen-rich tissues during mouse embryonic development

Aortic carboxypeptidase-like protein (ACLP) was originally identified in vascular smooth muscle cells and contains discoidin and catalytically inactive metallocarboxypeptidase domains. ACLP is a secreted protein that associates with the extracellular matrix and is essential for abdominal wall development and contributes to dermal wound healing. Because of these developmental and adult phenotypes, we examined the expression of ACLP by immunohistochemistry throughout mouse embryonic development. ACLP was not detected in 7.5 days post-coitum (dpc) embryos, however at 9.5 dpc low levels of expression were detected in the somites and dorsal aorta. Expression was detected in both the yolk sac and embryonic vasculature at 10.5d pc. ACLP expression increased in both large and small blood vessels at 11.5 and 13.5 dpc and intense expression was detected within the vascular smooth muscle layer in 16.5 dpc embryos. At later developmental time points, discrete areas of ACLP expression were detected in the mesenchymal cells in the dermal layer, developing skeletal structures, connective tissue, and in the umbilical ring and vessels. The predominance of ACLP immunoreactivity localized with collagen-rich regions including tendons and basement membranes. Overall, the developmental expression pattern is consistent with a regulatory or structural role in the abdominal wall, vasculature, and dermis.     

A novel BMP expressed in developing mouse limb, spinal cord, and tail bud is a potent mesoderm inducer in Xenopus embryos

The bone morphogenetic proteins (BMPs) play critical roles in patterning the early embryo and in the development of many organs and tissues. We have identified a new member of this multifunctional gene family, BMP-11, which is most closely related to GDF-8/myostatin. During mouse embryogenesis, BMP-11 is first detected at 9.5 dpc in the tail bud with expression becoming stronger as development proceeds. At 10.0 dpc, BMP-11 is expressed in the distal and posterior region of the limb bud and later localizes to the mesenchyme between the skeletal elements. BMP-11 is also expressed in the developing nervous system, in the dorsal root ganglia, and dorsal lateral region of the spinal cord. To assess the biological activity of BMP-11, we tested the protein in the Xenopus ectodermal explant (animal cap) assay. BMP-11 induced axial mesodermal tissue (muscle and notochord) in a dose-dependent fashion. At higher concentrations, BMP-11 also induced neural tissue. Interestingly, the activin antagonist, follistatin, but not noggin, an antagonist of BMPs 2 and 4, inhibited BMP-11 activity on animal caps. Our data suggest that in Xenopus embryos, BMP-11 acts more like activin, inducing dorsal mesoderm and neural tissue, and less like other family members such as BMPs 2, 4, and 7, which are ventralizing and anti-neuralizing signals. Taken together, these data suggest that during vertebrate embryogenesis, BMP-11 plays a unique role in patterning both mesodermal and neural tissues.     

Up-regulation of neural stem cell markers suggests the occurrence of dedifferentiation in regenerating spinal cord

Following tail amputation in urodele amphibians, an ependymal tube, that resembles a developing neural tube, forms from ependymal cells that migrate from the cord stump and elongates by cell proliferation. Expression of the keratin pair 8 and 18 has been observed in the developing urodele nervous system and is maintained in the ependymal cells of the mature cord. We show here that expression of these keratins is not unique to urodeles, but is also observed in the radial glia of the human spinal cord, suggesting that these proteins might play a role both in neural development and regeneration. Analysis of their expression in the regenerating spinal cord following tail amputation shows that their expression, as well as that of glial fibrillary acidic protein (GFAP), is maintained in the ependymal tube during regeneration, though differences in their levels of expression are observed along the anteroposterior axis and appear to be related to the progression of morphogenesis. In addition, we show that following tail amputation the ependymal tube expresses the neural stem cell markers nestin and vimentin, which are undetectable in normal urodele spinal cord. This up-regulation of neural stem cell markers shows that the ependymal cells undergo a phenotypic change. Whereas maintenance of keratin and GFAP expression in the adult ependyma may reflect a higher plasticity of these cells in adult urodeles than in other vertebrates, re-expression of markers of early neural development suggests the occurrence of a dedifferentiation process in the spinal cord in response to injury.Edited by J. Campos-Ortega     

Expression of the mouse PR domain protein Prdm8 in the developing central nervous system

