RA is an important modulator of cell survival, cellular proliferation and differentiation. Moreover, this signal plays important roles during pattern formation and organogenesis in vertebrate embryos (Glover et al., 2006; Mark et al., 2006). If vitamin A is excluded from the diet during pregnancy, congenital malformations can be observed in the offspring. These include hydrocephalus, spina bifida, anophthalmia, microphthalmia and patterning abnormalties of the CNS (Maden, 2002). At lower frequency cleft palate and lip, accessory external ears and arrested ascension of the kidney were reported (Hale et al., 1933). Experiments in the rat revealed additional defects in the developing genito-urinary tract, diaphragm, lung, aortic arch and heart (Wilson and Warkany, 1948; Wilson and Warkany, 1949).
Knockout experiments in mice, in which several RARs have been inactivated, phenocopy vitamin A deficiency in several aspects, but also reveal defects of the ocular and salivary glands and their ducts as well as skeletal abnormalities of forelimbs, hindlimbs and the cervical region of the axial skeleton. RAR# mutant mice display webbed digits on both forelimbs and hindlimbs, while RAR$ mutants show several congenital abnormalities including tracheal cartilage malformations and homeotic transformations along the cervical axial skeleton (Grondona et al., 1996).
On the other hand, an excess of RA in the diet of pregnant animals can cause exencephaly, cranium bifidum, microcephaly, microphthalmia and spina bifida (Cohlan,1953; Langman and Welch, 1967). A daily oral administration of RA to pregnant pigtail monkeys (Macaca nemestria) results in a high frequency of craniofacial and muscoskeletal malformations. Craniofacial defects including cleft palate were reported as well as ectrodactyly, kyphosis, and muscular-joint contractures. Less frequently, transposition of the great vessels of the heart, polycystic kidney and associated urogenital anomalies occurr (Fantel et al., 1977).
These studies indicate that RA plays important roles during embryonic development and that the concentration of this signalling molecule needs to be tightly regulated.
1.9 Retinoic acid signalling during organogenesis
Subsequent studies revealed that RA is involved in the formation of several embryonic structures and organs including limbs, somites, heart, pancreas, lung and the genito-urinary tract. In some cases, RA signalling acts through modulating other signalling pathways (Duester, 2008). Exogenous RA administration alters anterior-posterior patterning of the chick limb bud or proximodistal patterning of regenerating axolotl limbs (Tickle et al., 1982; Maden, 1983). Later, it was demonstrated that RA-bead implants ectopically induce Sonic Hedgehog (Shh) gene expression and that Shh controls limb anterior-posterior patterning (Riddle et al., 1993). Earlier studies in the chick limb bud suggest an RA morphogen gradient with highest concentration in the posterior zone of polarizing activity (Thaller and Eichele, 1987). However, studies in mouse embryos demonstrated that RA in early limb buds is distributed uniformly across the anterior-posterior axis but decrases from proximal to distal (Mic et al., 2004). In the absence of RA synthesis, forelimb buds do not develop and embryonic growth ceases prior to the stage when hindlimb buds are initiated (Niederreither et al., 2002; Mic et al., 2004). In zebrafish, the absence of RA synthesis blocks the induction of pectoral fin buds (Gilbert et al., 2006).
During somitogenesis, RA can repress caudal Fgf8 expression in mouse embryos (Vermot et al., 2005; Sirbu and Duester, 2006). In chick embryos, it could be shown that RA attenuates Fgf8 signalling in the neuroepithelium and paraxial mesoderm, in order to control somite boundary position. (Diez del Corral et al., 2003). Experiments in mouse, chick and zebrafish embryos demonstrated that RA is required to retain bilateral symmetry of the left and right columns of somites (Kawakami et al., 2005;
Vermot et al., 2005; Vermot and Pourquie, 2005, Sirbu and Duester, 2006).
RA is required for anterior-posterior patterning of the heart tube. In mice, loss of RA synthesis leads to a severe reduction of the atria/inflow tract domain. The outflow tract/ventricular domain forms an abnormal cavity that is distended medially rather than undergoing rightward looping and septation into right and left ventricles.
(Niederreither et al., 2001). Analysis of cardiac-specific genes suggested that the effects of RA on early heart development are mediated through repression of Fgf8 expression in the posterior region of the heart (Ryckebusch et al., 2008; Sirbu et al., 2008).
In the developing pancreas, studies have focused on the homeobox transcription factor Pdx1 which is required for pancreatic specification in the posterior foregut.
Evidence suggests that RA may be the signal required for Pdx1 initiation (Stafford and Prince, 2002). Zebrafish embryos, deficient in RA, lack Pdx1 expression and consequently fail to induce pancreas development. Similar results have been obtained in mouse and Xenopus embryos (Chen et al., 2004; Martin et al., 2005; Molotkov et al., 2005). In Xenopus embryos, an RAR antagonist induces misspecification of dorsal pancreatic tissue while ventral endodermal pancreatic tissue and the liver are not effected, demonstrating that only the dorsal pancreas requires RA activity (Chen et al., 2004). Furthermore, RA signalling influences both, mesodermal and endodermal germlayers, during pancreas development (Pan et al., 2007).
In the mouse, RA has been shown to be important for stimulation of posterior foregut endoderm to aquire a lung fate (Malpel et al., 2000). Embryos that lack RA signalling in the foregut fail to develop lungs (Wang et al., 2006).
RA controls some aspects of genito-urinary tract development. Analysis of RAR knockout mice showed that RA plays a key role in controlling epithelial / mesenchymal interactions during kidney development (Batourina et al., 2001). RA, generated in the urogenital sinus, stimulates apoptosis in the common nephric duct that is needed for the establishment of connections between the ureters and bladder (Batourina et al., 2005).
The involvement of RA in the generation of several different embryonic structures points towards the importance of this morphogen during development and explains the diversity of malformations observed upon abberant RA signalling.