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Retinoic acid regulation of the Mesp-Ripply feedback loop during vertebrate segmental patterning.

ABSTRACT: The Mesp bHLH genes play a conserved role during segmental patterning of the mesoderm in the vertebrate embryo by specifying segmental boundaries and anteroposterior (A-P) segmental polarity. Here we use a xenotransgenic approach to compare the transcriptional enhancers that drive expression of the Mesp genes within segments of the presomitic mesoderm (PSM) of different vertebrate species. We find that the genomic sequences upstream of the mespb gene in the pufferfish Takifugu rubripes (Tr-mespb) are able to drive segmental expression in transgenic Xenopus embryos while those from the Xenopus laevis mespb (Xl-mespb) gene drive segmental expression in transgenic zebrafish. In both cases, the anterior segmental boundary of transgene expression closely matches the expression of the endogenous Mesp genes, indicating that many inputs into segmental gene expression are highly conserved. By contrast, we find that direct retinoic acid (RA) regulation of endogenous Mesp gene expression is variable among vertebrate species. Both Tr-mespb and Xl-mespb are directly upregulated by RA, through a complex, distal element. By contrast, RA represses the zebrafish Mesp genes. We show that this repression is mediated, in part, by RA-mediated activation of the Ripply genes, which together with Mesp genes form an RA-responsive negative feedback loop. These observations suggest that variations in a direct response to RA input may allow for changes in A-P patterning of the segments in different vertebrate species.

SUBMITTER: Moreno TA

PROVIDER: S-EPMC4648629 | biostudies-other | 2008 Mar

REPOSITORIES: biostudies-other

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Retinoic acid regulation of the Mesp-Ripply feedback loop during vertebrate segmental patterning.

Moreno Tanya A TA Jappelli Roberto R Izpisúa Belmonte Juan Carlos JC Kintner Chris C

Developmental biology 20080103 2

The Mesp bHLH genes play a conserved role during segmental patterning of the mesoderm in the vertebrate embryo by specifying segmental boundaries and anteroposterior (A-P) segmental polarity. Here we use a xenotransgenic approach to compare the transcriptional enhancers that drive expression of the Mesp genes within segments of the presomitic mesoderm (PSM) of different vertebrate species. We find that the genomic sequences upstream of the mespb gene in the pufferfish Takifugu rubripes (Tr-mespb ...[more]

PMID: 18261720

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Project description:Xenopus laevis is among the few species that are capable of fully regenerating a lost lens de novo. This occurs upon removal of the lens, when secreted factors from the retina are permitted to reach the cornea epithelium and trigger it to form a new lens. Although many studies have investigated the retinal factors that initiate lens regeneration, relatively little is known about what factors support this process and make the cornea competent to form a lens. We presently investigate the role of Retinoic acid (RA) signaling in lens regeneration in Xenopus. RA is a highly important morphogen during vertebrate development, including the development of various eye tissues, and has been previously implicated in several regenerative processes as well. For instance, Wolffian lens regeneration in the newt requires active RA signaling. In contrast, we provide evidence here that lens regeneration in Xenopus actually depends on the attenuation of RA signaling, which is regulated by the RA-degrading enzyme CYP26. Using RT-PCR we examined the expression of RA synthesis and metabolism related genes within ocular tissues. We found expression of aldh1a1, aldh1a2, and aldh1a3, as well as cyp26a1 and cyp26b1 in both normal and regenerating corneal tissue. On the other hand, cyp26c1 does not appear to be expressed in either control or regenerating corneas, but it is expressed in the lens. Additionally in the lens, we found expression of aldh1a1 and aldh1a2, but not aldh1a3. Using an inhibitor of CYP26, and separately using exogenous retinoids, as well as RA signaling inhibitors, we demonstrate that CYP26 activity is necessary for lens regeneration to occur. We also find using phosphorylated Histone H3 labeling that CYP26 antagonism reduces cell proliferation in the cornea, and using qPCR we find that exogenous retinoids alter the expression of putative corneal stem cell markers. Furthermore, the Xenopus cornea is composed of an outer layer and inner basal epithelium, as well as a deeper fibrillar layer sparsely populated with cells. We employed antibody staining to visualize the localization of CYP26A, CYP26B, and RALDH1 within these corneal layers. Immunohistochemical staining of these enzymes revealed that all 3 proteins are expressed in both the outer and basal layers. CYP26A appears to be unique in also being present in the deeper fibrillar layer, which may contain cornea stem cells. This study reveals a clear molecular difference between newt and Xenopus lens regeneration, and it implicates CYP26 in the latter regenerative process.

Project description:BACKGROUND:The bat has strikingly divergent forelimbs (long digits supporting wing membranes) and hindlimbs (short, typically free digits) due to the distinct requirements of both aerial and terrestrial locomotion. During embryonic development, the morphology of the bat forelimb deviates dramatically from the mouse and chick, offering an alternative paradigm for identifying genes that play an important role in limb patterning. RESULTS:Using transcriptome analysis of developing Natal long-fingered bat (Miniopterus natalensis) fore- and hindlimbs, we demonstrate that the transcription factor Meis2 has a significantly higher expression in bat forelimb autopods compared to hindlimbs. Validation by reverse transcriptase and quantitative polymerase chain reaction (RT-qPCR) and whole mount in situ hybridisation shows that Meis2, conventionally known as a marker of the early proximal limb bud, is upregulated in the bat forelimb autopod from CS16. Meis2 expression is localised to the expanding interdigital webbing and the membranes linking the wing to the hindlimb and tail. In mice, Meis2 is also expressed in the interdigital region prior to tissue regression. This interdigital Meis2 expression is not activated by retinoic acid (RA) signalling as it is present in the retained interdigital tissue of Rdh10 (trex/trex) mice, which lack RA. Additionally, genes encoding RA-synthesising enzymes, Rdh10 and Aldh1a2, and the RA nuclear receptor Rar? are robustly expressed in bat fore- and hindlimb interdigital tissues indicating that the mechanism that retains interdigital tissue in bats also occurs independently of RA signalling. CONCLUSIONS:Mammalian interdigital Meis2 expression, and upregulation in the interdigital webbing of bat wings, suggests an important role for Meis2 in autopod development. Interdigital Meis2 expression is RA-independent, and retention of interdigital webbing in bat wings is not due to the suppression of RA-induced cell death. Rather, RA signalling may play a role in the thinning (rather than complete loss) of the interdigital tissue in the bat forelimb, while Meis2 may interact with other factors during both bat and mouse autopod development to maintain a pool of interdigital cells that contribute to digit patterning and growth.

Dataset Information

Retinoic acid regulation of the Mesp-Ripply feedback loop during vertebrate segmental patterning.

Publications

Retinoic acid regulation of the Mesp-Ripply feedback loop during vertebrate segmental patterning.

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