The murine Hox-2 genes display dynamic dorsoventral patterns of expression during central nervous system development.

This report demonstrates that the genes in the murine Hox-2 cluster display spatially and temporally dynamic patterns of expression in the transverse plane of the developing CNS. All of the Hox-2 genes exhibit changing patterns of expression that reflect events during the ontogeny of the CNS. The observed expression correlates with the timing and location of the birth of major classes of neurons in the spinal cord. Therefore, it is suggested that the Hox-2 genes act to confer rostrocaudal positional information on each successive class of newly born neurons. This analysis has also revealed a striking dorsal restriction in the patterns of Hox-2 expression in the spinal cord between 12.5 and 14.5 days of gestation, which does not appear to correlate with any morphological structure. The cellular retinol binding protein (CRBP) shows a complementary ventral staining pattern, suggesting that a number of genes are dorsoventrally restricted during the development of the CNS. The expression of Hox-2 genes has also been compared with the Hox-3.1 gene, which exhibits a markedly different dorsoventral pattern of expression. This suggests that, while genes in the different murine Hox clusters may have similar A-P domains of expression, they are responding to different dorsoventral patterning signals in the developing spinal cord.     

Human homeo box-containing genes located at chromosome regions 2q31----2q37 and 12q12----12q13

Four human homeo box-containing cDNAs isolated from mRNA of an SV40-transformed human fibroblast cell line have been regionally localized on the human gene map. One cDNA clone, c10, was found to be nearly identical to the previously mapped Hox-2.1 gene at 17q21. A second cDNA clone, c1, which is 87% homologous to Hox-2.2 at the nucleotide level but is distinct from Hox-2.1 and Hox-2.2, also maps to this region of human chromosome 17 and is probably another member of the Hox-2 cluster of homeo box-containing genes. The third cDNA clone, c8, in which the homeo box is approximately 84% homologous to the mouse Hox-1.1 homeo box region on mouse chromosome 6, maps to chromosome region 12q12----12q13, a region that is involved in chromosome abnormalities in human seminomas and teratomas. The fourth cDNA clone, c13, whose homeo box is approximately 73% homologous to the Hox-2.2 homeo box sequence, is located at chromosome region 2q31----q37. The human homeo box-containing cluster of genes at chromosome region 17q21 is the human cognate of the mouse homeo box-containing gene cluster on mouse chromosome 11. Other mouse homeo box-containing genes of the Antennapedia class (class I) map to mouse chromosomes 6 (Hox-1, proximal to the IgK locus) and 15 (Hox-3). A mouse gene, En-1, with an engrailed-like homeo box (class II) and flanking region maps to mouse chromosome 1 (near the dominant hemimelia gene). Neither of the class I homeo box-containing genes--c8 and c13--maps to a region of obvious homology to chromosomal positions of the presently known mouse homeo box-containing genes.     

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.     

Alcaligenes eutrophus hydrogenase genes (Hox)

Mutants of Alcaligenes eutrophus H16 lacking catalytically active soluble hydrogenase (Hos-) grew very slowly lithoautotrophically with hydrogen. Mutants devoid of particulate hydrogenase activity (Hop-) were not affected in growth with hydrogen. The use of Hos- and Hop- mutants as donors of hydrogen-oxidizing ability in crosses with plasmid-free recipients impaired in both hydrogenases (Hox-) resulted in transconjugants which had inherited the plasmid and the phenotype of the donor. This indicates that the structural genes which code for the hydrogenases reside on plasmid pHG1. The Hox function of one class of Hox- mutants could not be restored by conjugation. These mutants exhibited a pleiotropic phenotype since they were unable to grow with hydrogen and also failed to grow heterotrophically with nitrate (Hox- Nit-). Nitrate was scarcely utilized as electron acceptor or as nitrogen source. Hox- Nit- mutants did not act as recipients but could act as donors of the Hox character. Transconjugants derived from those crosses were Hox+ Nit+, indicating that the mutation which leads to the Hox- Nit- phenotype maps on the chromosome. Apparently, the product of a chromosomal gene is involved in the expression of plasmid-encoded Hox genes. We observed that the elimination of plasmid pHG1 coincided with the occurrence of multiple resistances to various antibiotics. Since Hox+ transconjugate retained the antibiotic-resistant phenotype, we conclude that this property is not directly plasmid associated.     

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.     

Identification of a retinoic acid response element upstream of the murine Hox-4.2 gene.

