Retinoic acid and development of the central nervous system.

We consider the evidence that RA, the vitamin A metabolite, is involved in three fundamental aspects of the development of the CNS: 1) the stimulation of axon outgrowth in particular neuronal sub-types; 2) the migration of the neural crest; and 3) the specification of rostrocaudal position in the developing CNS (forebrain, midbrain, hindbrain, spinal cord). The evidence we discuss involves RA-induction of neurites in cell cultures and explants of neural tissue; the teratological effects of RA on the embryo's nervous system; the observation that RA can be detected endogenously in the spinal cord; and the fact that the receptors and binding proteins for RA are expressed in precise domains and neuronal cell types within the nervous system.     

Segmental pattern of development of the hindbrain and spinal cord of the zebrafish embryo

In the ventral hindbrain and spinal cord of zebrafish embryos, the first neurones that can be identified appear as single cells or small clusters of cells, distributed periodically at intervals equal to the length of a somite. In the hindbrain, a series of neuromeres of corresponding length is present, and the earliest neurones are located in the centres of each neuromere. Young neurones within both the hindbrain and spinal cord were identified in live embryos using Nomarski optics, and histochemically by labelling for acetylcholinesterase activity and expression of an antigen recognized by the monoclonal antibody zn-1. Among them are individually identified hindbrain reticulospinal neurones and spinal motoneurones. These observations suggest that early development in these regions of the CNS reflects a common segmental pattern. Subsequently, as more neurones differentiate, the initially similar patterning of the cells in these two regions diverges. A continuous longitudinal column of developing neurones appears in the spinal cord, whereas an alternating series of large and small clusters of neurones is present in the hindbrain.     

CNS neurotrophins are biologically active and expressed by multiple cell types

Neutrotrophins are increasingly appreciated as potential modulators of neuronal function in the adult central nervous system (CNS). To describe the neurotrophin environment within the adult CNS, mRNA and protein expression patterns of neurotrophins-3 and –4 and of brain-derived neurotrophin were investigated in adult rat spinal cord and brain. Co-localization studies with CNS cell type-specific markers demonstrates that multiple cell types, including both neurons and glia, express these neurotrophins in the normal adult CNS. Although widely implicated in important CNS functions such as synaptic plasticity, biological activity of endogenous CNS neurotrophins has not been directly demonstrated. With a sensitive neurite outgrowth bioassay we demonstrate that CNS neurotrophins elicit neurite outgrowth and are biologically active. Moreover, antibody-blocking studies suggest that these three neurotrophins may comprise the bulk of adult CNS neurotrophic activity.     

Retinoic acid signalling in the zebrafish embryo is necessary during pre-segmentation stages to pattern the anterior-posterior axis of the CNS and to induce a pectoral fin bud

A number of studies have suggested that retinoic acid (RA) is an important signal for patterning the hindbrain, the branchial arches and the limb bud. Retinoic acid is thought to act on the posterior hindbrain and the limb buds at somitogenesis stages in chick and mouse embryos. Here we report a much earlier requirement for RA signalling during pre-segmentation stages for proper development of these structures in zebrafish. We present evidence that a RA signal is necessary during pre-segmentation stages for proper expression of the spinal cord markers hoxb5a and hoxb6b, suggesting an influence of RA on anteroposterior patterning of the neural plate posterior to the hindbrain. We report the identification and expression pattern of the zebrafish retinaldehyde dehydrogenase2 (raldh2/aldh1a2) gene. Raldh2 synthesises retinoic acid (RA) from its immediate precursor retinal. It is expressed in a highly ordered spatial and temporal fashion during gastrulation in the involuting mesoderm and during later embryogenesis in paraxial mesoderm, branchial arches, eyes and fin buds, suggesting the involvement of RA at different times of development in different functional contexts. Mapping of the raldh2 gene reveals close linkage to no-fin (nof), a newly discovered mutant lacking pectoral fins and cartilaginous gill arches. Cloning and functional tests of the wild-type and nof alleles of raldh2 reveal that nof is a raldh2 mutant. By treating nof mutants with RA during different time windows and by making use of a retinoic acid receptor antagonist, we show that RA signalling during pre-segmentation stages is necessary for anteroposterior patterning in the CNS and for fin induction to occur.     

