From the transcription of genes involved in ectodermal dysplasias to the understanding of associated dental anomalies.

Orodental anomalies are one aspect of rare diseases and are increasingly identified as diagnostic and predictive traits. To understand the rationale behind gene expression during tooth or other ectodermal derivative development and the disruption of odontogenesis or hair and salivary gland formation in human syndromes we analyzed the expression patterns of a set of genes (Irf6, Nfkbia, Ercc3, Evc2, Map2k1) involved in human ectodermal dysplasias in mouse by in situ hybridization. The expression patterns of Nfkbia, Ercc3 and Evc2 during odontogenesis had never been reported previously. All genes were indeed transcribed in different tissues/organs of ectodermal origin. However, for Nfkbia, Ercc3, Evc2, and Map2k1, signals were also present in the ectomesenchymal components of the tooth germs. These expression patterns were consistent in timing and localization with the known dental anomalies (tooth agenesis, microdontia, conical shape, enamel hypoplasia) encountered in syndromes resulting from mutations in those genes. They could also explain the similar orodental anomalies encountered in some of the corresponding mutant mouse models. Translational approaches in development and medicine are relevant to gain understanding of the molecular events underlying clinical manifestations.

Specific expression of the retinoic acid-synthesizing enzyme RALDH2 during mouse inner ear development.

Retinoid binding proteins and nuclear receptors are expressed in the developing mouse inner ear. Here, we report that the retinaldehyde dehydrogenase 2 (Raldh2) gene, whose product is involved in the enzymatic generation of retinoic acid (RA), exhibits a restricted expression pattern during mouse inner ear ontogenesis. The Raldh2 gene is first expressed at embryonic day (E) 10.5 in a V-shaped medio-dorsal region of the otocyst outer epithelium, which evolves as two separate domains upon otocyst morphogenesis. At E14.5, Raldh2 is expressed in two areas of the utricle epithelium and specific regions of the saccule and cochlear mesenchyme. Later, Raldh2 transcripts are restricted to two cochlear areas, the stria vascularis and Reissner membrane. Raldh2 mesenchymal expression did not correlate with migrating neural crest-derived melanoblasts. These restricted expression domains may correspond to specific sites of RA synthesis during inner ear morphogenesis.

A bidirectional promoter connects the poly(ADP-ribose) polymerase 2 (PARP-2) gene to the gene for RNase P RNA. structure and expression of the mouse PARP-2 gene.

Poly(ADP-ribose) polymerase 2 (PARP-2) is a DNA damage-dependent enzyme that belongs to a growing family of enzymes seemingly involved in genome protection. To gain insight into the physiological role of PARP-2 and to investigate mechanisms of PARP-2 gene regulation, we cloned and characterized the murine PARP-2 gene. The PARP-2 gene consists of 16 exons and 15 introns spanning about 13 kilobase pairs. Interestingly, the PARP-2 gene lies head to head with the gene encoding the mouse RNase P RNA subunit. The distance between the transcription start sites of the PARP-2 and RNase P RNA genes is 114 base pairs. This suggested that regulation of the expression of both genes may be coordinated through a bi-directional promoter. The PARP-2/RNase P RNA gene organization is conserved in the human. To our knowledge, this is the first report of a RNA polymerase II gene and an RNA polymerase III gene sharing the same promoter region and potentially the same transcriptional control elements. Reporter gene constructs showed that the 113-base pair intergenic region was indeed sufficient for the expression of both genes and revealed the importance of both the TATA and the DSE/Oct-1 expression control elements for the PARP-2 gene transcription. The expression of both genes is clearly independently regulated. PARP-2 is expressed only in certain tissues, and RNase P RNA is expressed in all tissues. This suggests that both genes may be subjected to multiple levels of control and may be regulated by different factors in different cellular contexts.

Expression of enzymes synthesizing (aldehyde dehydrogenase 1 and reinaldehyde dehydrogenase 2) and metabolizaing (Cyp26) retinoic acid in the mouse female reproductive system.

