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Several decades have passed since the discovery of Hox genes in the fruit fly Drosophila melanogaster. Their unique ability to regulate morphologies along the anteroposterior (AP) axis (Lewis, 1978) earned them well-deserved attention as important regulators of embryonic development. Phenotypes due to loss- and gain-of-function mutations in mouse Hox genes have revealed that the spatiotemporally controlled expression of these genes is critical for the correct morphogenesis of embryonic axial structures. Here, we review recent novel insight into the modalities of Hox protein function in imparting specific identity to anatomical regions of the vertebral column, and in controlling the emergence of these tissues concomitantly with providing them with axial identity. The control of these functions must have been intimately linked to the shaping of the body plan during evolution.

The Hox genes confer positional information to the axial and paraxial tissues as they emerge gradually from the posterior aspect of the vertebrate embryo. Hox genes are sequentially activated in time and space, in a way that reflects their organisation into clusters in the genome. Although this co-linearity of expression of the Hox genes has been conserved during evolution, it is a phenomenon that is still not understood at the molecular level. This review aims to bring together recent findings that have advanced our understanding of the regulation of the Hox genes during mouse embryonic development. In particular, we highlight the integration of these transducers of anteroposterior positional information into the genetic network that drives tissue generation and patterning during axial elongation.

Hox and Cdx transcription factors regulate embryonic positional identities. Cdx mutant mice display posterior body truncations of the axial skeleton, neuraxis, and caudal urorectal structures. We show that trunk Hox genes stimulate axial extension, as they can largely rescue these Cdx mutant phenotypes. Conversely, posterior (paralog group 13) Hox genes can prematurely arrest posterior axial growth when precociously expressed. Our data suggest that the transition from trunk to tail Hox gene expression successively regulates the construction and termination of axial structures in the mouse embryo. Thus, Hox genes seem to differentially orchestrate posterior expansion of embryonic tissues during axial morphogenesis as an integral part of their function in specifying head-to-tail identity. In addition, we present evidence that Cdx and Hox transcription factors exert these effects by controlling Wnt signaling. Concomitant regulation of Cyp26a1 expression, restraining retinoic acid signaling away from the posterior growth zone, may likewise play a role in timing the trunk-tail transition.

The highly homologous Rnf2 (Ring1b) and Ring1 (Ring1a) proteins were identified as in vivo interactors of the Polycomb Group (PcG) protein Bmi1. Functional ablation of Rnf2 results in gastrulation arrest, in contrast to relatively mild phenotypes in most other PcG gene null mutants belonging to the same functional group, among which is Ring1. Developmental defects occur in both embryonic and extraembryonic tissues during gastrulation. The early lethal phenotype is reminiscent of that of the PcG-gene knockouts Eed and Ezh2, which belong to a separate functional PcG group and PcG protein complex. This finding indicates that these biochemically distinct PcG complexes are both required during early mouse development. In contrast to the strong skeletal transformation in Ring1 hemizygous mice, hemizygocity for Rnf2 does not affect vertebral identity. However, it does aggravate the cerebellar phenotype in a Bmi1 nullmutant background. Together, these results suggest that Rnf2 or Ring1-containing PcG complexes have minimal functional redundancy in specific tissues, despite overlap in expression patterns. We show that the early developmental arrest in Rnf2-null embryos is partially bypassed by genetic inactivation of the Cdkn2a (Ink4a͞ARF) locus. Importantly, this finding implicates Polycomb-mediated repression of the Cdkn2a locus in early murine development. P olycomb Group (PcG) proteins and their genetic counterparts, the trithorax Group proteins (trxG), maintain Hox gene expression boundaries (1-4), which are critical for regional patterning along the antero-posterior (AP) axis (5-7). Based on biochemical characteristics, mammalian PcG proteins are currently grouped into at least two distinct functional groups: the first comprises Eed, Ezh1, and Ezh2 in the mouse (8, 9); the second consists of the highly related protein pairs Cbx2 (M33)͞Cbx4 (MPc2), Bmi͞Zfp144 (Mel18), and Edr1 and 2 (Rae28͞Mph1 and 2), respectively (10, 11). For ease of this discussion we refer to them as groups I and II, respectively. Group I and II homologs are evolutionarily conserved from Drosophila to humans, only group I homologs are found in plants and Caenorhabditis elegans as well, supporting the concept of separate function (12, 13). In addition, complex composition differs throughout development in a temporal and cell-type-specific manner (14, 15). Interaction of Eed with histone deacetylases and the intrinsic histone methyltransferase activity of Ezh proteins suggest mechanisms for repression by group I complexes (16)(17)(18)(19). Although a mammalian hPRC-H (group II) complex harbors an intrinsic capacity to stabilize a repressive chromatin structure and counteract SWI͞SNF chromatin remodeling complexes in vitro, its in vivo mode of action is not well understood (20). Association with histone methyl transferase activity may in part help explain the repressive action of some group II complexes (21).Rnf2 and Ring1 have been identified as in vivo interactors of the group II PcG protein Bmi1 by us and others (22). These Ring finger proteins...

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. Chem. 258, 4331 (1983)]. The enzyme was measured in 4M urea extracts of skin samples [H. M. Kagan and K. A. Sullivan, Methods Enzymol. 82A, 637 (1982)] by an enzyme-linked immunoassay. The net accumulation oflysyl oxidase in the extracellular matrix of dorsal skin (in micrograms per gram ± SEM) was reduced from 500± 123 to 146 ± 55 by week 8 (P < 0.01; for two replicates, n = 4 to 6 each). The lysyl oxidase antibody preparation and the enzyme-linked inumunoassay were done as described by H. Bode and H. Stegeman [J. Immunol. Methods 72,421 (1984) Carroll and B. D. Stallar [J. Biol. Chem. 258, 24 (1983)]. A preliminary report on the characterization of lysyl oxidase and its quantitation by enzyme-linked inmmunosorbent assay (ELISA) has been published by D. Tinker, N. Romero, and R. B.] and S. B.

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