Rab23 GTPase is expressed asymmetrically in Hensen's node and plays a role in the dorsoventral patterning of the chick neural tube.

The mouse Rab23 protein, a Ras-like GTPase, inhibits signaling through the Sonic hedgehog pathway and thus exerts a role in the dorsoventral patterning of the spinal cord. Rab23 mouse mutant embryos lack dorsal spinal cord cell types. We cloned the chicken Rab23 gene and studied its expression in the developing nervous system. Chick Rab23 mRNA is initially expressed in the entire neural tube but retracts to the dorsal alar plate. Unlike in mouse, we find Rab23 in chick already expressed asymmetrically during gastrulation. Ectopic expression of Rab23 in ventral midbrain induced dorsal genes (Pax3, Pax7) ectopically and reduced ventral genes (Nkx2.2 and Nkx6) without influencing cell proliferation or neurogenesis. Thus, in the developing brain of chick embryos Rab23 acts in the same manner as described for the caudal spinal cord in mouse. These data indicate that Rab23 plays an important role in patterning the dorso-ventral axis by dorsalizing the neural tube.     

Developmental regulation of activated ERK expression in the spinal cord and dorsal root ganglion of the chick embryo

Mitogen-activated protein kinases (MAPKs) are involved in the intracellular pathways that respond to various extracellular signals. Extracellular signal-regulated kinase (ERK) is a member of MAPKs and has various functions in neural development. However, the in vivo distribution of the activated form of ERK (p-ERK) in the developing nervous system is not well understood. Here, we investigated the expression of p-ERK in the spinal cord and dorsal root ganglion (DRG) of chick embryos. In the spinal cord, p-ERK-positive cells appeared in the ventral ventricular zone on embryonic day 4 (E4). From E6 onward, they appeared in the gray matter and in the white matter, suggesting migration from the ventricular zone. A double labeling method revealed that these p-ERK-positive cells included oligodendrocyte precursors. In the dorsal horn, p-ERK-positive small cells appeared on E6. Subsequently, the positive cells in the dorsal horn increased transiently in number and then decreased markedly by E10. Motoneurons also expressed p-ERK transiently on E7. In the DRG, weak p-ERK immunoreaction appeared in the ventrolateral region on E5. From E6, the immunoreactivity became stronger and by E9 intense p-ERK-positive cells were observed throughout the DRG. These data provide a neuroanatomical framework to begin to examine the in vivo role of ERK in neural development.     

背侧抑制性轴突导向蛋白的敲减对鸡胚后脑内23C10阳性神经纤维投射的影响

目的 探讨敲减鸡胚后脑内背侧抑制性轴突导向蛋白（draxin）的表达对23C10阳性神经纤维投射特性的影响。 方法 以碱性磷酸酶（ALP）融合蛋白与HH21~22阶段鸡胚后脑活组织切片体外孵育为对照组,以draxin-ALP融合蛋白与HH21~22阶段鸡胚后脑活组织切片体外孵育为实验组,每组10例,检测鸡胚后脑内的23C10阳性神经纤维是否具有与draxin直接结合的能力;以鸡胚后脑内空白质粒电穿孔组为对照,以针对draxin表达的小干扰RNA（siRNA）表达载体质粒电穿孔为实验组,每组18例,观察敲减draxin的效果,以及是否引起鸡胚后脑内23C10阳性神经纤维投射特性的改变。 结果 HH21~22阶段鸡胚后脑活组织切片内,大部分draxin阳性信号与23C10阳性神经纤维重叠;应用电穿孔方法导入draxin的siRNA敲减质粒后,draxin相对表达量显著降低（P<0.05）;HH25~26阶段的鸡胚后脑内,电穿孔一侧的23C10阳性神经纤维的背侧区域内投射纤维分布分散（P<0.05）。 结论 鸡胚后脑发育过程中,draxin可以与23C10阳性神经纤维直接结合,且其在鸡胚后脑内23C10阳性神经纤维成束投射的形成过程中发挥重要的调控作用。     

Identification of genes regulating sensory neuron genesis and differentiation in the avian dorsal root ganglia.

