Emily Gale

Opposing FGF and retinoid pathways control ventral neural pattern, neuronal differentiation, and segmentation during body axis extension.

Vertebrate body axis extension involves progressive generation and subsequent differentiation of new cells derived from a caudal stem zone; however, molecular mechanisms that preserve caudal progenitors and coordinate differentiation are poorly understood. FGF maintains caudal progenitors and its attenuation is required for neuronal and mesodermal differentiation and to position segment boundaries. Furthermore, somitic mesoderm promotes neuronal differentiation in part by downregulating Fgf8. Here we identify retinoic acid (RA) as this somitic signal and show that retinoid and FGF pathways have opposing actions. FGF is a general repressor of differentiation, including ventral neural patterning, while RA attenuates Fgf8 in neuroepithelium and paraxial mesoderm, where it controls somite boundary position. RA is further required for neuronal differentiation and expression of key ventral neural patterning genes. Our data demonstrate that FGF and RA pathways are mutually inhibitory and suggest that their opposing actions provide a global mechanism that controls differentiation during axis extension.

Vitamin A-deficient quail embryos have half a hindbrain and other neural defects

BACKGROUND Retinoic acid (RA) is a morphogenetically active signalling molecule thought to be involved in the development of severely embryonic systems (based on its effect when applied in excess and the fact that it can be detected endogenously in embryos). Here, we adopt a novel approach and use the vitamin A-deficient (A-) quail embryo to ask what defects these embryos show when they develop in the absence of RA, with particular reference to the nervous system. RESULTS We have examined the anatomy, the expression domains of a variety of genes and the immunoreactivity to several antibodies in these A- embryos. In addition to the previously documented cardiovascular abnormalities, we find that the somites are smaller in A- embryos, otic vesicle development is abnormal and the somites continue up to and underneath the otic vesicle. In the central nervous system, we find that neural crest cells need RA for normal development and survival, and the neural tube fails to extend any neurites into the periphery. Using general hindbrain morphology and the expression patterns of Hoxa-2, Hoxb-1, Hoxb-4, Krox-20 and FGF-3 as markers, we conclude that segmentation in the myelencephalon (rhombomeres 4-8) is disrupted. In contrast, the dorsoventral axis of the neural tube using Shh, islet-1 and Pax-3 as markers is normal. CONCLUSIONS These results demonstrate at least three roles for RA in central nervous system development: neural crest survival, neurite outgrowth and hindbrain patterning.

Retinoic acid signalling centres in the avian embryo identified by sites of expression of synthesising and catabolising enzymes

Retinoic acid is an important signalling molecule in the developing embryo, but its precise distribution throughout development is very difficult to determine by available techniques. Examining the distribution of the enzymes by which it is synthesised by using in situ hybridisation is an alternative strategy. Here, we describe the distribution of three retinoic acid synthesising enzymes and one retinoic acid catabolic enzyme during the early stages of chick embryogenesis with the intention of identifying localized retinoic acid signalling regions. The enzymes involved are Raldh1, Raldh2, Raldh3, and Cyp26A1. Although some of these distributions have been described before, here we assemble them all in one species and several novel sites of enzyme expression are identified, including Hensens node, the cardiac endoderm, the presumptive pancreatic endoderm, and the dorsal lens. This study emphasizes the dynamic pattern of expression of the enzymes that control the availability of retinoic acid as well as the role that retinoic acid plays in the development of many regions of the embryo throughout embryogenesis. This strategy provides a basis for understanding the phenotypes of retinoic acid teratology and retinoic acid–deficiency syndromes. Developmental Dynamics 227:114–127, 2003.

Hindbrain respecification in the retinoid-deficient quail.

