Comparative analysis of Pax-2 protein distributions during neurulation in mice and zebrafish.

Members of different vertebrate species share a number of developmental mechanisms and control genes, suggesting that they have similar genetic programs of development. We compared the expression patterns of the Pax-2 protein in Mus musculus and Brachydanio rerio to gain a better understanding of the evolution of developmental control genes. We found that the tissue specificity and the time course of Pax-2 expression relative to specific developmental processes are remarkably similar during the early development of the two organisms. The brain, the optic stalk, the auditory vesicle, the pronephros, and single cells in the spinal cord and the hindbrain express Pax-2 in both species. The Pax-2 expression domain in the prospective brain of E8 mouse embryos has not been described previously. Expression appears first during early neurulation at the junction between the midbrain and hindbrain. However, there are some differences in Pax-2 expression between the two species. Most notable, expression at the midbrain/hindbrain boundary is no longer detectable after E11 in the mouse. Using monoclonal antibodies, we could exclude that primary neurons express Pax-2 in the zebrafish spinal cord. Our results confirm that Pax genes are highly conserved both in sequences and in expression patterns, indicating that they may have a function during early development that has been conserved during vertebrate evolution.     

pax-6 gene expression in newt eye development

 pax-6 is thought to be a master control gene of eye development in species ranging from insects to mammals. We have isolated a pax-6 cDNA homolog of the newt, Cynops pyrrhogaster. RT-PCR and sequence analyses predicted four alternatively spliced forms derived from inclusion or exclusion of the region corresponding to exons 5a and 12 in the human pax-6 ortholog. This gene shared extensive sequence identitiy and similar expression patterns with those of mouse and zebrafish. pax-6 signal was first detected at the anterior ridge of the neural plate, and later at the eye and nasal primordium and in the central nervous system – except for the midbrain. The injection of sonic hedgehog (shh) RNA inhibited the expression of pax-6 within the optic vesicle and disturbed eye cup formation. A similar suppressive effect of shh was also observed in the conjugation of the animal caps preloaded with exogenous shh and noggin mRNA, which was used as an inducer of pax-6. In contrast, shh injection had no effect on the expression of pax-6 in the surface ectoderm overlying the optic cup, suggesting that the expression of pax-6 in the surface ectoderm is not regulated by shh in vivo. Moreover, we found transient activation of pax-6 in animal cap explants at the sibling stage of mid-late gastrula. This observation raises the possibility that the ectoderm is competent to the lens-inducing signal at a stage as early as mid gastrula. Received: 5 February 1997 / Accepted: 30 April 1997     

成年斑马鱼脊髓损伤修复中脑gdnf和nos基因的表达

成年斑马鱼(Danio rerio)具有很强的脊髓损伤后自主修复的能力, 但目前其机制不明。为了研究斑马鱼中脑组织对脊髓再生的影响, 文章应用成年斑马鱼脊髓损伤模型, 采用实时定量PCR方法和原位杂交技术, 检测了斑马鱼脑中胶质细胞源性神经营养因子(gdnf)和一氧化氮合酶(nos)基因在脊髓损伤后4 h、12 h、6 d、11 d的表达情况, 展示了这两种基因在斑马鱼脑内不同核团的动态表达变化。结果显示, 成年斑马鱼脊髓损伤后, 神经营养因子gdnf基因在损伤急性期(4 h、12 h)和神经修复期(6 d、11 d)于斑马鱼脑内呈现显著性升高(P<0.05),而一氧化氮合酶基因nos的表达于损伤急性期显著性升高 (P<0.05), 随后下降, 并在修复期 (11 d)显著降低(P<0.05)。这表明, 脊髓损伤后, 高表达gdnf基因同时低表达nos基因的脑环境给脊髓损伤提供了良好的神经再生微环境, 从而可能促进轴突的再生长及运动能力的恢复。     

The eyeless mutant gene (e) in the Mexican axolotl (Ambystoma mexicanum) affects pax-6 expression and forebrain axonogenesis.