Hox genes play an important role in the process of vertebrate pattern formation, and their expression is intricately regulated both temporally and spatially. All-trans-retinoic acid (RA), a physiologically active metabolite of vitamin A, affects the expression of a large number of Hox genes in vitro and in vivo. However, the regulatory mechanisms underlying the RA response of these genes have not been extensively studied, and no response element for RA receptors (RARs) has been characterized in a Hox regulatory region. The expression of murine Hox-4.2 and its human homolog, HOX4B, is increased in embryonal carcinoma (EC) cell lines upon RA treatment (M. S. Featherstone, A. Baron, S. J. Gaunt, M.-G. Mattei, and D. Duboule, Proc. Natl. Acad. Sci. USA 85:4760-4764, 1988; A. Simeone, D. Acampora, V. Nigro, A. Faiella, M. D'Esposito, A. Stornaiuolo, F. Mavilio, and E. Boncinelli, Mech. Dev. 33:215-228, 1991). Using transient expression assays, we showed that luciferase reporter gene constructs carrying genomic sequences located upstream of Hox-4.2 responded to RA in murine P19 EC cells. A 402-bp NcoI fragment was necessary for the RA responsiveness of reporter constructs. This fragment contained a regulatory element, 5'-AGGTGA(N)5AGGTCA-3', that closely resembles the consensus sequence for an RA response element. The Hox-4.2 RA response element was critical for the RA induction and specifically bound RARs. In addition, the response to RA could be inhibited by expressing a dominant negative form of RAR alpha in transfected P19 EC cells. These results suggested that Hox-4.2 is a target for RAR-mediated regulation by RA.     

Primary structure and embryonic expression pattern of the mouse Hox-4.3 homeobox gene

We report the cloning, genomic localization, primary structure and developmental expression pattern of the novel mouse Hox-4.3 gene. This gene is located within the HOX-4(5) complex, at a position which classifies it as a member of the Hox-3.1 and -2.4 subfamily, the DNA and predicted protein sequences further confirmed this classification. Hox-4.3 has a primary structure characteristic of a Hox gene but, in addition, contains several monotonic stretches of amino acids, one of the 'paired'-like type. As expected from its presence and position within the complex. Hox-4.3 is developmentally expressed in structures of either mesodermal or neurectodermal origin located or derived from below a precise craniocaudal level. However, a very important offset between anteroposterior boundaries within neuroectoderm versus mesoderm derivatives is observed. Like other genes of the HOX-4(5) complex, Hox-4.3 is expressed in developing limbs and gonads, suggesting that 'cluster specificity' could be a feature of the HOX network.     

The murine genes Hox-5.1 and Hox-4.1 belong to the same HOX complex on chromosome 2

Two different loci of Antennapedia-related homeobox-containing genes have been shown to map to mouse chromosome 2: the HOX-5 complex and the Hox-4.1 gene. These independently derived loci are likely to be parts of a single gene complex, although their close linkage has not yet been demonstrated. Since cosmid walks to extend the HOX-5 cluster and to potentially link the two loci were unsuccessful, we have used large restriction fragments separated by pulsed-field gel electrophoresis to demonstrate the linkage between probes from the HOX-5 region and sequences near Hox-4.1. To further define the distance between the two linked loci, we screened a NotI jumping library with sequences near the Hox-5.1 gene to obtain a marker within the region predicted to contain Hox-4.1. The jumping endpoint lies within genomic clones from a lambda phage walk extending from the 5' end of Hox-4.1, and thus provides clear evidence of linkage between the two Hox loci. Our results demonstrate that Hox-4.1 lies approximately 35 kb downstream of the Hox-5.1 gene and that the two loci do indeed thus constitute parts of the same HOX complex.     

Isolation and analysis of embryonic expression of Hox-4.9, a member of the murine labial-like gene family.

Two members of the murine labial (lab) subfamily of Antennapedia-like homeobox-containing genes, Hox-1.6 and Hox-2.9, have been identified previously. Here we describe a third member genetically linked to the Hox-4 cluster on chromosome 2. This gene, designated Hox-4.9, is similar in structure to the other lab subfamily members. However, little coding sequence other than the homeobox and sequences immediately upstream of it have been conserved. By in situ hybridization analysis, Hox-4.9 mRNA is first detected at the end of the late streak stage (E7.75) in presumptive lateral and extraembryonic mesoderm. During early neurogenesis (E8.0-8.5), Hox-4.9 is detected solely in lateral mesoderm; its lack of expression in somitic mesoderm and the neural tube makes it unique among the Hox genes. By late neurogenesis and through mid-gestation (E9.0-E11.5), Hox-4.9 is no longer detected in lateral mesoderm but is found instead in a restricted region of presumed trunk neural crest and in the dermatome. These data are discussed in comparison with what is known about expression of the other members of the lab subfamily.