We report here the development and rescue of the truncated hindbrain of retinoid-deprived quail embryos. The embryo is completely rescued by an injection of retinol into the egg; this confirms retinol, or a related retinoid, as a required molecule in hindbrain development. Staging the retinoid replacement enabled us to determine that the 3-4 somite stage is the period when retinoids are required for normal development. Analysis of the development of the retinoid-deprived hindbrain phenotype through somitogenesis has revealed a pathway of retinoid action in early hindbrain regionalization. The hindbrain of the retinoid-deprived embryo is normal in size, during early somitogenesis, but has a respecified pattern of Krox-20 expression. From the earliest expression of Krox-20, at the 5 somite stage, the rhombomere 3 stripe fills the caudal third of the developing hindbrain to the level of the first somite. Morphologically only 2, instead of the normal 5, rhombomere bulges form. These 2 bulges express genes and, later, develop morphology characteristic of rhombomeres 1 and 2 and rhombomere 3. Posterior hindbrain specific genes, Hoxb-1, Fgf3, MafB, and the rhombomere 5 stripe of Krox-20 are never expressed in the head neuroepithelium of these embryos. From the initial formation of the neural plate, there is no evidence of rhombomere 4-7 specific characteristics. These results indicate the specification of the posterior hindbrain is lost and its cells participate in the formation of an enlarged anterior hindbrain. In our previous study, we reported the absence of the posterior hindbrain in retinoid-deprived quails (Maden, M., Gale, E., Kostetskii, I., Zile, M., 1996. Vitamin A-deficient quail embryos have half a hindbrain and other neural defects. Curr. Biol. 6, 417-426). Here, we show this phenotype to be the result of respecification of the hindbrain cells. This provides evidence for a region specific response to a single stimulus, retinol, which suggests a pre-rhombomeric regionalization of the hindbrain.

Generating gradients of retinoic acid in the chick embryo: Cyp26C1 expression and a comparative analysis of the Cyp26 enzymes

We have cloned a novel retinoic acid (RA) catabolizing enzyme, Cyp26C1, in the chick and describe here its distribution during early stages of chick embryogenesis. It is expressed from stage 4 in the presumptive anterior (cephalic) mesoderm, in a subset of cephalic neural crest cells, the ventral otic vesicle, mesenchyme adjacent to the otic vesicle, the branchial pouches and grooves, a part of the neural retina, and the anterior telencephalon, and shows a dynamic expression in the hindbrain rhombomeres and neuronal populations within them. By examining the distribution of Cyp26C1 in the RA‐free quail embryo, we can determine which of these expression domains is dependent on RA, and it is only the rhombomeric sites that do not appear, suggesting a role for RA in this location. The most striking domain of Cyp26C1 distribution is in the anterior cephalic mesoderm, which is adjacent to the domain of Raldh2 in the trunk mesoderm, but separated from it by a gap dorsal to which the posterior hindbrain will develop. We suggest that a gradient of RA within the mesoderm generated by Raldh2 and catabolized by Cyp26C1 could be responsible for patterning the hindbrain. We have compared this distribution of Cyp26C1 with that of Cyp26A1 and Cyp26B1 in the chick and shown that they generally occupy nonoverlapping sites of expression in the embryo, and as a result, we suggest individual roles for each of the Cyp enzymes in the developing embryo. Developmental Dynamics 230:509–517, 2004.

Expression of the retinoic acid catabolising enzyme CYP26B1 in the chick embryo and its regulation by retinoic acid

We have cloned a fragment of Cyp26B1, a novel retinoic acid (RA) catabolising enzyme, and examined its expression pattern during early stages of chick embryogenesis. It is expressed from stage 7 in the tail bud, an anterior patch of mesenchyme, the heart, the endothelium of the vasculature, the eye, the limb bud, the hindgut and in a complex pattern in the rhombomeres of the hindbrain. As such it has a non-overlapping expression with chick Cyp26A1, the other RA catabolising enzyme, but shows a combination of features of mouse Cyp26A1 and Cyp26B1. We have also examined its expression in the quail embryo and in the RA-free quail embryo. In the absence of RA, Cyp26B1 is only expressed in the hindbrain and fails to be expressed in all the other regions of the embryo, most dramatically in the trunk. Adding back RA rescues Cyp26B1 expression.