This study tested the hypothesis that changes in the patterns of pax-6 expression disrupt the anatomy and axonogenesis of the diencephalic areas of the eyeless axolotl. Proper pax-6 expression is necessary for eye and hypothalamus morphogenesis. Since the expression boundaries of pax-6 also provide a permissive environment for axonal outgrowth, an extensive study examining the effects of the eyeless gene (e) in the Mexican axolotl upon pax-6 expression and forebrain axonogenesis was begun. This study used whole embryo in situ hybridization techniques to follow pax-6 expression and whole brain immunocytochemistry to examine axonogenesis and neural differentiation. These studies demonstrated that the mutant gene e in the axolotl alters the response of midanterior neural-plate tissue to signals from the prechordal plate. This response was hypothesized to be a hyper-response to signals (sonic hedgehog?) that suppressed pax-6 expression within the midanterior neural plate and later developmental stages. Alternatively, the affected neuroectoderm of the eyeless embryos may lack competence to express pax-6. Lowered pax-6 expression inhibited eye and forebrain morphogenesis as well as neural axonogenesis and differentiation. Differentiation defects were detected as the suppression of midline dopaminergic neurons within the suprachiasmatic nucleus of eyeless animals. Thus, lowered pax-6 expression by the midanterior neuroectoderm promotes the eyeless condition by inhibiting the role of pax-6 in eye formation. This lowered expression also leads to concurrent alterations in the hypothalamic terrain which disrupt axonogenesis and ultimately promote sterility.     

Paraxial mesoderm specifies zebrafish primary motoneuron subtype identity

We provide the first analysis of how a segmentally reiterated pattern of neurons is specified along the anteroposterior axis of the vertebrate spinal cord by investigating how zebrafish primary motoneurons are patterned. Two identified primary motoneuron subtypes, MiP and CaP, occupy distinct locations within the ventral neural tube relative to overlying somites, express different genes and innervate different muscle territories. In all vertebrates examined so far, paraxial mesoderm-derived signals specify distinct motoneuron subpopulations in specific anteroposterior regions of the spinal cord. We show that signals from paraxial mesoderm also control the much finer-grained segmental patterning of zebrafish primary motoneurons. We examined primary motoneuron specification in several zebrafish mutants that have distinct effects on paraxial mesoderm development. Our findings suggest that in the absence of signals from paraxial mesoderm, primary motoneurons have a hybrid identity with respect to gene expression, and that under these conditions the CaP axon trajectory may be dominant.     

Regional expression of three homeobox transcripts in the inner ear of zebrafish embryos.

The inner ear of all jawed vertebrates arises from the epithelium of the otic vesicle and contains three semicircular canals, otoliths, and sets of sensory neurons, all positioned precisely within the cranium to detect head orientation and movement. The msh-C gene and two new homebox genes, msh-D and a gene related to distal-less, dlx-3, are each expressed in distinct regions of the otic vesicle during its early development in zebrafish embryos. Cells in the ectoderm express dlx-3 before induction of the otic vesicle, suggesting that dlx-3 has an early function in this process. Later, cells aligned with the future axes of the semicircular canals specifically express either dlx-3 or msh-D. Even later, sensory hair cells express msh-C and msh-D, while other cells of the epithelium express dlx-3. The early expression of these genes could specify the orientation and morphogenesis of the inner ear, whereas their later expression could specify the fates of particular cell types.     