The dorsal root ganglia (DRG) derive from a population of migrating neural crest cells that coalesce laterally to the neural tube. As the DRG matures, discrete cell types emerge from a pool of differentiating progenitor cells. To identify genes that regulate sensory genesis and differentiation, we have designed screens to identify members from families of known regulatory molecules such as receptor tyrosine kinases, and generated full-length and subtractive cDNA libraries between immature and mature DRG for identifying novel genes not previously implicated in DRG development. Several genes were identified in these analyses that belong to important regulatory gene families. Quantitative PCR confirmed differential expression of candidate cDNAs identified from the subtraction/differential screening. In situ hybridization further validated dynamic expression of several cDNAs identified in our screens. Our results demonstrate the utility of combining specific and general screening approaches for isolating key regulatory genes involved in the genesis and differentiation of discrete cell types and tissues within the classic embryonic chick model system.     

Enforced expression of the transcription factor HOXD3 under the control of the Wnt1 regulatory element modulates cell adhesion properties in the developing mouse neural tube

HOX genes expressed in a specific spatial and temporal manner play a crucial role in determining the body plan during the early development of vertebrates. In adult tissues, many HOX genes participate in normal hematopoiesis and carcinogenesis. We previously found that overexpression of the homeobox gene HOXD3 alters expression levels of cell adhesion molecules in human cancer cell lines. Here, we have investigated whether HOXD3 expression is related to the cell adhesion processes during mouse development focusing on dorsal midline cells or roof-plate cells of the neural tube and neural crest cells. We created transgenic mouse embryos, in which HOXD3 is expressed in the dorsal midline under the control of the Wnt1 regulatory element, and analyzed these embryos at embryonic day 10.5-13.5. In HOXD3-expressing transgenic embryos, although neural crest-derived structures in the trunk region appeared to be normal, striking abnormalities were found in the neural tube. In transgenic embryos expressing the lacZ gene under the control of the Wnt1 regulatory element, expression of lacZ was restricted to roof-plate cells within the neural tube. By contrast, in HOXD3-expressing transgenic embryos, expression of HOXD3 was not only located in the dorsal neural tube, but also had spread inside the ventricular zone in more ventral regions of the neural tube. These findings show that the HOXD3 transgene is expressed more broadly than the Wnt1 gene is normally expressed. Expression of both Wnt1 and Msx1, marker genes in the roof plate, was further extended ventrally in HOXD3-expressing embryos than in normal embryos, suggesting that expression of the HOXD3 transgene expands the roof plate ventrally within the neural tube. In the ventricular zone of HOXD3-expressing embryos at embryonic day 10.5, we observed an increase in the number of mitotic cells and failure of interkinetic nuclear migration of progenitor cells. Furthermore, in HOXD3-expressing embryos at embryonic day 12.5, the ventricular zone, in which progenitor cells became more loosely connected to each other, was composed of a large number of cells that did not express N-cadherin. Our results indicate that expression of HOXD3 is closely associated with modulation of cell-adhesive properties during embryonic development.     

Retinoic acid generated by Raldh2 in mesoderm is required for mouse dorsal endodermal pancreas development.

Studies on nonmammalian vertebrate embryos have indicated that retinoic acid (RA) is required for pancreas development. We have analyzed mouse embryos carrying a null mutation of the gene encoding retinaldehyde dehydrogenase 2 (Raldh2), which controls RA synthesis. Raldh2-/- embryos specifically lack expression of Pdx1 (a homeobox gene required for pancreas development) and Prox1 in dorsal endodermal but not ventral endodermal pancreatic precursor tissues. Ventral endodermal expression of Hex is not affected in Raldh2-/- embryos, indicating that liver specification is not dependent upon RA. Also, expression of Foxa2 across the dorsoventral axis of the endoderm is not affected in Raldh2-/- embryos, indicating that a lack of RA does not cause a general defect in foregut endoderm development. Comparison of wild-type and Raldh2-/- embryos carrying an RA-reporter transgene demonstrates that RA activity is normally present throughout the endoderm except in the ventral-most region but is totally missing in endoderm of Raldh2-/- embryos. Thus, Raldh2 expressed in adjacent splanchnic lateral plate mesoderm provides an RA signal to dorsal endoderm. Dorsal Pdx1 expression is rescued in Raldh2-/- embryos by low-dose maternal administration of RA, which preferentially restores RA-reporter expression in the dorsal endoderm. Our findings demonstrate a specific role for RA in mouse embryos as a mesodermally synthesized signal needed for dorsal endodermal expression of Pdx1 during development of the dorsal pancreatic lineage.     