Vitamin A is required for female reproduction. Rodent uterine cells are able to synthesize retinoic acid (RA), the active vitamin A derivative, and express RA receptors. Here, we report that two RA-synthesizing enzymes [aldehyde dehydrogenase 1 (Aldh1) and retinaldehyde dehydrogenase 2 (Raldh2)] and a cytochrome P450 (Cyp26) that metabolizes vitamin A and RA into more polar metabolites exhibit dynamic expression patterns in the mouse uterus, both during the ovarian cycle and during early pregnancy. Aldh1 expression is up-regulated during diestrus and proestrus in the uterine glands, whereas Raldh2 is highly induced in the endometrial stroma in metestrus. Cyp26 expression, which is not detectable during the normal ovarian cycle, is strongly induced in the uterine luminal epithelium, 24 h after human CG hormonal administration. Raldh2 stromal expression also strongly responds to gonadotropin (PMSG and human CG) induction. Furthermore, Raldh2 expression can be hormonally induced in stromal cells of the vagina and cervix. All three enzymes exhibit differential expression profiles during early pregnancy. Aldh1 glandular expression is sharply induced at 2.5 gestational days, whereas Raldh2 stromal expression increases more steadily until the implantation phase. Cyp26 epithelial expression is strongly induced between 3.5-4.5 gestational days, i.e. when the developing blastocysts colonize the uterine lumen. These data suggest a need for precise regulation of RA synthesis and/or metabolism, in both cycling and pregnant uterus.

Gene dosage-dependent effects of the Hoxa-13 and Hoxd-13 mutations on morphogenesis of the terminal parts of the digestive and urogenital tracts.

Gene targeting experiments have shown that the murine Hoxa-13 and Hoxd-13 paralogous genes control skeletal patterning in the distal region of the developing limbs. However, both genes are also expressed in the terminal part of the digestive and urogenital tracts during embryogenesis and postnatal development. Here, we report the abnormalities occuring in these systems in Hoxa-13(-/-) and Hoxa-13/Hoxd-13 compound mutant mice. Hoxa-13(-/-) mutant fetuses show agenesis of the caudal portion of the Müllerian ducts, lack of development of the presumptive urinary bladder and premature stenosis of the umbilical arteries, which could account for the lethality of this mutation at mid-gestational stages. Due to such lethality, only Hoxa-13(+/-)/Hoxd-13(-/-) compound mutants can reach adulthood. These compound mutants display: (i) agenesis or hypoplasia of some of the male accessory sex glands, (ii) malpositioning of the vaginal, urethral and anal openings, and improper separation of the vagina from the urogenital sinus, (iii) hydronephrosis and (iv) anomalies of the muscular and epithelial layers of the rectum. Thus, Hoxa-13 and Hoxd-13 play important roles in the morphogenesis of the terminal part of the gut and urogenital tract. While Hoxa-13(-/-)/Hoxd-13(+/-) fetuses show severely impaired development of the urogenital sinus, double null (Hoxa-13[-/-]/Hoxd-13[-/-]) fetuses display no separation of the terminal (cloacal) hindgut cavity into a urogenital sinus and presumptive rectum, and no development of the genital bud, thereby demonstrating that both genes act, in a partly redundant manner, during early morphogenesis of posterior trunk structures.

Functional cooperation between the non-paralogous genes Hoxa-10 and Hoxd-11 in the developing forelimb and axial skeleton.

The Abdominal B-related Hoxa-10 gene displays similar expression patterns in the differentiating forelimbs and hindlimbs of the mouse, with preferential expression around the humeral and femoral cartilages and more diffuse expression in distal regions. We found that a targeted disruption of Hoxa-10 has almost no effect in the forelimbs, while it affects the proximal hindlimb skeleton. The alterations were located along the dorsolateral side of the femur (labium laterale), with an enlargement and distal shift of the third trochanter, a misshapen lateral knee sesamoid, a supernumerary 'ligament' connecting these structures and an occasional duplication of the femoral trochlea. Some Hoxa-10-/- mutant mice developed severe degenerative alterations of the knee articulation upon ageing. Viable Hoxa-10/Hoxd-11 double mutant mice were produced by genetic intercrosses. The compound mutation resulted in synergistic forelimb phenotypic alterations, consisting of: (i) an exacerbation of Hoxd-11-/- phenotypic traits in the carpal and digital region, e.g. more pronounced truncations of the ulna styloid, pyramidal and pisiform bones and of some metacarpal and phalangeal bones and (ii) marked alterations in a more proximal region which is nearly unaffected in Hoxd-11-/- single mutants; the entire radius and ulna were truncated and thickened, with deformations of the ulna proximal extremity. Thus, functional redundancy can occur even between non-paralogous Abdominal B-related Hox genes. The double Hoxa-10/Hoxd-11 mutation also conferred full penetrance to the sacral and caudal vertebrae transformations which are approximately 50% penetrant in Hoxd-11-/- single mutants, revealing that functional cooperation can also occur between non-paralogous Hox gene products in axial skeleton patterning.