Expression of estrogen-receptor related receptors in amphioxus and zebrafish: implications for the evolution of posterior brain segmentation at the invertebrate-to-vertebrate transition

Summary The evolutionary origin of vertebrate hindbrain segmentation is unclear since the amphioxus, the closest living invertebrate relative to the vertebrates, possesses a hindbrain homolog that displays no gross morphological segmentation. Three of the estrogen-receptor related (ERR) receptors are segmentally expressed in the zebrafish hindbrain, suggesting that their common ancestor was expressed in a similar, reiterated manner. We have also cloned and determined the developmental expression of the single homolog of the vertebrate ERR genes in the amphioxus (AmphiERR). This gene is also expressed in a segmented manner in a region considered homologous to the vertebrate hindbrain. In contrast to the expression of amphioxus islet (a LIM-homeobox gene that also labels motoneurons), AmphiERR expression persists longer in the hindbrain homolog and does not later extend to additional posterior cells. In addition, AmphiERR and one of its vertebrate homologs (ERRalpha) are expressed in the developing somitic musculature of amphioxus and zebrafish, respectively. Altogether, our results are consistent with fine structural evidence suggesting that the amphioxus hindbrain is segmented, and indicate that chordate ERR gene expression is a marker for both hindbrain and muscle segmentation. Furthermore, our data support an evolution model of chordate brain segmentation: originally, the program for anterior segmentation in the protochordate ancestors of the vertebrates resided in the developing axial mesoderm which imposed reiterated patterning on the adjacent neural tube; during early vertebrate evolution, this segmentation program was transferred to and controlled by the neural tube.     

NEFLb impairs early nervous system development via regulation of neuron apoptosis in zebrafish

Neurofilament light chain (NEFL), a subunit of neurofilament, has been shown to play an important role in pathogenic neurodegenerative disease and in radial axonal growth. However, information remains largely lacking regarding the function of NEFL in early development to date. In this study, we demonstrated the presence of two nefl genes, nefla and neflb, in zebrafish, generated by fish-specific third round genome duplication. These duplicated nefl genes were predominantly expressed in the nervous system with an overlapping and distinct expression pattern. Both gene knockdown and rescue experiments show that it was neflb rather than nefla that played an indispensable role in nervous system development. It was also found that neflb knockdown resulted in striking apoptosis of the neurons in the brain and spinal cord, leading to morphological defects such as brain structure disorder and trunk bending. Thus, we report a previously uncharacterized role of NEFL that NEFLb impairs the early development of zebrafish nervous system via regulation of the neuron apoptosis in the brain and spinal cord.     

Expression of protein kinase C genes during ontogenic development of the central nervous system.

We have analyzed the RNA expression of three protein kinase C (PKC) genes (alpha, beta, and gamma) in human and murine central nervous systems during embryonic-fetal, perinatal, and adult life. Analysis of human brain poly(A)+ RNA indicates that expression of PKC alpha and beta genes can be detected as early as 6 weeks postconception, undergoes a gradual increase until 9 weeks postconception, and reaches its highest level in the adult stage, and that the PKC gamma gene, although not expressed during embryonic and early fetal development, is abundantly expressed in the adult period. Similar developmental patterns were observed in human spinal cord and medulla oblongata. A detailed analysis of PKC gene expression during mammalian ontogeny was performed on poly(A)+ RNA from the brain cells of murine embryos at different stages of development and the brain cells of neonatal and adult mice. The ontogenetic patterns were similar to those observed for human brain. Furthermore, we observed that the expression of PKC gamma is induced in the peri- and postnatal phases. These results suggest that expression of PKC alpha, beta, and gamma genes possibly mediates the development of central neuronal functions, and expression of PKC gamma in particular may be involved in the development of peri- and postnatal functions.     

Expression of Zash-1a in the postembryonic zebrafish brain allows comparison to mouse Mash1 domains