Differential expression of beta3 integrin gene in chick and mouse cranial neural crest cells.

RNA in situ hybridization on early chicken embryos revealed that the beta3 integrin gene started to be expressed after Hamburger and Hamilton (HH) stage 6 in the presumptive epidermis adjacent to the neural plate, before closure of the neural tube. The beta3 integrin gene was also strongly expressed in cephalic neural crest cells at the same stage in which they begin their migration but disappeared progressively in these cells along the route they take to the branchial arches. The gene was weakly expressed in the differentiating cranial neural crest cells. The alphaVbeta3 integrin protein complex was also mainly detected in the migratory cephalic neural crest cells. However, during early mouse embryogenesis and in contrast to the chick, the beta3 integrin gene was expressed in the foregut diverticulum and in the heart and not in the cephalic neural crest cells. Therefore, the difference in the beta3 integrin expression suggests that mouse and chicken cranial neural crest cells may have distinct integrin requirements during their ontogenesis.     

Nociceptive sensory neurons derive from contralaterally migrating, fate-restricted neural crest cells

Neural crest cells (NCCs) are a transient population of multipotent progenitors that give rise to numerous cell types in the embryo. An unresolved issue is the degree to which the fate of NCCs is specified prior to their emigration from the neural tube. In chick embryos, we identified a subpopulation of NCCs that, upon delamination, crossed the dorsal midline to colonize spatially discrete regions of the contralateral dorsal root ganglia (DRG), where they later gave rise to nearly half of the nociceptor sensory neuron population. Our data indicate that before emigration, this NCC subset is phenotypically distinct, with an intrinsic lineage potential that differs from its temporally synchronized, but ipsilaterally migrating, cohort. These findings not only identify a major source of progenitor cells for the pain- and temperature-sensing afferents, but also reveal a previously unknown migratory pathway for sensory-fated NCCs that requires the capacity to cross the embryonic midline.     

Conservation of K1 immunoreactivity against early cortical neurones in the vertebrate telencephalon

A monoclonal antibody, K1, immunostains neurones generated earliest in the rat neocortex. The K1 immunoreactivity was found in both mouse and human embryos. In the human marginal zone, the subpial granular layer and the inner sublayer were stained at the 19th and 20th week of gestation, respectively. Western blot analysis revealed that the K1 immunoreactivity was conserved in a variety of vertebrates. While a protein of low molecular weight (200 kDa) reacted dominantly in an amphibian (Xenopus laevis) and a reptile (Agkistrodon blomhoffii), proteins of higher molecular weights (280 and 290 kDa) reacted dominantly in mammals (mouse, rat and macaque). In the brain of the reptile (Lacerta triliniata) embryo, K1 stained a marginal part of the superficial molecular layer in the dorsal cortex that is probably homologous to the mammalian marginal zone in the neocortex. In the chick embryo at the 8th day of incubation, the immunoreactivity was observed on neurones generated earliest in the dorsal cortex but not in the superficial molecular layer. The dorsal ventricular ridge and pallial thickening in either the reptile or chick were not stained. The K1 antigen could be a good marker for evolutional study of the mammalian neocortex.     

Establishment of synaptic connections between explants of embryonic neural tissue in culture: experimental ultrastructural studies