Insertion of a targeting construct in a Hoxd-10 allele can influence the control of Hoxd-9 expression.

A neomycin resistance (neo) gene driven by the phosphoglycerokinase (PGK) promoter was inserted into the Hoxd-10 homeobox by homologous recombination in embryonic stem (ES) cells. Chimeric mice derived from ES cell-injected blastocysts died shortly after birth. Craniofacial and axial abnormalities were found in the skeleton of these chimeras, resembling some of the previously described Hox gene gain-of-function phenotypes. The spatial expression patterns of various Hoxd gene transcripts were analysed in chimeric mutant embryos by in situ hybridization. Two main observations were made: (1) a wide ectopic expression domain of the Hoxd-9 gene was found in the spinal cord of these embryos, and (2) the neo gene exhibited a specific Hox-like expression domain which extended far more rostrally than that of the Hoxd-10 gene, showing that, in the context of this mutation, the PGK promoter could be regulated as a Hox promoter. These results provide the first evidence that a targeted insertion into a Hox gene coding sequence, in the context of its own cluster, could result in misexpression of a neighbour gene of the complex.

Developmental expression of murine retinoid X receptor (RXR) genes.

The developmental expression patterns of the three mouse retinoid X receptor genes (RXR alpha, beta and gamma) have been investigated by Northern blotting and in situ analysis of RNA transcript distribution, and compared to those of retinoic acid receptor (RAR) genes. RXR beta showed a diffuse and probably ubiquitous expression pattern at all developmental stages studied. RXR alpha also exhibited a diffuse expression at early developmental stages, but an enhanced in situ labelling was observed during late development in the epidermis and several other squamous epithelia. By contrast, RXR gamma apparently displayed a restricted expression in the myogenic lineage, i.e. in myotomes and subsequently in various differentiating muscles including those of the face and limbs. Apparently RXR gamma was not co-expressed with RAR beta and RAR gamma in these domains. RXR gamma transcripts were developmentally regulated in the otic epithelium, the retina, the pituitary and thyroid glands. In addition, RXR gamma was expressed in several discrete areas of the fetal central nervous system, namely in the diencephalon, the striatum and in part of the ventral horns of the spinal cord. In the two latter domains, there was a precise co-expression with RAR beta transcripts, although with quantitative differences, which suggests a possible preferential heterodimerization between these two retinoic acid receptors in the developing central nervous system.

Developmental expression of the mouse Evx-2 gene: relationship with the evolution of the HOM/Hox complex.

The mouse Evx-2 gene is located in the immediate vicinity of the Hoxd-13 gene, the most posteriorly expressed gene of the HOXD complex. While the Evx-1 gene is also physically linked to the HOXA complex, it is more distantly located from the corresponding Hoxa-13 gene. We have analysed the expression of Evx-2 during development and compared it to that of Evx-1 and Hoxd-13. We show that, even though Evx-2 is expressed in the developing CNS in a pattern resembling that of other Evx-related genes, the overall expression profile is similar to that of the neighbouring limbs and genitalia. We propose that the acquisition of expression features typical of Hox genes, together with the disappearance of some expression traits common to Evx genes, is due to the close physical linkage of Evx-2 to the HOXD complex, which results in Evx-2 expression being partly controlled by mechanisms acting in the HOX complex. This transposition of the Evx-2 gene next to the Hoxd-13 gene may have occurred soon after the large scale duplications of the HOX complexes. A scheme is proposed to account for the functional evolution of eve-related genes in the context of their linkage to the HOM/Hox complexes.