Four areas in the late embryonic murine forebrain, i.e. the subpallium (striatum), the preoptic region, the ventral thalamus, and the hypothalamus, have been described to express the basic helix-loop-helix (bHLH) gene mammalian achaete-scute homolog Mash1 (Ascl1, Mouse Genome Informatics) in a complementary fashion to another bHLH gene, neurogenin1 (ngn1) (Neurod3, Mouse Genome Informatics), which is expressed in directly adjacent forebrain regions. We report here that the four regions previously identified as subpallium, preoptic region, ventral thalamus and hypothalamus (i.e. ventral inferior lobe) in the postembryonic zebrafish brain show Zash-1a expression at 3 days postfertilization (dpf), whereas none of those areas express the bHLH gene neuroD (nrd) between 2 and 5 dpf. This indicates that two well established alternative genetic pathways involved in neurogenesis in the amniote (mammalian) brain are present in homologous phenotypic locations in the anamniote (zebrafish) brain as well and that these pathways possibly act similarly in the generation of different neuronal phenotypes (e.g. subpallial GABAergic interneurons versus pallial glutamatergic projection neurons, or dopaminergic neurons versus other neurotransmitter phenotypes). Furthermore, previous initial identification of early postembryonic brain subdivisions in the zebrafish is strongly corroborated by these expression patterns.     

Embryonic expression of three mouse genes with homology to the Drosophila melanogaster prickle gene

The Drosophila melanogaster gene prickle-spiny-legs (pk) functions in an intercellular feedback loop that is central to the establishment of planar cell polarity in the eye and epidermis of the fly, by modulating Frizzled-Disheveled signalling. Here we identify three mouse prickle-related genes (dyxin, testin and prickle) and describe their expression pattern during murine embryogenesis (E7.5-E15.5). We report that the three genes are expressed in restricted areas of the developing mouse brain: dyxin in the most ventral region of the neural tube and in some localized regions of the ventricular layer of the mesencephalon and rhombencephalon, prickle in the pons region, ventrolateral part of rhombencephalon and motoneurons in the spinal cord, and testin in differentiating neurons of the spinal cord and retina. At the stages analyzed, the main site of expression of testin is the migrating cranial neural crest, while the expression of dyxin is noticeable in myotomal cells and its derivatives, with prickle expression being reciprocally localized to some sclerotomal derivatives, like bone primordia. prickle is also expressed in the apical ectodermal ridge and the most distal mesenchyme of the forming limb buds.     

Embryonic expression of three mouse genes with homology to the Drosophila melanogaster prickle gene

The Drosophila melanogaster gene prickle-spiny-legs (pk) functions in an intercellular feedback loop that is central to the establishment of planar cell polarity in the eye and epidermis of the fly, by modulating Frizzled-Disheveled signalling. Here we identify three mouse prickle-related genes (dyxin, testin and prickle) and describe their expression pattern during murine embryogenesis (E7.5-E15.5). We report that the three genes are expressed in restricted areas of the developing mouse brain: dyxin in the most ventral region of the neural tube and in some localized regions of the ventricular layer of the mesencephalon and rhombencephalon, prickle in the pons region, ventrolateral part of rhombencephalon and motoneurons in the spinal cord, and testin in differentiating neurons of the spinal cord and retina. At the stages analyzed, the main site of expression of testin is the migrating cranial neural crest, while the expression of dyxin is noticeable in myotomal cells and its derivatives, with prickle expression being reciprocally localized to some sclerotomal derivatives, like bone primordia. prickle is also expressed in the apical ectodermal ridge and the most distal mesenchyme of the forming limb buds.     

Coordinate embryonic expression of three zebrafish engrailed genes.

We have identified three genes, expressed in zebrafish embryos, that are members of the engrailed gene family. On the basis of sequence comparisons and analyses of their expression patterns, we suggest that two of these genes, eng2 and eng3, are closely related to the En-2 gene of other vertebrates. The third gene, eng1, is probably the zebrafish homolog of En-1. Subsets of cells at the developing junction between the midbrain and hindbrain express three different combinations of these genes, revealing a previously unknown complexity of this region of the CNS. Other cells, for example, jaw and myotomal muscle precursors, express two of the three genes in combinations which, in the myotomal muscles, change during development. Cells in the developing hindbrain and fins express only a single engrailed gene. We propose that the fates and patterning of these cells may be regulated by the coordinate expression of particular combinations of these closely related homeoproteins.