It was first shown in the PR (PRDI-BF1 and RIZ homology) domain family proteins that the PR domain has homology to the SET (Su(var)3-9, Enhancer-of-zeste and Trithorax) domain, a catalytic domain of the histone lysine methyltransferases. Recently, there are many reports that the PR domain proteins have important roles in development and/or cell differentiation. In this report, we show the expression patterns of one of the mouse PR domain proteins, Prdm8, in the developing central nervous system. In the developing retina, Prdm8 expression was detected in postmitotic neurons in the inner nuclear layer and the ganglion cell layer, and its expression became restricted predominantly to the rod bipolar cells when retinogenesis was completed. In the developing spinal cord, Prdm8 was expressed first in the progenitor populations of ventral interneurons and motor neurons, and later in a subpopulation of interneurons. In the developing brain, Prdm8 expression was observed in postmitotic neurons in the intermediate zone and the cortical plate. In the postnatal brain, Prdm8 was expressed mainly in layer 4 neurons of the cerebral cortex. These results show that Prdm8 expression is tightly regulated in a spatio-temporal manner during neural development and mainly restricted to postmitotic neurons, except in the spinal cord.     

Expression pattern of BM88 in the developing nervous system of the chick and mouse embryo

We isolated a chick homologue of BM88 (cBM88), a cell-intrinsic nervous system-specific protein and examined the expression of BM88 mRNA and protein in the developing brain, spinal cord and peripheral nervous system of the chick embryo by in situ hybridization and immunohistochemistry. cBM88 is widely expressed in the developing central nervous system, both in the ventricular and mantle zones where precursor and differentiated cells lie, respectively. In the spinal cord, particularly strong cBM88 expression is detected ventrally in the motor neuron area. cBM88 is also expressed in the dorsal root ganglia and sympathetic ganglia. In the early neural tube, cBM88 is first detected at HH stage 15 and its expression increases with embryonic age. At early stages, cBM88 expression is weaker in the ventricular zone (VZ) and higher in the mantle zone. At later stages, when gliogenesis persists instead of neurogenesis, BM88 expression is abolished in the VZ and cBM88 is restricted in the neuron-containing mantle zone of the neural tube. Association of cBM88 expression with cells of the neuronal lineage in the chick spinal cord was demonstrated using a combination of markers characteristic of neuronal or glial precursors, as well as markers of differentiated neuronal, oligodendroglial and astroglial cells. In addition to the spinal cord, cBM88 is expressed in the HH stage 45 (embryonic day 19) brain, including the telencephalon, diencephalon, mesencephalon, optic tectum and cerebellum. BM88 is also widely expressed in the mouse embryonic CNS and PNS, in both nestin-positive neuroepithelial cells and post-mitotic betaIII-tubulin positive neurons.     

Dorsally derived netrin 1 provides an inhibitory cue and elaborates the 'waiting period' for primary sensory axons in the developing spinal cord

Dorsal root ganglion (DRG) neurons extend axons to specific targets in the gray matter of the spinal cord. During development, DRG axons grow into the dorsolateral margin of the spinal cord and projection into the dorsal mantle layer occurs after a ;waiting period' of a few days. Netrin 1 is a long-range diffusible factor expressed in the ventral midline of the developing neural tube, and has chemoattractive and chemorepulsive effects on growing axons. Netrin 1 is also expressed in the dorsal spinal cord. However, the roles of dorsally derived netrin 1 remain totally unknown. Here, we show that dorsal netrin 1 controls the correct guidance of primary sensory axons. During the waiting period, netrin 1 is transiently expressed or upregulated in the dorsal spinal cord, and the absence of netrin 1 results in the aberrant projection of sensory axons, including both cutaneous and proprioceptive afferents, into the dorsal mantle layer. Netrin 1 derived from the dorsal spinal cord, but not the floor plate, is involved in the correct projection of DRG axons. Furthermore, netrin 1 suppresses axon outgrowth from DRG in vitro. Unc5c(rcm) mutant shows abnormal invasion of DRG axons as observed in netrin 1 mutants. These results are the first direct evidence that netrin 1 in the dorsal spinal cord acts as an inhibitory cue for primary sensory axons and is a crucial signal for the formation of sensory afferent neural networks.