Early postembryonic neural development in the zebrafish: a 3-D reconstruction of forebrain proliferation zones shows their relation to prosomeres.

Based on a section-by-section analysis of the morphology (combined silver/Nissl stain) and of the distribution of proliferation zones (immunohistochemical detection of the proliferating cell nuclear antigen) in the zebrafish (Danio rerio) forebrain at 5 days postfertilization, we created a three-dimensional reconstruction of proliferation zones of that developmental stage. The resulting model visualizes the size, number, location and morphology of forebrain proliferation zones. The latter foreshadow closely adult neuroanatomical forebrain entities. Furthermore, the detailed distribution of proliferation zones in the posterior forebrain - but not in the more anterior secondary prosencephalon - supports a segmental prosomeric organization.     

Acquisition of regional and cellular identities in the developing zebrafish nervous system.

In the past year, several new techniques have been used with great success in the study of nervous system development in the zebrafish. Perhaps the most exciting results have come from experiments in which single identified cells or small groups of cells have been transplanted between embryos in order to examine cell determination and the site of action of genetic mutations.     

Expression of collapsin response mediator proteins in the nervous system of embryonic zebrafish

Collapsin response mediator proteins (CRMPs also known as TUC, Drp, Ulip, TOAD-64) are cytosolic phosphoproteins that are involved in signal transduction during axon growth and in cytoskeletal dynamics. Here we report cloning and mRNA expression patterns of CRMP-1, -2, -3, -4 and, owing to a genome duplication in teleosts, two homologs of CRMP-5 (CRMP-5a and -5b) in embryonic zebrafish at 16 and 24 h post-fertilization (hpf). CRMPs are evolutionarily conserved and zebrafish CRMPs show amino acid identities of 76–90% with their homologs in humans, with the exception of CRMP-3, which shows only 67% homology. Between 16 and 24 hpf, expression of CRMPs generally increased in many regions of the CNS undergoing neuronal differentiation and axonogenesis, but not in the proliferative ventricular zone. Structures that were typically labeled by most, but not all the CRMP probes were the telencephalon, the nucleus of the tract of the post-optic commissure, the epiphysis, the nucleus of the medial longitudinal fascicle, clusters of hindbrain neurons, cranial ganglia, as well as Rohon-Beard neurons. No expression of CRMP mRNAs was observed outside the nervous system. Thus, expression patterns of different CRMP family members correlate with neuronal differentiation and axonogenesis in embryonic zebrafish.     

Expression patterns of chick Musashi-1 in the developing nervous system

Vertebrate homologues of musashi have recently been referred to as neural stem cell markers because of their expression patterns and RNA-binding interactions. In the context of the notch signaling pathway, Musashi-1 (Msi-1) is a regulator of neural cell generation, cooperating with notch to maintain mitosis. In an effort to identify definitive stem cell markers of the neural retina, a portion of the Msi-1 cDNA was cloned, and the expression of Msi-1 in the chick eye was analyzed. Using an Msi-1-specific antibody and RNA probe, we show that expression of Msi-1 in the early neural tube is consistent with neural stem identity. In the neural retina, expression starts shortly before embryonic day 3 (E3) and continues up to and including E18. A BrdU incorporation assay shows Msi-1 to be found in both proliferating and differentiating cells of E5 neural retina. At E8 (when proliferation is complete in the fundus of the retina) and E18 (mature retina) Msi-1 expression was found in the ciliary marginal zone (CMZ) as well as in a subpopulation of differentiated cells, including photoreceptors and ganglion cells.     

Expression of isotocin-neurophysin mRNA in developing zebrafish

Neurohypophysial peptides are important regulators of homeostasis, reproduction and behavior. We have sequenced a zebrafish cDNA representing isotocin-neurophysin (IT-NP) mRNA. The developmental expression pattern of zebrafish IT-NP mRNA was determined by whole-mount in situ hybridization histochemistry. At 32 h post fertilization (hpf) no IT-NP mRNA is detected. However, by 36 hpf, staining for IT-NP mRNA is detected in a tight bilateral cluster of cells located in the anterior hypothalamus. The IT-NP mRNA expression pattern remains remarkably stable throughout further development at least until 120 hpf.     

