Role of Retinoic Acid Signaling, FGF Signaling and Meis Genes in Control of Limb Development

Abstract

The function of retinoic acid (RA) during limb development is still debated, as loss and gain of function studies led to opposite conclusions. With regard to limb initiation, genetic studies demonstrated that activation of FGF10 signaling is required for the emergence of limb buds from the trunk, with Tbx5 and RA signaling acting upstream in the forelimb field, whereas Tbx4 and Pitx1 act upstream in the hindlimb field. Early studies in chick embryos suggested that RA as well as Meis1 and Meis2 (Meis1/2) are required for subsequent proximodistal patterning of both forelimbs and hindlimbs, with RA diffusing from the trunk, functioning to activate Meis1/2 specifically in the proximal limb bud mesoderm. However, genetic loss of RA signaling does not result in loss of limb Meis1/2 expression and limb patterning is normal, although Meis1/2 expression is reduced in trunk somitic mesoderm. More recent studies demonstrated that global genetic loss of Meis1/2 results in a somite defect and failure of limb bud initiation. Other new studies reported that conditional genetic loss of Meis1/2 in the limb results in proximodistal patterning defects, and distal FGF8 signaling represses Meis1/2 to constrain its expression to the proximal limb. In this review, we hypothesize that RA and Meis1/2 both function in the trunk to initiate forelimb bud initiation, but that limb Meis1/2 expression is activated proximally by a factor other than RA and repressed distally by FGF8 to generate proximodistal patterning.

Keywords: FGF; Meis; limb development; retinoic acid.

Figure 1

Generation of retinoic acid (RA) and molecular mechanism used by RA to control transcriptional activation or repression of target genes. (A) RA is synthesized by sequential conversion of retinol (vitamin A) to retinaldehyde by retinol dehydrogenase 10 (RDH10), followed by metabolism of retinaldehyde to RA. RA can be degraded by CYP26 enzymes. (B,C) The heterodimer formed by nuclear RA receptors (RARs) complexed with retinoid X receptors (RXRs) binds noncoding DNA sequences called RA response elements (RAREs). Binding of RA to RAR creates conformational changes that alter the recruitment of nuclear receptor coactivators (NCOAs) or nuclear receptor corepressors (NCORs) leading to a change in the appearance of epigenetic marks on histone H3, i.e., lysine 27 acetylation (K27ac) associated with gene activation or lysine 27 trimethylation (K27me3) associated with gene repression. In the presence of RA, RARE enhancers stimulate recruitment of NCOAs that result in a nearby increase in H3K27ac and/or decrease in H3K27me3 leading to increased target gene expression such as Meis1 and Meis2 in embryonic trunk tissue (B). In the presence of RA, RARE silencers stimulate recruitment of NCORs that result in a nearby decrease in H3K27ac and/or increase in H3K27me3 leading to decreased target gene expression such as Fgf8 in embryonic trunk tissue (C).

Figure 2

Role of RA signaling, fibroblast growth factor (FGF) signaling, and Meis1/2 genes in forelimb initiation. In the E8.5 mouse embryo, the forelimb field (yellow) resides within an RA-rich trunk domain (blue) where the RA-generating enzymes encoded by Rdh10 and Aldh1a2 are expressed, thus positioned between two domains of FGF8 signaling in the heart and caudal progenitors (red). Trunk RA signaling represses Fgf8 at the borders of these two domains to permit activation of Tbx5 in the forelimb field, which then activates FGF10 signaling to stimulate limb outgrowth. Thus, RA acts permissively to activate forelimb Tbx5 expression. Genetic studies in zebrafish showing that loss of forelimb bud initiation in RA-deficient embryos can be fully rescued by reducing FGF signaling provides evidence that RA is not required to function instructively to activate forelimb Tbx5 expression. Meis1/2 genes are required for forelimb bud initiation, but it remains unclear if they activate forelimb Tbx5 or function in another manner.

Figure 3

Role of Meis1/2 genes and FGF signaling in limb proximodistal patterning. In the E10.5 mouse embryo, Meis1/2 expression that already exists in trunk lateral plate mesoderm (LPM) prior to limb bud initiation extends into the proximal regions of both forelimbs and hindlimbs as they undergo outgrowth from the trunk. Meis1/2 expression in the trunk LPM and proximal limb does not require RA signaling; the factor(s) that activate Meis1/2 expression in trunk LPM and limb are unknown. A boundary of Meis1/2 expression is formed by distal FGF8 (and other FGFs) secreted by the apical ectodermal ridge (AER) that represses Meis1/2 to limit expression to a proximal limb domain.