Spatially restricted patterns of expression of the homeobox-containing gene Hox 2.1. during mouse embryogenesis

The mouse Hox 2.1 gene contains a homeobox sequence and is therefore a candidate for a vertebrate gene involved in the control of embryonic patterning or positional specification. To investigate this possibility, we have used in situ hybridization to determine the pattern of Hox 2.1 expression during mouse embryogenesis. At 8.5 days post coitum, Hox 2.1 is expressed at a low level in the posterior neuroectoderm and mesoderm, and in the neuroectoderm of the presumptive hindbrain. At 12.5 days p.c., Hox 2.1 is expressed in an anteroposterior restricted domain extending from the hindbrain throughout the length of the spinal cord, predominantly in the dorsal region. Between 12.5 and 13.5 days p.c. the domain becomes localized to the occipital and cervical regions. We also detect Hox 2.1 RNA in the embryonic lung, stomach, mesonephros and metanephros, as well as in myenteric plexus, dorsal root ganglia and the nodose ganglion, and in mature granulocytes. The embryonic expression of Hox 2.1 in neural tissue is compared with that of Hox 3.1, which also shows anteroposterior restricted domains of gene expression. These patterns of expression are not clearly consistent with Hox 2.1 or Hox 3.1 having roles in segmental patterning. However, the data are consistent with these genes having regulatory roles in anteroposterior positional specification in the neuroectoderm and mesoderm, and suggest that Hox 2.1 may also have functions during organogenesis.     

A regulatory region from the mouse Hox-2.2 promoter directs gene expression into developing limbs

To characterize cis-acting regulatory elements of the murine homeobox gene, Hox-2.2, transgenic mouse lines were generated that contained the LacZ reporter gene under the control of different fragments from the presumptive Hox-2.2 promoter. A promoter region of 3600 base pairs (bp) was identified, which reproducibly directed reporter gene expression into specific regions of developing mouse embryos. At 8.5 days postcoitum (p.c.) reporter gene activity was detected in posterior regions of the lateral mesoderm and, in subsequent developmental stages, expression of the LacZ gene was restricted to specific regions of the developing limb buds and the mesenchyme of the ventrolateral body region. This pattern of Hox-2.2-LacZ expression was found in all transgenic embryos that have been generated with the 3.6 kb promoter fragment (two founder embryos and embryos from five transgenic lines). In addition, embryos from two transgenic mouse lines expressed the reporter gene at low levels in the developing central nervous system (CNS). Our results are consistent with the idea that in addition to their presumptive role in CNS and vertebrae development, Hox-2.2 gene products are involved in controlling pattern formation in developing limbs.     

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.     

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.     

The mouse Dlx-2 (Tes-1) gene is expressed in spatially restricted domains of the forebrain, face and limbs in midgestation mouse embryos

The pattern of RNA expression of the murine Dlx-2 (Tes-1) homeobox gene is described in embryos ranging in age from E8.5 through E11.5. Dlx-2 is a vertebrate homologue of the Drosophila Distal-less (Dll) gene. Dll expression in the Drosophila embryo is principally limited to the primordia of the brain, head and limbs. Dlx-2 is also expressed principally in the primordia of the forebrain, head and limbs. Within these regions it is expressed in spatially restricted domains. These include two discontinuous regions of the forebrain (basal telencephalon and ventral diencephalon), the branchial arches, facial ectoderm, cranial ganglia and limb ectoderm. Several mouse and human disorders have phenotypes which potentially are the result of mutations in the Dlx genes.     

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.     

Mice doubly deficient for the Polycomb Group genes Mel18 and Bmi1 reveal synergy and requirement for maintenance but not initiation of Hox gene expression

Polycomb group genes were identified as a conserved group of genes whose products are required in multimeric complexes to maintain spatially restricted expression of Hox cluster genes. Unlike in Drosophila, in mammals Polycomb group (PcG) genes are represented as highly related gene pairs, indicative of duplication during metazoan evolution. Mel18 and Bmi1 are mammalian homologs of Drosophila Posterior sex combs. Mice deficient for Mel18 or Bmi1 exhibit similar posterior transformations of the axial skeleton and display severe immune deficiency, suggesting that their gene products act on overlapping pathways/target genes. However unique phenotypes upon loss of either Mel18 or Bmi1 are also observed. We show using embryos doubly deficient for Mel18 and Bmi1 that Mel18 and Bmi1 act in synergy and in a dose-dependent and cell type-specific manner to repress Hox cluster genes and mediate cell survival of embryos during development. In addition, we demonstrate that Mel18 and Bmi1, although essential for maintenance of the appropriate expression domains of Hox cluster genes, are not required for the initial establishment of Hox gene expression. Furthermore, we show an unexpected requirement for Mel18 and Bmi1 gene products to maintain stable expression of Hox cluster genes in regions caudal to the prospective anterior expression boundaries during subsequent development.     

