Retinoic acid and hindbrain patterning

Retinoid signaling plays an important role in the developmental patterning of the hindbrain. Studies of the teratogenic effects of retinoids showed early on that the hindbrain suffered patterning defects in cases of retinoid excess or deficiency. Closer examination of these effects in animal models suggested that retinoids might play a physiological role in specifying the antero-posterior axis of the hindbrain. This idea was supported by the localization of retinoid synthetic and degradative enzymes, binding proteins, and receptors to the hindbrain and neighboring regions of the neuroepithelium and the mesoderm. In parallel, it became clear that the molecular patterning of the hindbrain, in terms of the regionalized expression of Hox genes and other developmental regulatory genes, is profoundly influenced by retinoid signaling.     

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。     

Retinoic acid synthesis and hindbrain patterning in the mouse embryo

Targeted disruption of the murine retinaldehyde dehydrogenase 2 (Raldh2) gene precludes embryonic retinoic acid (RA) synthesis, leading to midgestational lethality (Niederreither, K., Subbarayan, V., Dolle, P. and Chambon, P. (1999). Nature Genet. 21, 444-448). We describe here the effects of this RA deficiency on the development of the hindbrain and associated neural crest. Morphological segmentation is impaired throughout the hindbrain of Raldh2-/- embryos, but its caudal portion becomes preferentially reduced in size during development. Specification of the midbrain region and of the rostralmost rhombomeres is apparently normal in the absence of RA synthesis. In contrast, marked alterations are seen throughout the caudal hindbrain of mutant embryos. Instead of being expressed in two alternate rhombomeres (r3 and r5), Krox20 is expressed in a single broad domain, correlating with an abnormal expansion of the r2-r3 marker Meis2. Instead of forming a defined r4, Hoxb1- and Wnt8A-expressing cells are scattered throughout the caudal hindbrain, whereas r5/r8 markers such as kreisler or group 3/4 Hox genes are undetectable or markedly downregulated. Lack of alternate Eph receptor gene expression could explain the failure to establish rhombomere boundaries. Increased apoptosis and altered migratory pathways of the posterior rhombencephalic neural crest cells are associated with impaired branchial arch morphogenesis in mutant embryos. We conclude that RA produced by the embryo is required to generate posterior cell fates in the developing mouse hindbrain, its absence leading to an abnormal r3 (and, to a lesser extent, r4) identity of the caudal hindbrain cells.     

13-cis-retinoic acid causes patterning defects in the early embryonic rostral hindbrain and abnormal development of the cerebellum in the macaque

BACKGROUND: We have previously reported that exposure of embryos to 13-cis-retinoic acid (cRA) results in an abnormal phenotype of the fetal cerebellum. In this study, we analyzed early changes in the cerebellar anlagen (midbrain-hindbrain junction) as well as lesions of the fetal cerebellar vermis after a teratogenic dosing regimen of cRA in the macaque model. METHODS: We examined embryo coronal sections of the midbrain-hindbrain junction immunostained for Pax-2, Engrailed-2 (En-2) and Krox-20. To characterize the cerebellum foliation and fissure formation processes, we analyzed vermal cortical cell layer development and the number and depth of the major fissures on sagittal sections of fetal vermis. We also examined Purkinje cell development in vermal sections immunostained for CD3. RESULTS: Compared with controls, there was a consistent truncation of the midbrain-hindbrain region of early embryos exposed to cRA. The cRA-induced fetal vermis lesions included inhibition in its anteroposterior growth, altered folial patterning, a general loss of prominence of the fissures accompanied by a total loss of sublobular fissures, and changes in cortical cell layer development. CD3(+) Purkinje cells were abnormally dispersed deep into the molecular layer in the vermis. CONCLUSIONS: Our findings indicate that the effects of cRA on the developing cerebellum involve interference with the hierarchy of complex cellular and genetic interactions that lead to the growth and subdivision of the cerebellum into smaller units. The regional vermal defects may be related to early postnatal functional deficits.     

Retinoic acid affects left-right patterning.

Our understanding of the means by which the left-right axis is patterned is not fully understood, although a number of key intermediaries have been recently described. We report here that retinoic acid (RA) excess affects heart situs concomitant with alterations in the expression of genes implicated in the establishment of the left-right axis. Specifically, RA exposure during a specific developmental window evoked bilateral expression of lefty-1, lefty-2, nodal, and pitx-2 in the lateral plate mesoderm. Time course experiments, together with analysis of midline markers, suggest that nascent mesoderm constitutes a predominant RA target involved in this process. These events are likely to underlie the perturbations of heart looping provoked by excess RA and suggest a means by which retinoids influence the early steps in establishment of the left-right embryonic axis.     

