Ectopic expression of Hox-2.3 induces craniofacial and skeletal malformations in transgenic mice.

To better understand the role of the Hox-2.3 murine homeobox gene during development, a dominant gain-of-function mutation was generated. The developmental malformations that resulted when the chicken beta-actin promoter was used to direct widespread expression of the Hox-2.3 gene in transgenic mice included early postnatal death as well as craniofacial abnormalities, including open eyes and cleft palate. Ventricular septal defects were also observed in the hearts of three transgenic mice. Skeletal malformations were seen in the bones of the craniocervical transition, with the occipital, basisphenoid, and atlas bones deficient or misshapen. Interestingly, one mutant exhibited an extra pair of ribs as well as alterations in cervical vertebrae identities. Some of the malformations observed in Hox-2.3 gain-of-function mutants overlap with those seen in Hox-1.1 and Hox-2.2 misexpression mutants which suggests functional similarities between paralogous homeobox genes. The results of these experiments are consistent with a role for Hox-2.3 in specifying positional information during development.     

The murine Hox-2 cluster of homeo box containing genes maps distal on chromosome 11 near the tail-short (Ts) locus

Two probes derived from a mouse recombinant lambda-clone (H24.1), that contains a sequence closely homologous to the Drosophila antennapedia homeo box, were mapped to mouse chromosome (MMU) 11 by filter hybridization of somatic cell hybrid DNA. This sequence is highly homologous to a human homeo box gene (HOX2) and appears to represent one of the two genes in the Hox-2 cluster previously assigned to MMU 11. To regionally map the Hox-2 cluster, we have carried out in situ hybridization of the two H24.1 probes and of an independently isolated Hox-2 probe. The autoradiographic silver grain distributions were similar in all three experiments with a peak over band 11D. This region contains the locus for the tail-short (Ts) mutation which causes skeletal abnormalities in heterozygotes and early embryonic death in homozygotes.     

Sequence and embryonic expression of the murine Hox-3.5 gene.

The murine Hox-3.5 gene has been mapped and linked genomically to a position 18 kb 3' of its most 5' locus neighbour, Hox-3.4, on chromosome 15. The sequence of the Hox-3.5 cDNA, together with the position of the gene within the locus, show it to be a paralogue of Hox-2.6, Hox-1.4 and Hox-4.2. The patterns of embryonic expression for the Hox-3.5 gene are examined in terms of three rules, proposed to relate a Hox gene's expression pattern to its position within the locus. The anterior boundaries of Hox-3.5 expression in the hindbrain and prevertebral column lie anterior to those of Hox-3.4 and all other, more 5'-located Hox-3 genes. Within the hindbrain, the Hox-3.5 boundary is seen to lie posterior to that of its paralogue, Hox-2.6, by a distance equal to about the length of one rhombomere. Patterns of Hox-3.5 expression within the oesophagus and spinal cord, but not the testis, are similar to those of other Hox-3 genes, Hox-3.3 and Hox-3.4.     

Differential expression of Hox 3.1 protein in subregions of the embryonic and adult spinal cord

Synthetic oligopeptides derived from the predicted Hox 3.1 protein coding sequence were used for the production of antibodies (anti-aa2) that specifically recognize Hox 3.1 protein in tissue sections. These antibodies were applied in immunohistochemical studies to monitor the expression of Hox 3.1 protein within the central nervous system (CNS) of embryonic and adult mice. We demonstrate congruency between the distinct Hox 3.1 RNA and protein expression patterns in the developing spinal cord by direct comparison of in situ hybridization and immunohistochemical staining in frozen sagittal sections from embryos of 12.5 days of gestation. A distinct pattern of spatially restricted expression of Hox 3.1 protein within the spinal cord was first detected at around 10.5 days of embryonic development. Within certain anteroposterior limits the geometries of this expression pattern change drastically during subsequent embryonic stages, concomitant with important cytoarchitectural changes in the developing spinal cord. Analyses on subcellular levels indicate predominant accumulation of Hox 3.1 protein within nuclei of neuronal cells. In addition to the nuclear localization in subsets of embryonic cells, persistent accumulation of Hox 3.1 protein was shown in nuclei of fully differentiated and mature neuronal cells of the adult CNS.     

Homeotic transformations in the mouse induced by overexpression of a human Hox3.3 transgene.

A permanent transgenic mouse line was generated carrying 40 copies of the human Hox3.3 gene. The resulting mice express large amounts of Hox3.3 protein in posterior regions of the embryo where this homeodomain protein is normally not expressed. The transgene causes homeotic transformations of the skeleton, in particular the appearance of an extra pair of ribs in the lumbar region, transformation of the shape of posterior ribs into that of more anterior ones, and the joining of an additional pair of ribs to the sternum. The phenotype of this line resembles that obtained by the targeted loss-of-function mutation of Hox3.1 (Le Mouellic et al., 1992). In transient assays, the human Hox3.3 transgene leads to the formation of additional ribs in more posterior vertebrae as well.     

Hox-3.6: isolation and characterization of a new murine homeobox gene located in the 5' region of the Hox-3 cluster.

