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.     

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.     

Cell-surface changes induced by ectopic expression of the murine homeobox gene Hox-3.3.

Murine homeobox-containing genes (Hox genes) are postulated as playing key roles in the establishment of the anterior-posterior embryonic body axis, possibly providing cells with positional cues. Little is known, however, concerning how cells might respond to homeobox gene expression to interpret these cues. Since changes in the cell-surface are central to many processes in early development we reasoned that cells expressing different complements of Hox genes might have different surface properties. In order to investigate this we have used the sensitive, non-disruptive technique of multiple two-phase aqueous partition, which is able to detect small differences on the surface of intact cells. Using this technique we have found that ectopic expression of the murine Hox-3.3 gene in cultured cells induces reproducible changes in the cell surface. Changes only occurred above a threshold level of gene expression, but above this level a correlation between surface change and gene expression was seen. The implications for the establishment of a 'Hox' code of homeobox genes acting to specifically change cell-surface properties are discussed.     

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 homeobox-containing exon of the murine Hox-4.3 homeogene.

A homeobox-containing gene * was detected by Southern analysis of a cosmid spanning a region of the murine HOX-4 complex between Hox-4.4 (Hox-5.2) and Hox-4.2 (Hox-5.1) with a probe derived from the Hox-4.2 homeobox. The sequence of a cross-hybridizing region revealed an open reading frame encoding an Antennapedia (Antp) class homeodomain highly homologous to the products of human HOX4C (Hox-5.4/HOX4E), mouse Hox-3.1 and Hox-2.4. This, together with strong conservation of sequences 3' to the homoebox, indicates that we have cloned the murine Hox-4.3 gene. No other homeobox sequences were detected in this screen suggesting that the HOX-4 complex lacks paralogous genes represented in the equivalent regions of other HOX loci.     

Chromosome assignment of the murine Hox-4.1 gene

The murine homebox gene 4.1 was assigned to chromosome 2 by Southern analysis of somatic cell hybrids and by in situ hybridization. This assignment and the report of Featherstone et al. (M. S. Featherstone, A. Baron, S. J. Gaunt, M. G. Mattei, and D. Duboule, 1988, Proc. Natl. Acad. Sci. USA, 85, 4760-4764) indicate that a fourth group of homeobox genes is located on chromosome 2 in the mouse (in addition to the homeobox gene clusters on chromosomes 6, 11, and 15).     

Dominant mutation of the murine Hox-2.2 gene results in developmental abnormalities.

Genes carrying the homeobox were originally identified in Drosophila, in which they are now known to play key roles in establishing segmentation patterns and in determining segment identities. A number of genes with striking homology to the Drosophila homeobox genes have now been found in the mouse genome, and mutational analysis is beginning to shed light on their function in mammalian development. To understand better the developmental significance of the murine Hox-2.2 gene, we have generated gain of function mutants by using the chicken beta-actin promoter to drive ubiquitous expression in transgenic mice. The resulting Hox-2.2 misexpression produces early postnatal lethality as well as craniofacial and axial skeletal perturbations that include open eyes at birth, cleft palate, micrognathia, microtia, skull bone deficiencies, and structural and positional alterations in the vertebral column. We repeatedly observe complete or partial absence of the supraoccipital bone and malformations of the exoccipital and the basioccipital bones. These results suggests a role for the Hox-2.2 gene in specifying positional identity along the anterior-posterior axis.     

Sequence of a novel chicken genomic DNA fragment that hybridizes to the murine Hox-3.1 homeobox.

A chicken genomic library was screened for novel homeobox-like elements. We describe a chicken genomic DNA fragment which hybridizes to the murine Hox-3.1 homeobox. The fragment is a single-copy sequence which seems to be transcribed without spacial or temporal restrictions. The sequence presented here is not representative of anything yet in the databases; however, it contains a very probable open reading frame which would encode a peptide slightly homologous to the Hox-3.1 protein.     

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.     

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.     

Differentiation of murine embryonic stem cells induces progesterone receptor gene expression

The role of steroid hormone receptors in very early embryonic development remains unknown. Clearly, expression during organogenesis is important for tissue-specific development. However, progesterone receptor (PR) and estrogen receptors (ERalpha, ERbeta) are expressed during early development through the blastocyst stage in mice and other species, and yet are not essential for embryonic viability. We have utilized the mouse embryonic stem (mES) cell model to investigate the regulated expression of these receptors during differentiation. Surprisingly, one of the earliest changes in gene expression in response to a differentiation signal observed is PR gene induction. It parallels the time course of expression for the patterning genes Hoxb1 and Hoxa5. Unexpectedly, PR gene expression is not regulated in an estrogen-dependent manner by endogenous ERs or by transiently overexpressed ERalpha. Our results suggest a potentially novel mechanism of PR gene regulation within mES cells compared to adult tissues and the possibility of unique targets of PR action during early mES cell differentiation.