Explant cultures of teleost spinal cord: source of neurite outgrowth

The source of neurite outgrowth in explant cultures of normal adult Apteronotus spinal cord was examined. Explants which contained the central region of spinal cord, including ependyma, showed neurite outgrowth in culture. Explants which did not contain ependyma showed no neurite outgrowth. It is concluded that the ependymal region is necessary for neurite outgrowth in these cultures of adult teleost spinal cord. In addition, our failure to observe axon outgrowth clearly attributable to fluorescently back-labeled electromotor neurons in these cultures suggests that the exuberant neurite outgrowth in vitro is most probably due to cells other than the electromotor neurons. This explant culture system provides a unique opportunity to study neuronal differentiation, regeneration, and neurogenesis in vitro.     

Gli2 and Gli3 play distinct roles in the dorsoventral patterning of the mouse hindbrain

Sonic Hedgehog (Shh) signaling plays a critical role during dorsoventral (DV) patterning of the developing neural tube by modulating the expression of neural patterning genes. Overlapping activator functions of Gli2 and Gli3 have been shown to be required for motoneuron development and correct neural patterning in the ventral spinal cord. However, the role of Gli2 and Gli3 in ventral hindbrain development is unclear. In this paper, we have examined DV patterning of the hindbrain of Shh(-/-), Gli2(-/-) and Gli3(-/-) embryos, and found that the respective role of Gli2 and Gli3 is not only different between the hindbrain and spinal cord, but also at distinct rostrocaudal levels of the hindbrain. Remarkably, the anterior hindbrain of Gli2(-/-) embryos displays ventral patterning defects as severe as those observed in Shh(-/-) embryos suggesting that, unlike in the spinal cord and posterior hindbrain, Gli3 cannot compensate for the loss of Gli2 activator function in Shh-dependent ventral patterning of the anterior hindbrain. Loss of Gli3 also results in a distinct patterning defect in the anterior hindbrain, including dorsal expansion of Nkx6.1 expression. Furthermore, we demonstrate that ventral patterning of rhombomere 4 is less affected by loss of Gli2 function revealing a different requirement for Gli proteins in this rhombomere. Taken together, these observations indicate that Gli2 and Gli3 perform rhombomere-specific function during DV patterning of the hindbrain.     

Effects of GnRH on Neurite Outgrowth,Neurofilament and Spinophilin Proteins Expression in Cultured Spinal Cord Neurons of Rat Embryos

It has been previously described the presence of GnRH receptor in spinal cord neurons of rat embryos and adult rats. However, the functional role of these receptors has not been studied. In this work, the effect of GnRH on neurite outgrowth and cytoskeletal protein expression in cultured spinal cord neurons of rat embryos was analyzed. Specifically, neurofilaments of 68 and 200 kDa by immunoblot assays and spinophilin mRNA expression by RT-PCR. Results show that GnRH stimulates neurite outgrowth in addition to an increase in neurofilaments and spinophilin expression. These findings suggest that GnRH may play a role as neuromodulator in neuronal plasticity and that could be considered as a potential factor for neuronal regeneration in spinal cord injuries.     

A retinoic acid-Hox hierarchy controls both anterior/posterior patterning and neuronal specification in the developing central nervous system of the cephalochordate amphioxus

Retinoic acid (RA) mediates both anterior/posterior patterning and neuronal specification in the vertebrate central nervous system (CNS). However, the molecular mechanisms downstream of RA are not well understood. To investigate these mechanisms, we used the invertebrate chordate amphioxus, in which the CNS, although containing only about 20,000 neurons in adults, like the vertebrate CNS, has a forebrain, midbrain, hindbrain, and spinal cord and is regionalized by RA-signaling. Here we show, first, that domains of genes with expression normally limited to diencephalon and midbrain are generally not affected by altered RA-signaling, second, that contrary to previous reports, not only Hox1, 3, and 4, but also Hox2 and Hox6 are collinearly expressed in the amphioxus CNS, and third, that collinear expression of all these Hox genes is controlled by RA-signaling. Finally, we show that Hox1 is involved in mediating both the role of RA-signaling in regionalization of the hindbrain and in specification of hindbrain motor neurons. Thus, morpholino knock-down of the single amphioxus Hox1 mimics the effects of treatments with an RA-antagonist. This analysis establishes RA-dependent regulation of collinear Hox expression as a feature common to the chordate CNS and indicates that the RA-Hox hierarchy functions both in proper anterior/posterior patterning of the developing CNS and in specification of neuronal identity.     

