TIPOS DE CLIMA ORGANIZACIONAL.

Vertebrate homeobox genes were initiaUy isolated on the basis that they encoded DNA sequences which were identical to the homeobox o f the Drosophila Antennapaedia gene. Four homologous clusters o f Antp-VîkQ homeogenes have been identified in chick, Hoxa, Hoxb, Hoxc and Hoxd. These clusters are thought to have arisen by duphcation during the course o f evolution and exhibit a high degree o f homology both with each other and with the Drosophila homeogenes. In both vertebrates and Drosophila, the anterior boundary of expression of each gene corresponds to the relative order o f genes in each chromosome, with (3') genes exhibiting the most anterior expression and the (5') genes sequentially exhibiting more posterior expression.

Expression domains of homeobox genes in 5' regions o f the Hoxa and Hoxd clusters appear to correlate with regions that give rise to distract skeletal elements along the proximo-distal and antero-posterior axis o f developing limb buds. Specifically, the expression domain of 5* Hoxa genes appear to identify different proximo-distal segments of the limb while the Hoxd genes are expressed hi domains that correlate with pattern along both proximo-distal and antero­ posterior axes (reviewed by Izpisua-Belmonte and Duboule, 1992). Both Hoxa and Hoxd genes are activated sequentially and are expressed in nested proximo- distal domains along the limb buds, the most 5'-located gene transcripts being restricted to the tips of the limb buds. CeUs at the posterior tip o f the early buds express Hoxd-9 through Hoxd-13 and Hoxa-9 through Hoxa-13, whereas ceUs at

the anterior base o f the bud express Hoxd-9 and Hoxa-9 only. As the limb grows, expression expands anteriorly so that the final boundaries o f Hox expression domains are perpendicular to the proximo-distal axis (Yokouchi et al., 1991). Hox genes are activated in response to signals that specify limb pattern. Hoxd genes are induced in anterior mesenchyme by apphcation o f RA, polarising region grafts or SHH expressing cehs (Izpisua-Belmonte et al., 1991; Francis et al., 1994; Laufer et al., 1994). Importantly, induction oîH oxd gene expression takes place in the same 3' to 5' sequence as in normal development with Hoxd-11 being turned on before Hoxd-13. In addition, FGF-4 apphcation mamtains Hoxd-13 expression in the distal posterior mesenchyme after apical ridge removal. In contrast, expression of Hoxd-11 is relatively stable after ridge removal (Vogel et al., 1995a). This is consistent with the observed expression pattern o f H oxdl3 and Hoxd-11 in the developing hmb. Expression of Hoxd-13 remains at the tip o f the bud near the ridge (Figure 1.3), whereas expression o f Hoxd-11 is locahsed to regions o f the mesenchyme that do not appear to be influenced by the ridge. The requirement of apical ridge signals for proper Hoxd expression is also shown by the fact that Hoxd genes can be activated in anterior cehs in the absence o f the ridge only by applying two beads, one soaked in RA and one soaked in FGF-4 (Niswander et al, 1994).

Additional evidence supporting the crucial role of Hox genes in patterning and growth o f the hmb comes fi*om experiments in which a particular gene is overexpressed or fimctionahy inactivated. When Hoxd-11 is expressed throughout chick hmb buds fohowing retroviral expression, anterior leg digits come to resemble posterior digits and additional wmg digits develop (Morgan et al, 1992). In addition, misexpression of Hoxa-13 induces carthage homeotic transformation and changes ceh adhesiveness in chick hmb buds (Yokouchi et a l, 1995). When individual members of Hox clusters are fimctionahy inactivated, the position o f the resulting hmb deformities reflects the temporal and structural

colinearity of the Hox genes, such that inactivation of the 3' genes has a more proximal phenotypic boundary (afiFecting both the zeugopod and autopod of the limb) than that of the more 5' genes (affecting only the autopod). For example, Hoxd-11 targeted disruption results in limb defects including malformations o f the radius and ulna, inappropriate fusions between wrist bones, and regional malformations at the distal end of the forelimb (Davis and Capecchi, 1994). Mice with mutation in the Hoxd-13 gene exhibit more distal abnormahties such as growth retardation of several metacarpals and phalanges and the presence o f an extra posterior digit (DoUe et al., 1993). More recently, double "knock-outs" have been produced in which two paralogous genes in two different clusters have been inactivated in mice and more severe defects locahsed to predicted regions o f the limb have resulted. Thus, inactivation of Hoxd- 11 and Hoxa-11 leads to almost complete absence of zeugopod while proximal and distal structures are unaffected (Davis et al., 1995). However, functional redundancy can also occur even between non-paralogous Hox genes (for example between Hoxd-11 and Hoxa-10\

see Favier et al., 1996).

Recently, a transcription factor Lmx-1, has been shown to be activated in dorsal limb ectoderm by Wnt-7a signalling (Riddle et al., 1995; Vogel et al., 1995b; Figure 1.2B). When Lmx-1 is ventraUy expressed in chick limb buds this leads to ectopic dorsal structures. It was suggested that the limb pattern could be specified by the combination of expression of Hox genes and Lmx-1.