Overexpression of Fyn tyrosine kinase causes abnormal development of primary sensory neurons in Xenopus laevis embryos

The expression and function of the Src family protein tyrosine kinase Fyn in Xenopus laevis embryos have been examined. In situ hybridization analysis demonstrated nervous system-specific expression of Fyn mRNA in tail-bud embryos. However, a class of primary sensory neurons; that is, Rohon-Beard (RB) neurons, which is positive for immunoglobulin superfamily cell adhesion molecules (CAM), neural cell adhesion molecule (N-CAM) and contactin, is devoid of Fyn expression. Injection of Fyn mRNA into one of the blastomeres at the 2-cell stage led to overexpression of Fyn in the injected half of the tail-bud embryos. Immunolabeling of the embryos with anti-HNK-1 antibody revealed that the peripheral axons of RB neurons were partially misguided and bound to each other to form abnormal subcutaneous fascicles. Similar abnormality was induced by injection of the Fyn overexpression vector. The incidence of abnormality appeared dose-dependent, being 68-92% of the injected embryos at 50-400 pg of mRNA. Co-injection of the contactin antisense vector depleted contactin mRNA accumulation without affecting Fyn overexpression and reduced the incidence of the abnormal RB-cell phenotype. However, the N-CAM antisense was ineffective in reducing this abnormality. These results suggest that Fyn can modify signals regulating axonal guidance or fasciculation in the developing X. laevis nervous system and that contactin may affect this action of Fyn.     

Molecular mechanisms of segmental patterning in the vertebrate hindbrain and neural crest

Recent work has shown that segmentation underlies the patterning of the vertebrate hindbrain and its neural crest derivatives. Several genes have been identified with segment-restricted expression, and evidence is now emerging regarding their function and regulatory relationships. The expression patterns of Hox genes and the phenotype of null mutants indicate roles in specifying segment identity. A zinc finger gene Krox-20 is a segment-specific regulator of Hox expression, and it seems probable that retinoic acid receptors also regulate Hox genes in the hindbrain. The receptor tyrosine kinase gene Sek may mediate cell-cell interactions that lead to segmentation. These studies provide a starting point for understanding the molecular basis of segmental patterning in the hindbrain.     

Regulation of segmental patterning by retinoic acid signaling during Xenopus somitogenesis

Somites, the segmented building blocks of the vertebrate embryo, arise one by one in a patterning process that passes wavelike along the anteroposterior axis of the presomitic mesoderm (PSM). We have studied this process in Xenopus embryos by analyzing the expression of the bHLH gene, Thylacine1, which is turned on in the PSM as cells mature and segment, in a pattern that marks both segment boundaries and polarity. Here, we show that this segmental gene expression involves a PSM enhancer that is regulated by retinoic acid (RA) signaling at two levels. RA activates Thylacine1 expression in rostral PSM directly. RA also activates Thylacine1 expression in the caudal PSM indirectly by inducing the expression of MKP3, an inhibitor of the FGF signaling pathway. RA signaling is therefore a major contributor to segmental patterning by promoting anterior segmental polarity and by interacting with the FGF signaling pathway to position segmental boundaries.     

Retinoic acid metabolizing factor xCyp26c is specifically expressed in neuroectoderm and regulates anterior neural patterning in Xenopus laevis

Anterior-posterior neural patterning is determined during gastrulation when head structure is induced. Induction of anterior neural structures requires inhibition of Wnt signaling by several Wnt antagonists. We performed microarray analysis to isolate genes regulated by canonical Wnt signaling and abundantly expressed in the anterior neuroectoderm at the early neurula stage. We identified xCyp26c, a Cyp26 (RA-metabolizing protein)-family gene. In situ hybridization showed xCyp26c expression restricted to the anterior region of neurula, while xCyp26a was expressed in both anterior and posterior regions. At the tadpole stage, xCyp26c was also expressed in restricted sets of cranial nerves. Microarray, RT-PCR and in situ hybridization analyses revealed decreased xCyp26c expression with overexpression of beta-catenin, suggesting regulation by Wnt/beta-catenin signaling. We also assessed the effects of retinoic acid (RA) on xCyp26c expression. Embryos treated with 10(-7) M RA showed an anterior shift in the spatial expression of xCyp26c, reflecting a posteriorization effect. Conversely, expression patterns in embryos treated with more than 10(-6) M RA were less affected and remained restricted to the most anterior region. Moreover, injection of xCyp26c mRNA into animal poles caused head defects, and exogenous expression of xCyp26c rescued the posteriorizing effect of RA treatment. Taken together, these results implicated a role for xCyp26c in anterior patterning via RA signaling.     

Regulation and function of FGF8 in patterning of midbrain and anterior hindbrain.

