Distinct roles for Fgf,Wnt and retinoic acid in posteriorizing the neural ectoderm

Early neural patterning in vertebrates involves signals that inhibit anterior (A) and promote posterior (P) positional values within the nascent neural plate. In this study, we have investigated the contributions of, and interactions between, retinoic acid (RA), Fgf and Wnt signals in the promotion of posterior fates in the ectoderm. We analyze expression and function of cyp26/P450RAI, a gene that encodes retinoic acid 4-hydroxylase, as a tool for investigating these events. Cyp26 is first expressed in the presumptive anterior neural ectoderm and the blastoderm margin at the late blastula. When the posterior neural gene hoxb1b is expressed during gastrulation, it shows a strikingly complementary pattern to cyp26. Using these two genes, as well as otx2 and meis3 as anterior and posterior markers, we show that Fgf and Wnt signals suppress expression of anterior genes, including cyp26. Overexpression of cyp26 suppresses posterior genes, suggesting that the anterior expression of cyp26 is important for restricting the expression of posterior genes. Consistent with this, knock-down of cyp26 by morpholino oligonucleotides leads to the anterior expansion of posterior genes. We further show that Fgf- and Wnt-dependent activation of posterior genes is mediated by RA, whereas suppression of anterior genes does not depend on RA signaling. Fgf and Wnt signals suppress cyp26 expression, while Cyp26 suppresses the RA signal. Thus, cyp26 has an important role in linking the Fgf, Wnt and RA signals to regulate AP patterning of the neural ectoderm in the late blastula to gastrula embryo in zebrafish.     

A dual requirement for Iroquois genes during Xenopus kidney development

The Iroquois (Irx) genes encode evolutionary conserved homeoproteins. We report that Xenopus genes Irx1 and Irx3 are expressed and required during different stages of Xenopus pronephros development. They are initially expressed during mid-neurulation in domains extending over most of the prospective pronephric territory. Expression onset takes place after kidney anlage specification, but before pronephric organogenesis occurs. Later, during nephron segmentation, expression becomes restricted to the intermediate tubule region of the proximal-distal axis. Loss- and gain-of-function analyses, performed with specific morpholinos and inducible wild-type and dominant-negative constructs, reveal a dual requirement for Irx1 and Irx3 during pronephros development. During neurula stages, these genes maintain the specification of the pronephric territory and define its size. This seems to occur, at least in part, through positive regulation of Bmp signalling. Subsequently, Irx genes are required for proper formation of the intermediate tubule. Finally, we find that retinoic acid signalling activates both Irx1 and Irx3 genes in the pronephros.     

Somatic motoneurone specification in the hindbrain: the influence of somite-derived signals,retinoic acid and Hoxa3

We have investigated the mechanisms involved in generating hindbrain motoneurone subtypes, focusing on somatic motoneurones, which are confined to the caudal hindbrain within rhombomeres 5-8. Following heterotopic transplantation of rhombomeres along the rostrocaudal axis at various developmental stages, we have found that the capacity of rhombomeres to generate somatic motoneurones is labile at the neural plate stage but becomes fixed just after neural tube closure, at stage 10-11. Grafting of somites or retinoic acid-loaded beads beneath the rostral hindbrain induced the formation of somatic motoneurones in rhombomere 4 only, and Hox genes normally expressed more caudally (Hoxa3, Hoxd4) were induced in this region. Targeted overexpression of Hoxa3 in the rostral hindbrain led to the generation of ectopic somatic motoneurones in ventral rhombomeres 1-4, and was accompanied by the repression of the dorsoventral patterning gene Irx3. Taken together, these observations suggest that the somites, retinoic acid and Hox genes play a role in patterning somatic motoneurones in vivo.     

The Xenopus Irx genes are essential for neural patterning and define the border between prethalamus and thalamus through mutual antagonism with the anterior repressors Fezf and Arx

The Iroquois (Irx) genes encode homeoproteins conserved during evolution. Vertebrate genomes contain six Irx genes organized in two clusters, IrxA (which harbors Irx1, Irx2 and Irx4) and IrxB (which harbors Irx3, Irx5 and Irx6). To determine the precise role of these genes during development and their putative redundancies, we conducted a comparative expression analysis and a comprehensive loss-of-function study of all the early expressed Irx genes (Irx1-5) using specific morpholinos in Xenopus. We found that the five Irx genes display largely overlapping expression patterns and contribute to neural patterning. All Irx genes are required for proper formation of posterior forebrain, midbrain, hindbrain and, to a lesser an extent, spinal cord. Nevertheless, Irx1 and Irx3 seem to have a predominant role during regionalization of the neural plate. In addition, we find that the common anterior limit of Irx gene expression, which will correspond to the future border between the prethalamus and thalamus, is defined by mutual repression between Fezf and Irx proteins. This mutual repression is likely direct. Finally, we show that Arx, another anteriorly expressed repressor, also contribute to delineate the anterior border of Irx expression.     

Anterior–posterior patterning of neural differentiated embryonic stem cells by canonical Wnts,Fgfs, Bmp4 and their respective antagonists

Embryonic stem (ES) cells are pluripotent and can differentiate into every cell type of the body. Next to their potential in regenerative medicine, they are excellent tools to study embryonic development. In this work the processes of neural induction and neural patterning along the antero‐posterior (A/P) body axis are studied and evidence suggests a two step mechanism for these events. First, neural induction occurs by default in the primitive ectoderm, forming anterior neural tissue and thereafter, a series of factors can posteriorize this anterior neurectoderm. In a gain‐of‐function/loss‐of‐function approach using mouse ES cells, we show that Fgf2 has the strongest caudalizing potential of all Fgfs tested. Furthermore, Bmp4 and Wnt3a, but not Wnt1, can caudalize the neurectodermal cells. The effect of the antagonists of these factors was also examined and though Dkk1 and Noggin clearly have an effect that opposes that of Wnt3a and Bmp4 respectively, they fail to anteriorize the neurectoderm. The patterning effect of SU5402, an Fgf receptor inhibitor, was rather limited. These data confirm that in the mouse, two steps are involved in neural patterning and we show that while Fgf4, Fgf8 and Wnt1 have no strong patterning effect, Fgf2, Wnt3a and Bmp4 are strong posteriorizing factors.     

Zebrafish Dkk1, induced by the pre-MBT Wnt signaling, is secreted from the prechordal plate and patterns the anterior neural plate

mRNA injection into the ventral blastomeres of Xenopus embryos of mRNA encoding Wnt pathway genes induces a secondary axis with complete head structures. To identify target genes of the pre-MBT dorsalization pathway that might be responsible for head formation in zebrafish, we have cloned zebrafish dickkopf1 (dkk1), which is expressed in tissues implicated in head patterning. We found that dkk1 blocks the post-MBT Wnt signaling and dkk1 is a target of the pre-MBT Wnt signaling. Dkk1 overexpression in the prechordal plate suggests that Dkk1, secreted from the prechordal plate, expands the forebrain at the expense of the midbrain in the anterior neural plate. Furthermore, dkk1 acts in parallel to the homeobox gene bozozok and bozozok is required for the maintenance of dkk1 expression. The nodal gene squint is also required for the maintenance of dkk1 expression. Among the mutually dependent target genes of the pre-MBT Wnt signaling, dkk1 plays an important role in patterning the anterior head of zebrafish.