Most members of the murine Hox gene system can be grouped into two subclasses based on their structural similarity to either one of the Drosophila homeotic genes Antennapedia (Antp) or Abdominal B (AbdB). All the AbdB-like genes reported thus far are located in the 5' region of their respective cluster. We describe here the isolation, structural characterization and spatio-temporal expression pattern of a new AbdB-like homeobox gene designated Hox-3.6 that is located in the 5' region of the Hox-3 cluster. Hox-3.6 has an extreme posterior expression domain in embryos of 12.5 days of gestation, a feature that has thus far only been observed for the 5' most genes of the Hox-4 cluster. Like the other members of the AbdB subfamily, Hox-3.6 exhibits spatially restricted expression in the hindlimb bud, but the expression domain is antero-proximal in contrast to the postero-distal domain reported for its cognate gene Hox-4.5. Structural analysis of the 5' region revealed the presence of a 35 bp sequence which shares homology and relative 5' position with an upstream sequence present in its two nearest downstream neighbors, Hox-3.2 and -3.1.     

Mapping of theHox-3.1 andMyc-1.2 genes on chromosome 15 of the mouse by restriction fragment length variations

Restriction endonuclease fragment length variations (RFLV) were detected by use of the cDNA probeHox-3.1 for the homeo box-3.1 gene and also thec-myc oncogene probe for exon 2. RFLV ofHox-3.1 were found inHindIII restriction patterns, and RFLV of theMyc-1.2 gene inEcoRV patterns. From the RFLV, theHox-3.1 andMyc-1.2 genes were mapped on chromosome 15. Three-point cross test data showed that the frequency of recombination is 26.4% betweenMyc-1.2 andGpt-1, 30.2% betweenGpt-1 andGdc-1, and 9.4% betweenGdc-1 andHox-3.1. The following order of these genes is proposed,Myc-1.2—Gpt-1—Gdc-1—Hox-3.1. All laboratory strains carry theHox-3.1 ^a andMyc-1.2 ^a alleles. Among strains of wild origin,domesticus strains carry only theHox-3.1 ^a andMyc-1.2 ^a alleles, as do the laboratory strains. One strain ofbrevirostris carries theHox-3.1 ^a andMyc-1.2 ^b alleles. Other wild subspecies from Europe and Asia,M. m. musculus, M. m. castaneus, M. m. molossinus, Chinese mice of wild origin, andM. m. yamashinai carry theHox-3.1 ^b andMyc-1.2 ^b alleles.     

Craniofacial abnormalities induced by ectopic expression of the homeobox gene Hox-1.1 in transgenic mice

Hox-1.1 is a murine homeobox-containing gene expressed in a time- and cell-specific manner during embryogenesis. We have generated transgenic mice that ectopically express Hox-1.1 from the chicken beta-actin promoter. In these mice Hox-1.1 expression was changed to an almost ubiquitous pattern. Ectopic expression of Hox-1.1 leads to death of the transgenic animals shortly after birth and is associated with multiple craniofacial anomalies, such as cleft palate, open eyes at birth, and nonfused pinnae. This phenotype is similar to the effects seen after systemic administration of retinoic acid during gestation. This suggests that retinoic acid embryopathy and the specific developmental defects caused by ectopic expression of a potential developmental control gene share a common pathogenic mechanism.     

An autoregulatory element of the murine Hox-4.2 gene.

Hox-4.2 promoter activity was assayed by transient expression assays in P19 embryonal carcinoma (EC) cells. Cotransfection of a luciferase reporter gene construct driven by Hox-4.2 upstream sequences with an expression vector for the Hox-4.2 gene product resulted in a 20-fold increase in luciferase activity. This activity was specific in that the Hox-1.6 gene product had no effect in the same assay. Mutational analysis defined a cis-acting element with enhancer function which conferred most of this increase. Activation was largely dependent on two TAAT/ATTA motifs within this 217 bp fragment and HOX-4.2 bound specifically to both of these motifs. The 217 bp element maps within a highly conserved region of the human Hox-4.2 gene (HOX4B) which has been shown to display spatial enhancer activity in mice and flies. These findings suggest a conserved autoregulatory mechanism for the control of Hox-4.2 expression.     

Sequence analysis of the murine Hox-2.2, -2.3, and -2.4 homeo boxes: evolutionary and structural comparisons

We have determined the nucleotide sequences and deduced the amino acid sequences of three tandemly arranged murine boxes of the Hox-2 homeo box gene complex on mouse chromosome 11 (Hox-2.2, -2.3, and -2.4). The type and position of differences with other sequenced homeo boxes were analyzed. Hox-2.2 is nearly identical with its cognate human homeo box Hu-2. Hox-2.3 shares 59 of 61 amino acids with the Antennapedia homeo domain of Drosophila and the MM-3 homeo domain of Xenopus and shows 60 of 61 amino acid identity with human HuC1. Hox-2.3, MM-3, and HuC1 also share a stretch of six glutamic acid residues followed by a stop codon 15-20 amino acids 3' of the homeo domain. Hox-2.4 is relatively divergent from most of the other homeo boxes sequenced to date; however, it matches the Hox-3.1 murine homeo domain at 60 of 61 positions. Sequence comparisons with other murine homeo domains, together with previous studies of their genomic organization and chromosomal location, provide support for the hypothesis of a large-scale duplication resulting in the two major murine homeo box gene complexes Hox-1 and Hox-2.