Retinoic acid modifies the pattern of cell differentiation in the central nervous system of neurula stage Xenopus embryos.

Neural cell markers have been used to examine the effect of retinoic acid (RA) on the development of the central nervous system (CNS) of Xenopus embryos. RA treatment of neurula stage embryos resulted in a concentration-dependent perturbation of anterior CNS development leading to a reduction in the size of the forebrain, midbrain and hindbrain. In addition the overt segmental organization of the hindbrain was abolished by high concentrations of RA. The regional expression of two cell-specific markers, the homeobox protein Xhox3 and the neurotransmitter serotonin was also examined in embryos exposed to RA. Treatment with RA caused a concentration-dependent change in the pattern of expression of Xhox3 and serotonin and resulted in the ectopic appearance of immunoreactive neurons in anterior regions of the CNS, including the forebrain. Collectively, our results extend previous studies by showing that RA treatment of embryos at the neurula stage inhibits the development of anterior regions of the CNS while promoting the differentiation of more posterior cell types. The relevance of these findings to the possible role of endogenous retinoids in the determination of neural cell fate and axial patterning is discussed.     

Retinoic acid-metabolizing enzyme Cyp26a1 is essential for determining territories of hindbrain and spinal cord in zebrafish

Retinoic acid (RA) plays a critical role in neural patterning and organogenesis in the vertebrate embryo. Here we characterize a mutant of the zebrafish named giraffe (gir) in which the gene for the RA-degrading enzyme Cyp26a1 is mutated. The gir mutant displayed patterning defects in multiple organs including the common cardinal vein, pectoral fin, tail, hindbrain, and spinal cord. Analyses of molecular markers suggested that the lateral plate mesoderm is posteriorized in the gir mutant, which is likely to cause the defects of the common cardinal vein and pectoral fin. The cyp26a1 expression in the rostral spinal cord was strongly upregulated in the gir mutant, suggesting a strong feedback control of its expression by RA signaling. We also found that the rostral spinal cord territory was expanded at the expense of the hindbrain territory in the gir mutant. Such a phenotype is the opposite of that of the mutant for Raldh2, an enzyme that synthesizes RA. We propose a model in which Cyp26a1 attenuates RA signaling in the prospective rostral spinal cord to limit the expression of hox genes and to determine the hindbrain-spinal cord boundary.     

Retinoic acid induces changes in the localization of homeobox proteins in the antero-posterior axis of Xenopus laevis embryos.

We have studied the localization of the proteins of Xeb1 and Xeb2, two homeobox (hbx)-containing genes that are expressed during the early development of Xenopus laevis. Both proteins are expressed in juxtaposed and partially overlapping domains along the antero-posterior axis of Xenopus laevis embryos, with clearly defined anterior boundaries. Xeb2 is predominantly expressed in the caudal region of the hindbrain, whereas the Xeb1 protein is located in the most rostral region of the spinal cord. Furthermore, both proteins are expressed in single cells dispersed in the lateral flanks of the embryo in positions that correlate with the expression domains in the neural tube. We suggest that these cells are migratory neural crest cells that have acquired positional information in the neural tube prior to migration. The Xeb2 protein was also detected in the most posterior branchial arches and the pronephros. In stage 45 embryos, nuclei of the IX-X cranial ganglia, the lung buds and cells spreading into the forelimb rudiment express the Xeb2 antigen. The Xeb1 protein was also detected in the lung buds and the forelimb rudiment. To examine the effect of retinoic acid on expression, gastrula embryos were treated with all-trans retinoic acid (RA). Increasing concentrations of RA caused progressive truncation of anterior structures. The most severely affected embryos lacked eyes, nasal pits, forebrain, midbrain and otic vesicles, and the anterior boundary of the hindbrain seemed to be displaced rostrally. This alteration correlates with a progressive displacement of the anterior boundary of the expression domain of Xeb2. On the other hand, 10(-6) M RA induces an ectopic site of Xeb1 expression at the anterior end of the central nervous system, located just anterior to the extended domain of Xeb2 whereas expression in the spinal cord remains unaffected.     