Expression zones of three novel genes abut the developing anterior neural plate of Xenopus embryo

We identified three novel genes that were expressed within the anterior non-neural ectoderm of Xenopus early neurula embryos. The expression of these genes was observed in the different areas complementary to the expression zone of a homeodomain gene Xanf-1 in the anterior neural plate. One of these genes, a Ras-like GTP-ase Ras-dva, marked the anterior placodal ectoderm area; a second, an Agr family homologous gene, XAgr2, was expressed in the anterior-most ectoderm in the cement gland primordium, and a third, novel gene Nlo was expressed in the lateral neural folds. The genes were transiently expressed in the developing cement and hatching gland primordia, and repressed in the mature cement and hatching glands. XAgr2 and Nlo were also expressed in the otic vesicles, and Ras-dva was expressed in the dorso-lateral column of the neural tube.     

中间丝蛋白Nestin在不同病程糖尿病大鼠肾组织中的表达

目的检测中间丝蛋白Nestin(巢蛋白)在不同病程糖尿病大鼠肾组织中的表达,探讨Nestin表达变化与糖尿病肾病发生发展的关系。方法腹腔注射链脲佐菌素(STZ)复制糖尿病(DM)大鼠模型,分别于第2、4、8、12和16周检测血糖、血尿素氮及24h尿蛋白量,HE染色观察肾脏病理学改变,免疫组织化学及流式细胞术检测Nestin表达水平。结果 HE染色可见,与对照组相比,DM组大鼠从第2周起出现肾小球体积增大;至8周时系膜基质明显增多,系膜区增宽;12、16周时肾小球呈分叶状,肾小管上皮细胞可见明显空泡变性及坏死。免疫组织化学和流式细胞术结果显示,DM各组Nestin表达水平均高于正常对照组,且在第8周时达高峰,而后逐渐下降。结论在不同病程糖尿病大鼠模型中,中间丝蛋白Nestin的表达先升高,而后降低,可能参与了糖尿病肾损害的发生与发展。     

Expression pattern of LINGO-1 in the developing nervous system of the chick embryo

We isolated a chick homologue of LINGO-1 (cLINGO-1), a novel component of the Nogo-66 receptor (NgR)/p75 neurotrophin receptor (NTR) signaling complex, and examined the expression of cLINGO-1 in the developing brain and spinal cord of the chick embryo by in situ hybridization and immunohistochemistry. cLINGO-1 was expressed broadly in the spinal cord, including the ventral portion of the ventricular zone, and motor neurons. cLINGO-1 was also expressed in the dorsal root ganglion and boundary cap cells at dorsal and ventral roots. In the early embryonic brain, cLINGO-1 was first expressed in the prosencephalon and the ventral mesencephalon, and later in the telencephalon, the rostral part of the mesencephalon and some parts of the hindbrain. cLINGO-1 was also expressed in the ventral part of the neural retina and trigeminal and facial nerves. We also found that cLINGO-1, cNgR1 and p75NTR were expressed in overlapped patterns in the spinal cord and the dorsal root ganglion, but that these genes were expressed in distinct patterns in the early embryonic brain.     

Expression of PKC in the developing zebrafish, Danio rerio

Protein kinase C (PKC) is a family of enzymes involved in a wide range of biological functions. We investigated the expression of PKC-positive cells in zebrafish embryos and larvae within the first week of development to determine the developmental profile of PKC-containing cells. Our other goal was to determine if PKC alpha was associated with Rohon-Beard neurons during the first 5 days of development, when they are reported to undergo apoptosis. First, we confirmed the specificity of the antibodies by Western blotting zebrafish brain homogenates with anti-PKC and anti-PKC alpha, and detected single protein bands of approximately 78-82 kDa in size. Immunohistochemistry showed that several types of neurons were labeled, including neurons in the trigeminal ganglia, the dorsal spinal cord, and the dorsal root ganglia. Double-labeling with anti-PKC alpha and both anti-Islet-1 and zn12 confirmed the identity of the PKC-positive cells in the brain as trigeminal neurons, and in the spinal cord as Rohon-Beard cells. Some Rohon-Beard cells were labeled with anti-PKC alpha up to 7 days post fertilization (dpf). We performed TUNEL labeling and found no correlation between TUNEL-labeled and PKC alpha-labeled Rohon-Beard cells, suggesting that PKC alpha is not involved in Rohon-Beard apoptosis. Only approximately 40% of the approximately 130 Rohon-Beard cells at 24 h postfertilization (hpf) were positively labeled for PKC. Mauthner cells were labeled by anti-PKC, but not anti-PKC alpha, suggesting that the major form of PKC within these cells was not PKC alpha.     

