*3.2.2 Increased RA signaling in embryonic caudal region*

RA signaling in the genesis of SML is well established in experimental animals. RA, the active metabolite of vitamin A, is degraded by the enzyme Cyp26a1, which is specifically expressed in the embryonic caudal region and the developing vascular network. [38, 39]. A deficiency of Cyp26a1 is demonstrated to result in excessive RA activity in the embryonic caudal region and induce multiple caudal defects, including that of SML with a reported 20% penetrance. This enzyme's lack or deficiency also results in diminished bone morphogenetic protein signaling in the caudal region of the embryo. Cdx2 is a transcription factor that encodes and activates the Cyp26a1 promoter, and Por encodes an enzyme that is required for the function of the Cyp26 family of enzymes. [1, 40, 41]. Even the disruption of these related factors Cdx2, and Por, are noted to result in SML. The relationship is further corroborated by the fact that a Cyp26a1 phenotype can be rescued by decreasing the production of RA by reducing the level of the enzyme haploinsufficiency of Raldh2, which is necessary for its production. [41].

Embryologically, the developing cells in caudal structures are especially sensitive to RA signaling during gastrulation. Its levels are tightly controlled by the expression of its metabolizing enzymes [1] A multitude of experiments that modified the level of RA signaling by genetic and nutritional means have demonstrated that the embryo is particularly sensitive to deviations from normal levels of RA during

gastrulation [1, 42–44]. The expression of Cyp26a1 takes place at the early gastrula stage in the primitive streak and the nascent mesoderm; and at the late gastrula stage in the neuropore, hindgut endoderm, and tailbud mesoderm [37, 39, 40]. RA signaling is generated by Raldh2 in the somites in association with specific growth factors. RA plays a key role in the proliferation and differentiation of precursor cells, and Cyp26a1 expression at the caudal area is pivotal in maintaining the physiologically appropriate RA levels. [45, 46] RA excess is shown to negatively regulate endothelial cell proliferation and impede vascular remodeling by inducing premature coalescence and differentiation of precursor cells. In animal models, abnormal development of umbilical and vitelline arteries in embryos similar to those in human SML was noted when pregnant rats were given RA. [47] However, despite strong evidence of Cyp26a1 being instrumental in the generation of SML phenotype, a mutational screening of the Cyp26a1 gene has not confirmed its involvement in caudal dysplasia in humans [48].

Even though both Bmp and RA signaling pathways are critical participants in the development of caudal structural during the early embryonic stage, it is unclear if their effects are synergistic or antagonistic and whether they modulate each other's roles. RA had been shown to decrease Bmp signal duration by reducing the level of phosphorylated Smad1, an intracellular component of the Bmp signaling pathway in the developing neural tube [1, 49], whereas Bmp signaling has been demonstrated to adversely regulates RA signaling during chondrogenesis [50]. However, such effects have not been studied or confirmed to be operative in the generation of SML phenotype. The detection of possible crosstalk between the two signaling cascades might provide helpful information regarding the pathogenesis of SML phenotype in humans.
