Wim de Graaff

Ectopic expression of Hoxb-8 causes duplication of the ZPA in the forelimb and homeotic transformation of axial structures.

Transgenic embryos were generated carrying a Hoxb-8 transgene under control of the mouse RAR beta 2 promoter, which extends the normal expression domain to more anterior regions of the embryo. These embryos showed mirror-image duplications in the forelimb, analogous to the duplications observed in chick in response to transplantation of a ZPA to the anterior margin of the limb bud. Examination of Sonic hedgehog, Fgf-4, and Hoxd-11 gene expression confirmed that a second ZPA had been generated at the anterior side of the limb bud. Besides other alterations, posterior homeotic transformations of axial structures were observed, involving the first spinal (Frorieps) ganglion and several cervical vertebrae.

Cdx and Hox Genes Differentially Regulate Posterior Axial Growth in Mammalian Embryos

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 Cdx4 mutation affects axial development and reveals an essential role of Cdx genes in the ontogenesis of the placental labyrinth in mice.

Caudal related homeobox (Cdx) genes have so far been shown to be important for embryonic axial elongation and patterning in several vertebrate species. We have generated a targeted mutation of mouse Cdx4, the third member of this family of transcription factor encoding genes and the last one to be inactivated genetically. Cdx4-null embryos were born healthy and appeared morphologically normal. A subtle contribution of Cdx4 to anteroposterior (AP) vertebral patterning was revealed in Cdx1/Cdx4 and Cdx2/Cdx4 compound mutants. Neither Cdx4-null nor Cdx1/Cdx4 double mutants are impaired in their axial elongation, but a redundant contribution of Cdx4 in this function was unveiled when combined with a Cdx2 mutant allele. In addition, inactivation of Cdx4 combined with heterozygous loss of Cdx2 results in embryonic death around E10.5 and reveals a novel function of Cdx genes in placental ontogenesis. In a subset of Cdx2/Cdx4 compound mutants, the fully grown allantois failed to fuse with the chorion. The remaining majority of these mutants undergo successful chorio-allantois fusion but fail to properly extend their allantoic vascular network into the chorionic ectoderm and do not develop a functional placental labyrinth. We present evidence that Cdx4 plays a crucial role in the ontogenesis of the allantoic component of the placental labyrinth when one Cdx2 allele is inactivated. The axial patterning role of Cdx transcription factors thus extends posteriorly to the epiblast-derived extra-embryonic mesoderm and, consequent upon the evolution of placental mammals, is centrally involved in placental morphogenesis. The relative contribution of Cdx family members in the stepwise ontogenesis of a functional placenta is discussed, with Cdx2 playing an obligatory part, assisted by Cdx4. The possible participation of Cdx1 was not documented but cannot be ruled out until allelic combinations further decreasing Cdx dose have been analyzed. Cdx genes thus operate in a redundant way during placentogenesis, as they do during embryonic axial elongation and patterning, and independently from the previously reported early Cdx2-specific role in the trophectoderm at implantation.

Loss of Hoxb8 alters spinal dorsal laminae and sensory responses in mice

Although Hox gene expression has been linked to motoneuron identity, a role of these genes in development of the spinal sensory system remained undocumented. Hoxb genes are expressed at high levels in the dorsal horn of the spinal cord. Hoxb8 null mutants manifest a striking phenotype of excessive grooming and hairless lesions on the lower back. Applying local anesthesia underneath the hairless skin suppressed excessive grooming, indicating that this behavior depends on peripheral nerve activity. Functional ablation of mouse Hoxb8 also leads to attenuated response to nociceptive and thermal stimuli. Although spinal ganglia were normal, a lower postmitotic neural count was found in the dorsalmost laminae at lumbar levels around birth, leading to a smaller dorsal horn and a correspondingly narrowed projection field of nociceptive and thermoceptive afferents. The distribution of the dorsal neuronal cell types that we assayed, including neurons expressing the itch-specific gastrin-releasing peptide receptor, was disorganized in the lumbar region of the mutant. BrdU labeling experiments and gene-expression studies at stages around the birth of these neurons suggest that loss of Hoxb8 starts impairing development of the upper laminae of the lumbar spinal cord at approximately embryonic day (E)15.5. Because none of the neuronal markers used was unexpressed in the adult dorsal horn, absence of Hoxb8 does not impair neuronal differentiation. The data therefore suggest that a lower number of neurons in the upper spinal laminae and neuronal disorganization in the dorsal horn underlie the sensory defects including the excessive grooming of the Hoxb8 mutant.

Randomly inserted and targeted Hox/reporter fusions transcriptionally silenced in Polycomb mutants

Polycomb-group (Pc-G) proteins ensure late maintenance of transcriptional repression outside the expression domain of target genes in flies and vertebrates. They act in complexes, presumably by modulating chromatin structure. In Drosophila, they have been found to be associated with transcriptionally inactive loci but seem to be present in association with actively transcribed promoters as well, a feature which is not yet understood. In the mouse, mutations in several Pc-G genes result in an often subtle, local derepression of only a subset of the Hox genes rostral to their expression domains. We report here that Hox/reporter fusion genes, either randomly integrated as transgenes or as insertions within endogenous loci, are transcriptionally silenced in two mouse Pc-G-null mutants, Mel18 and rae28. Transcriptional silencing of Hox/reporter transgenes in Pc-G mutants was accompanied by increased DNA methylation in the promoter region. Gene silencing was observed at early developmental stages, long before Pc-G and trithorax-group proteins exert their function in maintenance of the Hox patterns. Although all five Hox genes tested as Hox/reporter fusions were silenced in the Pc-G mutants, transcription of the endogenous loci was mildly decreased in a subset of these Hox genes, and Hoxb1 was the most strongly affected. We discuss the possibilities that the observed negative effect of Pc-G mutations on Hox and Hox/reporter expression may reflect a positive involvement of the Pc-G epigenetic repressors in initial Hox gene transcription and that this requirement is exacerbated by the reporter insertion.

Regulation of Expression of the Hox 2.3 Gene

Only few vertebrate genes have been described, that can be unambiguously linked to developmental processes. For this reason, investigators of vertebrate embryology concentrate their attention on genes for which the evidence implicating them in pattern formation is only circumstantial. The homeobox containing genes (1), found throughout the animal kingdom including mice and humans, exemplify such genes. In the genetically well-characterized fruitfly Drosophila melanogaster their involvement in various stages of the segmentation of the embryo and in neuronal development (1, 2) is beyond doubt, although their precise function still has to be established.