Novel retinoic acid generating activities in the neural tube and heart identified by conditional rescue of Raldh2 null mutant mice

Retinoid control of vertebrate development depends upon tissue-specific metabolism of retinol to retinoic acid (RA). The RA biosynthetic enzyme RALDH2 catalyzes much, but not all, RA production in mouse embryos, as revealed here with Raldh2 null mutants carrying an RA-responsive transgene. Targeted disruption of Raldh2 arrests development at midgestation and eliminates all RA synthesis except that associated with Raldh3 expression in the surface ectoderm of the eye field. Conditional rescue of Raldh2(-/-) embryos by limited maternal RA administration allows development to proceed and results in the establishment of additional sites of RA synthesis linked to Raldh1 expression in the dorsal retina and to Raldh3 expression in the ventral retina, olfactory pit and urinary tract. Unexpectedly, conditionally rescued Raldh2(-/-) embryos also possess novel sites of RA synthesis in the neural tube and heart that do not correspond to expression of Raldh1-3. RA synthesis in the mutant neural tube was localized in the spinal cord, posterior hindbrain and portions of the midbrain and forebrain, whereas activity in the mutant heart was localized in the conotruncus and sinus venosa. In the posterior hindbrain, this novel RA-generating activity was expressed during establishment of rhombomeric boundaries. In the spinal cord, the novel activity was localized in the floorplate plus in the intermediate region where retinoid-dependent interneurons develop. These novel RA-generating activities in the neural tube and heart fill gaps in our knowledge of how RA is generated spatiotemporally and may, along with Raldh1 and Raldh3, contribute to rescue of Raldh2(-/-) embryos by producing RA locally.     

RGMa inhibition promotes axonal growth and recovery after spinal cord injury

Repulsive guidance molecule (RGM) is a protein implicated in both axonal guidance and neural tube closure. We report RGMa as a potent inhibitor of axon regeneration in the adult central nervous system (CNS). RGMa inhibits mammalian CNS neurite outgrowth by a mechanism dependent on the activation of the RhoA-Rho kinase pathway. RGMa expression is observed in oligodendrocytes, myelinated fibers, and neurons of the adult rat spinal cord and is induced around the injury site after spinal cord injury. We developed an antibody to RGMa that efficiently blocks the effect of RGMa in vitro. Intrathecal administration of the antibody to rats with thoracic spinal cord hemisection results in significant axonal growth of the corticospinal tract and improves functional recovery. Thus, RGMa plays an important role in limiting axonal regeneration after CNS injury and the RGMa antibody offers a possible therapeutic agent in clinical conditions characterized by a failure of CNS regeneration.     

Diffusible signals and fasciculated growth in reticulospinal axon pathfinding in the hindbrain

We have addressed the control of longitudinal axon pathfinding in the developing hindbrain, including the caudal projections of reticular and raphe neurons. To test potential sources of guidance signals, we assessed axon outgrowth from embryonic rat hindbrain explants cultured in collagen gels at a distance from explants of midbrain-hindbrain boundary (isthmus), caudal hindbrain, or cervical spinal cord. Our results showed that the isthmus inhibited caudally directed axon outgrowth by 80% relative to controls, whereas rostrally directed axon outgrowth was unaffected. Moreover, caudal hindbrain or cervical spinal cord explants did not inhibit caudal axons. Immunohistochemistry for reticular and raphe neuronal markers indicated that the caudal, but not the rostral projections of these neuronal subpopulations were inhibited by isthmic explants. Companion studies in chick embryos showed that, when the hindbrain was surgically separated from the isthmus, caudal reticulospinal axon projections failed to form and that descending pioneer axons of the medial longitudinal fasciculus (MLF) play an important role in the caudal reticulospinal projection. Taken together, these results suggest that diffusible chemorepellent or nonpermissive signals from the isthmus and substrate-anchored signals on the pioneer MLF axons are involved in the caudal direction of reticulospinal projections and might influence other longitudinal axon projections in the brainstem.     

The retinoic acid signaling pathway regulates anterior/posterior patterning in the nerve cord and pharynx of amphioxus,a chordate lacking neural crest