Summary The development of synaptic interconnections between co-cultured explants of central and peripheral nervous tissue from chick embryos has been investigated by light and electron microscopy. Two sets of co-cultured explants were used: (a) dorsal root ganglion (DRG) and spinal cord and (b) retina and tectum. Both sets of co-cultured explants became linked by bundles of fibres but the most consistent results were obtained with the DRG-spinal cord explants. Thus axons from the DRG extended large distances across the culture substrate to reach and enter mainly the dorsal horn region of the spinal cord explants. In contrast retina-tectum links were less frequently established and were less extensive, possibly because there are fewer cells in retinal explants capable of establishing contacts in tectal explants than there are cells in DRG explants capable of establishing contacts in the spinal cord. In order to distinguish between synapses involving only neuronal elements within an expiant and those involving ingrowing fibres, fibre bundles linking adjacent explants were transected and the preparations fixed two to six hours later. Electron microscope study of such cultures revealed degenerating neurites and terminals in the spinal cord explants receiving DRG fibres but none in the corresponding DRG explants. Retinal explants contain numerous synapses of many types but degenerating terminals could not be found within the retinal explants after nerve fibre transections. Degenerating neurites and terminals were found within tectal explants but they were fewer and more difficult to locate than those found within spinal cord explants. The reasons for such differences are discussed.     

组织外植块对培养的鸡胚背根节神经突起生长的影响

为了解神经元的发育和周围组织的关系,本文分别用了:(1)10天鸡胚背根节与10天鸡胚心脏、皮肤、角膜、骨骼肌、肠、大脑、脊髓外植块联合培养;(2)不同时期的鸡角膜与不同胚龄的鸡胚背根节联合培养;(3)10天鸡胚背根节与雏鸡角膜内、外层联合培养。实验结果表明鸡胚心脏、皮肤和角膜外植块对背根节神经突起生长具较强的促进作用且对生长方向有明显诱导作用;骨骼肌、肠和大脑对神经突起生长也有不同程度促进作用,但脊髓却无明显作用。14、16、18天鸡胚角膜对8、10、12、14天鸡胚背根节神经突起生长有明显促进作用;12、14天鸡胚背根节在各时期角膜作用下神经突起生长都较丰富。含上皮层的雏鸡角膜外植块促进背根节神经突起生长的作用比含内皮层的角膜外植块强。     

Identification of a lipid kinase as a host factor involved in hepatitis C virus RNA replication

A functional screen of an adenovirus-delivered shRNA library that targets ∼ 4500 host genes was performed to identify cellular factors that regulate hepatitis C virus (HCV) sub-genomic RNA replication. Seventy-three hits were further examined by siRNA oligonucleotide-directed knockdown, and silencing of the PI4KA gene was demonstrated to have a significant effect on the replication of a HCV genotype 1b replicon. Using transient siRNA oligonucleotide transfections and stable shRNA knockdown clones in HuH-7 cells, the PI4KA gene was shown to be essential for the replication of all HCV genotypes tested (1a, 1b and 2a) but not required for bovine viral diarrhea virus (BVDV) RNA replication.     

Slit2/Robo1信号对鸡胚早期神经管及体节发育的影响

目的: 探讨Slit2/Robo1对鸡胚早期神经管和体节发育的影响。 方法: 显微注射法将质粒注射入HH10期胚胎神经管内,活体胚胎细胞电穿孔方法转染胚胎半侧神经管,以另一侧神经管为对照侧,原位杂交及免疫荧光方法观察转染10 h后神经管的发育和神经嵴细胞迁移至体节的情况。结果: 下调Robo1侧神经管发育较正常对照侧异常,同时发现Slug表达和神经嵴细胞迁移至体节路线发生改变。结论: Slit2/Robo1信号可能通过影响Slug基因表达,对胚胎早期神经管闭合、神经嵴细胞正常产生及迁移方向以及体节分化有重要作用。     

RhoA is highly up-regulated in the process of early heart development of the chick and important for normal embryogenesis.

We have used molecular techniques, combined with classic embryological methods, to identify up-regulated genes associated with early heart development. One of the cDNAs identified and isolated by screening a chick lambda cDNA library was the small guanosine triphosphatase RhoA. RhoA has at least three different length mRNA species, each varying in the length of the 3' untranslated region. In situ hybridisation and immunocytochemistry analysis of RhoA expression show marked up-regulation in the heart-forming region. In other systems, RhoA signalling has been shown to be important for both gene expression and morphology. To investigate the function of RhoA in early heart development, we used small interfering RNAs (siRNA) in early chick embryos. Disruption of RhoA expression by siRNA treatment resulted in lack of heart tube fusion and abnormal head development. These data indicate that RhoA is important for normal embryogenesis.