Endothelin-3 regulates neural crest cell proliferation and differentiation in the hindgut enteric nervous system

Neural crest cells (NCC) migrate, proliferate, and differentiate within the wall of the gastrointestinal tract to give rise to the neurons and glial cells of the enteric nervous system (ENS). The intestinal microenvironment is critical in this process and endothelin-3 (ET3) is known to have an essential role. Mutations of this gene cause distal intestinal aganglionosis in rodents, but its mechanism of action is poorly understood. We find that inhibition of ET3 signaling in cultured avian intestine also leads to hindgut aganglionosis. The aim of this study was to determine the role of ET3 during formation of the avian hindgut ENS. To answer this question, we created chick-quail intestinal chimeras by transplanting preganglionic quail hindguts into the coelomic cavity of chick embryos. The quail grafts develop two ganglionated plexuses of differentiated neurons and glial cells originating entirely from the host neural crest. The presence of excess ET3 in the grafts results in a significant increase in ganglion cell number, while inhibition of endothelin receptor-B (EDNRB) leads to severe hypoganglionosis. The ET3-induced hyperganglionosis is associated with an increase in enteric crest cell proliferation. Using hindgut explants cultured in collagen gel, we find that ET3 also inhibits neuronal differentiation in the ENS. Finally, ET3, which is strongly expressed in the ceca, inhibits the chemoattraction of NCC to glial-derived neurotrophic factor (GDNF). Our results demonstrate multiple roles for ET3 signaling during ENS development in the avian hindgut, where it influences NCC proliferation, differentiation, and migration.     

Expression of arginine vasotocin receptors in the developing zebrafish CNS

Vasotocin/vasopressin is a neuropeptide that regulates social and reproductive behaviors in a variety of animals including fish. Arginine vasotocin (AVT) is expressed by cells in the ventral hypothalamic and preoptic areas in the diencephalon during embryogenesis in zebrafish suggesting that vasotocin might mediate other functions within the CNS prior to the development of social and reproductive behaviors. In order to examine potential early roles for vasotocin we cloned two zebrafish vasotocin receptors homologous to AVPR_1a. The receptors are expressed primarily in the CNS in similar but generally non-overlapping patterns. Both receptors are expressed in the forebrain, midbrain and hindbrain by larval stage. Of note, AVTR_1a-expressing neurons in the hindbrain appear to be contacted by the axons of preoptic neurons in the forebrain that include avt+ neurons and sensory axons in the lateral longitudinal fasciculus (LLF). Furthermore, AVTR_1a-expressing hindbrain neurons extend axons into the medial longitudinal fasciculus (MLF) that contains axons of many neurons thought to be involved in locomotor responses to sensory stimulation. One hypothesis consistent with this anatomy is that AVT signaling mediates or gates sensory input to motor circuits in the hindbrain and spinal cord.     

Expression of arginine vasotocin receptors in the developing zebrafish CNS

Vasotocin/vasopressin is a neuropeptide that regulates social and reproductive behaviors in a variety of animals including fish. Arginine vasotocin (AVT) is expressed by cells in the ventral hypothalamic and preoptic areas in the diencephalon during embryogenesis in zebrafish suggesting that vasotocin might mediate other functions within the CNS prior to the development of social and reproductive behaviors. In order to examine potential early roles for vasotocin we cloned two zebrafish vasotocin receptors homologous to AVPR_1a. The receptors are expressed primarily in the CNS in similar but generally non-overlapping patterns. Both receptors are expressed in the forebrain, midbrain and hindbrain by larval stage. Of note, AVTR_1a-expressing neurons in the hindbrain appear to be contacted by the axons of preoptic neurons in the forebrain that include avt+ neurons and sensory axons in the lateral longitudinal fasciculus (LLF). Furthermore, AVTR_1a-expressing hindbrain neurons extend axons into the medial longitudinal fasciculus (MLF) that contains axons of many neurons thought to be involved in locomotor responses to sensory stimulation. One hypothesis consistent with this anatomy is that AVT signaling mediates or gates sensory input to motor circuits in the hindbrain and 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.