Regulation of Hoxb3 expression in the hindbrain and pharyngeal arches by rae28, a member of the mammalian Polycomb group of genes

During animal development, Hox genes are expressed in characteristic, spatially restricted patterns and specify regional identities along the anterior-posterior (A-P) axis. Polycomb group (PcG) proteins in Drosophila repress Hox expression and maintain the expression patterns during development. Mice deficient for homologues of the Drosophila PcG genes, such as M33, bmi1, mel18, rae28 and eed, show altered Hox expression patterns. In this study, we examined the time course of Hoxb3 expression during late gastrulation and early segmentation of rae28-deficient mice. Hoxb3 was expressed ectopically in pharyngeal arch and hindbrain from embryonic day (E) 9.5 and 10.5, respectively. The anterior boundary of ectopic expression in the hindbrain extended gradually in the rostral direction as development proceeded from E10.5 to E12.5. Expression of kreisler and Krox20, which function as positive regulators of Hoxb3 expression, was not affected in rae28-deficient embryos. Analysis of a neural crest marker, p75, in rae28-deficient mice revealed that the neural crest cells begin to ectopically express Hoxb3 after leaving the hindbrain. Our results suggest that rae28 is not required for the establishment but maintenance of Hoxb3 expression.     

Expression of transforming growth factor beta 2 RNA during murine embryogenesis

We have studied the temporal and spatial expression of transforming growth factor beta 2 (TGF beta 2) RNA in mouse embryos from 10.5 days post coitum (p.c.) to 3 days post partum (p.p.) by in situ hybridization analysis. TGF beta 2 RNA is expressed in a variety of tissues including bone, cartilage, tendon, gut, blood vessels, skin and fetal placenta, and is in general found in the mesenchymal component of these tissues. The expression of TGF beta 2 RNA changes during development in a manner consistent with a role for the gene product in mediating mesenchymal-epithelial interactions.     

The homeobox gene Hox 7.1 has specific regional and temporal expression patterns during early murine craniofacial embryogenesis, especially tooth development in vivo and in vitro.

Hox 7.1 is a murine homeobox-containing gene expressed in a range of neural-crest-derived tissues and areas of putative epithelial-mesenchymal interactions during embryogenesis. We have examined the expression of Hox 7.1 during craniofacial development in the mouse embryo between days 8 and 16 of development. Whereas facial expression at day 10 of gestation is broadly localised in the neural-crest-derived mesenchyme of the medial nasal, lateral nasal, maxillary and mandibular processes, by day 12 expression is restricted to the mesenchyme immediately surrounding the developing tooth germs in the maxillary and mandibular processes. Hox 7.1 expression in the mesenchyme of the dental papilla and follicle is maximal at the cap stage of development and progressively declines in the bell stage prior to differentiation of odontoblasts and ameloblasts. Hox 7.1 expression in tooth germs is independent of overall embryonic stage of development but is dependent on stage of development of the individual tooth. Similar patterns of transient Hox 7.1 expression can also be detected in tooth germs in vitro in organ cultures of day 11 first branchial arch explants cultured for up to 7 days. Hox 7.1 is also expressed early in development (days 10/11) in the epithelium of the developing anterior pituitary (Rathke's pouch), the connective tissue capsule and meninges of the developing brain, and specific regions of neuroepithelium in the developing brain.     

Desmin and titin expression in early postimplantation mouse embryos

The expression of the intermediate filament (IF) constituents desmin, vimentin and keratin, as well as the striated-muscle-specific marker titin, was studied in mouse embryos of 8.0 to 9.5 days post coitum (d.p.c.), using the indirect immunofluorescence technique in combination with polyclonal and monoclonal antibodies. During the development of the embryo, desmin was first detected at 8.25 d.p.c. in the ectoderm, where it was transiently coexpressed with keratin and vimentin. At later stages, the ectoderm contained only keratin and to a certain extent also vimentin IF. At 8.5 d.p.c., desmin was found exclusively in the heart rudiment, and remained present with increasing intensity in the myocardial cells during later cardiogenesis. Striation of desmin in the heart muscle cells was observed in 9.5 d.p.c. embryos. At these stages (8.5-9.5 d.p.c.), triple expression of the IF proteins desmin, vimentin and keratin was evident in these cells. From 9.0 d.p.c. onwards, desmin could be detected in the myotomes as well. Immunoblotting studies of 9.5 d.p.c. mouse embryos confirmed the immunohistochemical data. Titin was found in the early heart anlage at stage 8.25 d.p.c., when no desmin expression was observed in this tissue. At this stage the titin appeared in a punctate pattern, similar to that observed in cardiac myofibrils of early chicken embryos (Tokuyasu and Maher, 1987; J. Cell Biol. 105, 2781-2793). In 8.5 d.p.c. mouse embryos, this punctate titin staining pattern was still observed, while, at this stage, a filamentous staining reaction could be seen with the desmin antibodies. During further development, cross-striation was detected within myocardial cells using the polyclonal titin antibody from 9.0 d.p.c. onwards, i.e. before such striation could be detected with the desmin antibodies. From these data, we conclude that titin synthesis may anticipate desmin expression in the developing mouse myocard, although the level of expression of the former protein remains low until 9.0 d.p.c.