Hoxd4 and Rarg interact synergistically in the specification of the cervical vertebrae.

We show that, relative to single null mutants, mice bearing mutations in both Hoxd4 and Rarg display malformations of the basioccipital bone, and first (C1) and second cervical vertebrae (C2) at increased penetrance and expressivity, demonstrating synergy between Hoxd4 and Rarg in the specification of the cervical skeleton. In contrast to Rarg mutants, retinoic acid (RA) treatment on embryonic day 10.5 of Hoxd4 single or Hoxd4;Rarg double mutants does not rescue normal development of C2. Somitic expression of Hoxd4 is not altered in wild-type or Rarg mutant animals before or after RA treatment on day 10.5, suggesting that Hoxd4 and Rarg act in parallel to regulate the expression of target genes directing skeletogenesis.

Elements both 5' and 3' to the murine Hoxd4 gene establish anterior borders of expression in mesoderm and neurectoderm.

In this report, we show that a lacZ reporter spanning 12.5 kb of murine Hoxd4 genomic DNA contains the major regulatory elements controlling Hoxd4 expression in the mouse embryo. Mutational analysis revealed multiple regulatory regions both 5' and 3' to the coding region. These include a 3' enhancer region required for expression in the central nervous system (CNS) and setting the anterior border in the paraxial mesoderm, and a 5' mesodermal enhancer that directs expression in paraxial and lateral plate mesoderm. A previously defined retinoic acid response element (RARE) is a component of the 5' mesodermal enhancer. Our results support a model in which retinoic acid receptors (RARs) and HOX proteins mediate the initiation and maintenance of Hoxd4 expression.

Characterization and retinoic acid responsiveness of the murine Hoxd4 transcription unit.

We have characterized the transcription unit of a murine Hox gene in the fourth paralogous group, Hoxd4. We have identified two Hoxd4 transcription start sites by S1 analysis. The upstream promoter (P2) is 5.2 kilobase pairs upstream from the coding region, while the downstream promoter (P1) is 1.1 kilobase pairs distant. Both promoters bear a cluster of start sites. Multiple transcripts were identified by Northern blot, originating from both promoters and multiple polyadenylation signals. Expression of P1 transcripts in the neural tube shows an anterior border at the rhombomere 6/7 boundary, corresponding to previous reports (Gaunt, S. J., Krumlauf, R., and Duboule, D. (1989) Development 107, 131-141; Morrison, A., Moroni, M. C., Ariza-McNaughton, L., Krumlauf, R., and Mavilio, F. (1996) Development 122, 1895-1907). A more posterior boundary in the central nervous system was observed for P2 transcripts. We observed strong expression up to somite 6 and weak expression in somite 5, correlating with the phenotype of Hoxd4 null mutant mice (Horan, G. S. B., Nagy Kovàcs, E., Behringer, R. R., and Featherstone, M. S. (1995) Dev. Biol. 169, 359-372). In response to retinoic acid, expression from P1 in the hindbrain was anteriorized after 4 or 24 h of treatment. P2 transcripts seemed to be less responsive and/or to have an indirect response to retinoic acid. The long 5'-untranslated region found in all Hoxd4 transcripts suggests that translation does not occur by a classical ribosome scanning mechanism.

Mutations in paralogous Hox genes result in overlapping homeotic transformations of the axial skeleton: evidence for unique and redundant function.

Hoxd-4 (previously known as Hox-4.2 and -5.1) is a mouse homeobox-containing gene homologous to the Drosophila homeotic gene Deformed. During embryogenesis, Hoxd-4 is expressed in the presumptive hindbrain and spinal cord, prevertebrae, and other tissues. In the adult, Hoxd-4 transcripts are expressed predominantly in the testis and kidney, and to a lesser extent in intestine and heart. To understand the role of Hoxd-4 during mouse embryogenesis, we generated Hoxd-4 mutant mice. Mice heterozygous or homozygous for the Hoxd-4 mutation exhibit homeotic transformations of the second cervical vertebrae (C2) to the first cervical vertebrae (C1) and malformations of the neural arches of C1 to C3 and of the basioccipital bone. The phenotype was incompletely penetrant and showed variable expressivity on both an F2 hybrid and 129 inbred genetic background. The mutant phenotype was detected in the cartilaginous skeleton of 14.5-day (E14.5) mutant embryos but no apparent differences were detected in the somites of E9.5 mutant embryos, suggesting that the abnormalities develop after E9.5 perhaps during or after resegmentation of the somites to form the prevertebrae. These results suggest that Hoxd-4 plays a role in conferring position information along the anteroposterior axis in the skeleton. The phenotypic similarities and differences between Hoxd-4 and previously reported Hoxa-4 and Hoxb-4 mutant mice suggest that Hox gene paralogs have both redundant and unique functions.

A proline-rich transcriptional activation domain in murine HOXD-4 (HOX-4.2).

The product of the murine Hoxd-4 (Hox-4.2) gene is a transcription factor that acts upon an autoregulatory element in Hoxd-4 upstream sequences (1). Using this activity as an assay in transient transfections of P19 embryonal carcinoma (EC) cells, we performed a mutational analysis to map functional domains in the HOXD-4 protein. The importance of the homeodomain was shown by a single amino acid change in this region that abolished activity. Deletion analysis revealed that many evolutionarily conserved regions outside of the homeodomain were dispensable for activation in our assay. Fusions to the GAL4 DNA-binding domain mapped a transcriptional activation function to the HOXD-4 proline-rich N-terminus. The proline-rich transcription factor AP2 squelched activation by HOXD-4 and by GAL4/HOXD-4 N-terminus fusion proteins. Together, these results suggest that HOXD-4 harbors a transcriptional activation domain of the proline-rich type.