The role of retinoic acid in the morphogenesis of the neural tube

We have examined the role of the signalling molecule, retinoic acid, in the process of neurulation and the subsequent growth and differentiation of the central nervous system using quail embryos that have developed in the absence of retinoic acid. Such retinoic acid‐free embryos undergo abnormal neural tube formation in terms of its shape and structure, but the embryos do not display spina bifida or exencephaly. The neural tubes have a wider floor plate, a thicker roof plate and a different dorsoventral shape. Phalloidin staining and electron microscopy revealed alterations in the actin filaments and the junctional complexes of the cell layer lining the lumen. Initially the neural tubes proliferated at the same rate as normal, but later the proliferation rate declined drastically and neuronal differentiation was highly deficient. There were very few motoneurons extending neurites into the periphery, and within the neural tube axon trajectories were chaotic. These results reveal several functions for retinoic acid in the morphogenesis and growth of the neural tube, many of which can be explained by defective notochord signalling, but they do not suggest that this molecule plays a role in neural tube closure.

Deficits in the posterior pharyngeal endoderm in the absence of retinoids.

Recent studies have shown that the pharyngeal endoderm plays a critically important role in directing the development of the pharyngeal region of the vertebrate embryo. We have, however, had few insights into how the pharyngeal endoderm itself is patterned. Recently, several studies have suggested that retinoic acid is required for the development of the pharyngeal endoderm. To study this proposal in greater depth, we have examined the development of the pharyngeal endoderm in the absence of retinoid signalling, by using the vitamin A– deficient (VAD) quail model system. We find in early stages that, in the absence of retinoids, this territory extends further caudally than normal. Furthermore, as development proceeds, we find that the first pouch invariably forms, that the second pouch is abnormal, and that the third and fourth pharyngeal pouches never form. We do find, however, that dorsoventral patterning of the pharyngeal endoderm is unaffected. Finally, we have examined the expression patterns of RALDH2 before and during early development of the pharyngeal pouches. We find that this enzyme is expressed adjacent to the pharyngeal endoderm in tissues around the regressing anterior intestinal portal and that from stage 12 onward its anterior limit of expression lies at the level of the second pouch. This finding helps explain why the first pouch always forms in the absence of retinoids, and why defects are seen starting with the second and most evidently in the caudal pouches.

Doublesex and mab-3–related transcription factor 5 promotes midbrain dopaminergic identity in pluripotent stem cells by enforcing a ventral-medial progenitor fate

Understanding the control of cell-fate choices during embryonic stem cell (ESC) differentiation is crucial for harnessing strategies for efficient production of desired cell types for pharmaceutical drug screening and cell transplantation. Here we report the identification of the zinc finger-like doublesex and mab-3–related transcription factor 5 (Dmrt5) as a marker for mammalian ventral-medial mesencephalic neuroepithelium that give rise to dopamine neurons. Gain- and loss-of-function studies in ESC demonstrate that Dmrt5 is critically involved in the specification of ventral-medial neural progenitor cell fate and the subsequent generation of dopamine neurons expressing essential midbrain characteristics. Genome-wide analysis of Dmrt5-mediated transcriptome changes and expression profiling of ventral-medial and ventral-lateral mesencephalic neuroepithelium revealed suppressive and inductive regulatory roles for Dmrt5 in the transcription program associated with the ventral-medial neural progenitor fates. Together, these data identify Dmrt5 as an important player in ventral mesencephalic neural fate specification.

P1 Regionalisation of the brain as an evolutionarily conserved developmental mechanism.

Comparative studies of chordate neural connectivity and gene families have provided evidence for evolutionary conservation of the patterning mechanisms in brain development (review Holland & Holland, Curr. Opin. Neurobiol.9, 1999). Based on expression patterns of ascidian and amphioxus homologues of the Otx gene and the Hox1 gene and of the ascidian Pax‐2/5/8, the chordate brain has been suggested to have tripartite development (Wada et al., Development125, 1998; Kozmik et al., Development126, 1999). Primitively, the chordates have regions homologous to the vertebrate forebrain, anterior midbrain and posterior hindbrain while the posterior midbrain/anterior hindbrain region seems to be a vertebrate innovation. The extent of the homologies within each of these regions between the vertebrates and their ancestors is not fully determined but the similarity of Hox gene expression patterns suggests organisational constants over evolutionary time within the posterior hindbrain region.