In this article, an adjunct to a platform presentation at the Winternational 2000 Symposium, we summarize the recent findings of this group concerning the regulation and functions of FGF8 expressed at the isthmus of the developing brain. We show that several different FGF8 isoforms, ectopically expressed in midbrain or posterior forebrain, are able to mimic the proliferative and patterning functions previously attributed to the isthmus in tissue grafting studies. Moreover, we also show that FGF8 protein is sufficient to induce an ectopic isthmic organiser (Fgf-8+, Gbx2+) in anterior midbrain. We also provide evidence that isthmic FGF8 patterns anterior hindbrain, repressing Hox-a2 expression and setting aside a territory of the brain that includes the cerebellar anlage. We show that these effects of FGF8 are likely to be mediated via FGFR1 and be modulated by the putative FGF antagonist, Sprouty2, identified using a differential display screen. Finally, we provide evidence that the onset of Fgf8 expression is regulated by En1 and that its expression at the isthmus is subsequently maintained by a specific and direct interaction between rhombomere 1 and midbrain.     

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.     

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 in the anteroposterior patterning of the zebrafish trunk

Retinoic acid has been linked to pattern formation in the vertebrate anteroposterior axis. This report describes the spatial and temporal distributions of both endogenous retinoic acid and retinoic acid synthase activity along the anteroposterior axis of neurulating zebrafish embryos, as detected by a transient transgenic assay and by a zymography bioassay. Both retinoic acid levels and synthase activity were found to be highest in anterior regions of the trunk at all of the stages which were analysed. The drug disulfiram inhibited retinoic acid synthase activity in the zebrafish trunk both in vitro and in vivo, and reduced retinoic acid levels in vivo. Disulfiram treatment of neurulating embryos resulted in larvae with hypertrophic wavy notochords, shortened spinal cords and deformed pectoral fins. The results support the hypothesis that retinoic acid plays a role in the coordination of axial patterning at the developing node/zone of involution, as well as in the subsequent development of anterior trunk structures such as the fins.     

维甲酸对斑马鱼中枢神经系统及软骨发生的影响

脂溶性的维甲酸可通过固醇类激素作用途径启动ＨＯＸ族及ＬＩＭ族调节基因,进而影响脊椎动物胚胎的形态发生、再生组织的分化。本文通过不同浓度系列表明,维甲酸对早期鱼胚发育的影响程度依赖于其作用浓度与作用时间,１０＾－６￣１０＾－７Ｍ的浓度范围为作用敏感区。维甲酸对中枢神经系统前后轴上的影响尤其显著,主要表现为小头畸形、无眼或无心脏。作者首次用Ｂｒｄｕ－Ａｎｔｉ－Ｂｎｄｕ标记处于Ｓ期的细胞核表明,ＲＡ作用     

Retinoic acid modifies development of the midbrain-hindbrain border and affects cranial ganglion formation in zebrafish embryos.

Considerable evidence now suggests that retinoic acid (RA) is an important modulator of patterning events in early neuronal development in vertebrates. In this paper, we describe the effects of exogenously applied RA on early neural development in the zebrafish embryo. Neural anatomy is assessed by immunocytochemical and histochemical analysis of the developing embryo in whole mounts at 24 h post-fertilization. RA was applied for one hour at concentrations ranging from 10(-9) to 10(-6) M to embryos at 50% epiboly, the midgastrula stage, and at 10(-7) M to embryos at early and late gastrula stages. The neuroanatomical analysis shows that 10(-7) M RA causes a defined lesion to the developing central nervous system which corresponds to a loss of a region of the brain in the caudal midbrain-rostral hindbrain area, the precursor of the cerebellum and associated neural structures. The region that fails to develop corresponds to the cranial expression domain of the engrailed protein as assessed by the monoclonal antibody 4D9 (Patel et al. 1989: Expression of engrailed proteins in arthropods, annelids and chordates. Cell 58, 955-968). Structures caudal to rhombomere 4 are unaffected by 10(-7) M RA, as are the cranial midbrain and forebrain: 10(-7) M RA also affects the development of cranial ganglia, principally the Vth, anterior lateral line and VIIIth ganglia, suggesting that RA affects normal development of the cranial neural crest. Effects of RA at stages immediately prior to and after gastrulation show some similar and some distinct features. Results are discussed in terms of the possible role of RA as an endogenous moderator of normal head development.     

Retinoic acid-mediated patterning of the pre-pancreatic endoderm in Xenopus operates via direct and indirect mechanisms

Early patterning of the endoderm as a prerequisite for pancreas specification involves retinoic acid (RA) as a critical signalling molecule in gastrula stage Xenopus embryos. In extension of our previous studies, we made systematic use of early embryonic endodermal and mesodermal explants. We find RA to be sufficient to induce pancreas-specific gene expression in dorsal but not ventral endoderm. The differential expression of retinoic acid receptors (RARs) in gastrula stage endoderm is important for the distinct responsiveness of dorsal versus ventral explants. Furthermore, BMP signalling, that is repressed dorsally, prevents the formation of pancreatic precursor cells in the ventral endoderm of gastrula stage Xenopus embryos. An additional requirement for mesoderm suggests the production of one or more further pancreas inducing signals by this tissue. Finally, recombination of manipulated early embryonic explants, and also inhibition of RA activity in whole embryos, reveal that RA signalling, as it is relevant for pancreas development, operates simultaneously on both mesodermal and endodermal germ layers.