RETINOIC ACID ALTERS HINDBRAIN HOX CODE AND INDUCES TRANSFORMATION OF RHOMBOMERES 2-3 INTO A 4-5 IDENTITY

IT has been suggested that Hox genes play an important part in the patterning of limbs1-3, vertebrae4-6 and craniofacial structures5,7-9 by providing an ordered molecular system of positional values, termed the Hox code10,11. Little is known about the nature of the signals that govern the establishment and regulation of Hox genes, but retinoic acid can affect the expression of these genes in cell lines12-14 and in embryonic tissues2,11,15-17. On the basis of experimental and clinical evidence, the hindbrain and branchial region of the head are particularly sensitive to the effects of retinoic acid 15,18-21, but the phenotypes are complex and hard to interpret, and how and if they relate to Hox expression has not been clear. Here we follow the changes induced by retinoic acid to hindbrain segmentation and the branchial arches using transgenic mice which contain lacZ reporter genes that reveal the endogenous segment-restricted expression of the Hox-B1 (Hox-2.9), Hox-B2(Hox-2.8) and Krox-20 genes. Our results show that these genes rapidly respond to exposure to retinoic acid at preheadfold stages and undergo a progressive series of changes in segmental expression that are associated with specific phenotypes in hindbrain of first branchial arch. Together the molecular and anatomical alterations indicate that retinoic acid has induced changes in the hindbrain Hox code which result in the homeotic transformation of rhombomeres (r) 2/3 to an r4/5 identity. A main feature of this rhombomeric phenotype is that the trigeminal motor nerve is transformed to a facial identity. Furthermore, in support of this change in rhombomeric identity, neural crest cells derived from r2/3 also express posterior Hox markers suggesting that the retinoic acid-induced transformation extends to multiple components of the first branchial arch.