Amphioxus, the closest living invertebrate relative of the vertebrates, has a notochord, segmental axial musculature, pharyngeal gill slits and dorsal hollow nerve cord, but lacks neural crest. In amphioxus, as in vertebrates, exogenous retinoic acid (RA) posteriorizes the embryo. The mouth and gill slits never form, AmphiPax1, which is normally downregulated where gill slits form, remains upregulated and AmphiHox1 expression shifts anteriorly in the nerve cord. To dissect the role of RA signaling in patterning chordate embryos, we have cloned the single retinoic acid receptor (AmphiRAR), retinoid X receptor (AmphiRXR) and an orphan receptor (AmphiTR2/4) from amphioxus. AmphiTR2/4 inhibits AmphiRAR-AmphiRXR-mediated transactivation in the presence of RA by competing for DR5 or IR7 retinoic acid response elements (RAREs). The 5' untranslated region of AmphiTR2/4 contains an IR7 element, suggesting possible auto- and RA-regulation. The patterns of AmphiTR2/4 and AmphiRAR expression during embryogenesis are largely complementary: AmphiTR2/4 is strongly expressed in the cerebral vesicle (homologous to the diencephalon plus anterior midbrain), while AmphiRAR expression is high in the equivalent of the hindbrain and spinal cord. Similarly, while AmphiTR2/4 is expressed most strongly in the anterior and posterior thirds of the endoderm, the highest AmphiRAR expression is in the middle third. Expression of AmphiRAR is upregulated by exogenous RA and completely downregulated by the RA antagonist BMS009. Moreover, BMS009 expands the pharynx posteriorly; the first three gill slit primordia are elongated and shifted posteriorly, but do not penetrate, and additional, non-penetrating gill slit primordia are induced. Thus, in an organism without neural crest, initiation and penetration of gill slits appear to be separate events mediated by distinct levels of RA signaling in the pharyngeal endoderm. Although these compounds have little effect on levels of AmphiTR2/4 expression, RA shifts pharyngeal expression of AmphiTR2/4 anteriorly, while BMS009 extends it posteriorly. Collectively, our results suggest a model for anteroposterior patterning of the amphioxus nerve cord and pharynx, which is probably applicable to vertebrates as well, in which a low anterior level of AmphiRAR (caused, at least in part, by competitive inhibition by AmphiTR2/4) is necessary for patterning the forebrain and formation of gill slits, the posterior extent of both being set by a sharp increase in the level of AmphiRAR. Supplemental data available on-line     

维生素A缺乏致斑马鱼胚胎体节不对称及后脑图式形成异常

视黄酸(RA)在脊椎动物胚胎发生过程中发挥着关键作用。但是脊椎动物不能从头合成RA, 而必须以维生素A为前体通过视黄醇脱氢酶和视黄醛脱氢酶(Aldh1A)先将其氧化为视黄醛再氧化成RA。已知维生素A缺乏(VAD)会导致多种动物出现维生素A缺乏综合征, 但有关VAD对斑马鱼胚胎发育的影响尚未见报道。文章通过用不含维生素A及其他视黄类前体的饲料饲喂斑马鱼获得斑马鱼VAD胚胎。分析表明, 缺乏维生素A可导致斑马鱼胚胎体节出现不对称发育、胚胎的后脑图式形成异常。这些表型虽与aldh1a2基因敲落的及经醛脱氢酶抑制剂处理的斑马鱼胚胎表型类似, 但远不及后二者的严重, 提示VAD胚胎可能只是缺少而不是完全没有维生素A, 且可能存在不依赖视黄醛脱氢酶的RA合成途径。     

Cyp26 enzymes generate the retinoic acid response pattern necessary for hindbrain development

Retinoic acid (RA) is essential for normal vertebrate development, including the patterning of the central nervous system. During early embryogenesis, RA is produced in the trunk mesoderm through the metabolism of vitamin A derived from the maternal diet and behaves as a morphogen in the developing hindbrain where it specifies nested domains of Hox gene expression. The loss of endogenous sources of RA can be rescued by treatment with a uniform concentration of exogenous RA, indicating that domains of RA responsiveness can be shaped by mechanisms other than the simple diffusion of RA from a localized posterior source. Here, we show that the cytochrome p450 enzymes of the Cyp26 class, which metabolize RA into polar derivatives, function redundantly to shape RA-dependent gene-expression domains during hindbrain development. In zebrafish embryos depleted of the orthologs of the three mammalian CYP26 genes CYP26A1, CYP26B1 and CYP26C1, the entire hindbrain expresses RA-responsive genes that are normally restricted to nested domains in the posterior hindbrain. Furthermore, we show that Cyp26 enzymes are essential for exogenous RA to rescue hindbrain patterning in RA-depleted embryos. We present a ;gradient-free' model for hindbrain patterning in which differential RA responsiveness along the hindbrain anterior-posterior axis is shaped primarily by the dynamic expression of RA-degrading enzymes.