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.

Expression patterns of the Ets transcription factors from the PEA3 group during early stages of mouse development.

erm, er81 and pea3 are three related genes that define a novel Ets-related subfamily of transcription factors. The expression patterns of these genes has been previously characterized in the mouse from embryonic day (E) 9.5 to birth (Oncogene 15 (1997) 937). In this study, we report differential expression patterns of the PEA3 group genes during early mouse post-implantation development. erm and pea3 expression patterns were partly overlapping. erm was activated prior to pea3 in the distal tip of the embryonic epiblast but, at primitive streak-stages, both genes were coexpressed in the posterior region of the embryo in epiblast, primitive streak and adjacent mesoderm. Similar erm and pea3 expression patterns were seen later in posterior neural plate, presomitic and lateral mesoderm, mesonephros, and tail bud. Only erm, however, was expressed in specific brain regions corresponding to prospective midbrain and ventral forebrain. erm was also strongly expressed as early as E8 in the developing branchial region, especially at the level of branchial pouches, whereas pea3 transcripts appeared later in frontonasal and first arch mesenchyme. er81 transcripts were not detected prior to E9.0-9.5, suggesting that this gene may not be involved in early developmental events.

Net, an Ets ternary complex transcription factor, is expressed in sites of vasculogenesis, angiogenesis, and chondrogenesis during mouse development.

The Net gene encodes an Ets transcription factor belonging to the ternary complex factor subfamily. We studied Net expression during mouse development (E7.5-E18.5) by in situ hybridization. Net is expressed at E7.5-8.5 in developing vascular primordia, including the allantoic vessels, heart endocardium and dorsal aortae. Vascular endothelial cell expression persists throughout development. Additional sites of expression appear at E9.5-E10.5, especially in facial, branchial arch and distal limb-bud mesenchyme. Later, expression is most conspicuous in developing cartilage and becomes progressively restricted to perichondrium. Net expression during mouse development correlates with vasculogenesis, angiogenesis and cartilage ontogeny.

Inactivation of the Friedreich ataxia mouse gene leads to early embryonic lethality without iron accumulation.

Friedreich ataxia (FRDA), the most common autosomal recessive ataxia, is caused in almost all cases by homozygous intronic expansions resulting in the loss of frataxin, a mitochondrial protein conserved through evolution, and involved in mitochondrial iron homeostasis. Yeast knockout models, and histological and biochemical data from patient heart biopsies or autopsies indicate that the frataxin defect causes a specific iron-sulfur protein deficiency and mitochondrial iron accumulation leading to the pathological changes. Affected human tissues are rarely available to further examine this hypothesis. To study the mechanism of the disease, we generated a mouse model by deletion of exon 4 leading to inactivation of the Frda gene product. We show that homozygous deletions cause embryonic lethality a few days after implantation, demonstrating an important role for frataxin during early development. These results suggest that the milder phenotype in humans is due to residual frataxin expression associated with the expansion mutations. Surprisingly, in the frataxin knockout mouse, no iron accumulation was observed during embryonic resorption, suggesting that cell death could be due to a mechanism independent of iron accumulation.

Retinoic acid synthesis and hindbrain patterning in the mouse embryo.

Targeted disruption of the murine retinaldehyde dehydrogenase 2 (Raldh2) gene precludes embryonic retinoic acid (RA) synthesis, leading to midgestational lethality (Niederreither, K., Subbarayan, V., Dolle, P. and Chambon, P. (1999). Nature Genet. 21, 444-448). We describe here the effects of this RA deficiency on the development of the hindbrain and associated neural crest. Morphological segmentation is impaired throughout the hindbrain of Raldh2-/- embryos, but its caudal portion becomes preferentially reduced in size during development. Specification of the midbrain region and of the rostralmost rhombomeres is apparently normal in the absence of RA synthesis. In contrast, marked alterations are seen throughout the caudal hindbrain of mutant embryos. Instead of being expressed in two alternate rhombomeres (r3 and r5), Krox20 is expressed in a single broad domain, correlating with an abnormal expansion of the r2-r3 marker Meis2. Instead of forming a defined r4, Hoxb1- and Wnt8A-expressing cells are scattered throughout the caudal hindbrain, whereas r5/r8 markers such as kreisler or group 3/4 Hox genes are undetectable or markedly downregulated. Lack of alternate Eph receptor gene expression could explain the failure to establish rhombomere boundaries. Increased apoptosis and altered migratory pathways of the posterior rhombencephalic neural crest cells are associated with impaired branchial arch morphogenesis in mutant embryos. We conclude that RA produced by the embryo is required to generate posterior cell fates in the developing mouse hindbrain, its absence leading to an abnormal r3 (and, to a lesser extent, r4) identity of the caudal hindbrain cells.

Embryonic retinoic acid synthesis is essential for early mouse post-implantation development.

A number of studies have suggested that the active derivative of vitamin A, retinoic acid (RA), may be important for early development of mammalian embryos. Severe vitamin A deprivation in rodents results in maternal infertility, precluding a thorough investigation of the role of RA during embryogenesis. Here we show that production of RA by the retinaldehyde dehydrogenase-2 (Raldh2) enzyme is required for mouse embryo survival and early morphogenesis. Raldh2 is an NAD-dependent aldehyde dehydrogenase with high substrate specificity for retinaldehyde. Its pattern of expression during mouse development has suggested that it may be responsible for embryonic RA synthesis. We generated a targeted disruption of the mouse Raldh2 gene and found that Raldh2-/- embryos, which die at midgestation without undergoing axial rotation (body turning), exhibit shortening along the anterioposterior axis and do not form limb buds. Their heart consists of a single, medial, dilated cavity. Their frontonasal region is truncated and their otocysts are severely reduced. These defects result from a block in embryonic RA synthesis, as shown by the lack of activity of RA-responsive transgenes, the altered expression of an RA-target homeobox gene and the near full rescue of the mutant phenotype by maternal RA administration. Our data establish that RA synthesized by the post-implantation mammalian embryo is an essential developmental hormone whose lack leads to early embryo death.

Expression of T:G mismatch-specific thymidine-DNA glycosylase and DNA methyl transferase genes during development and tumorigenesis.

In situ hybridization was used to characterize the expression pattern of the T:G mismatch-specific thymidine-DNA glycosylase (TDG) gene, encoding a DNA repair enzyme which corrects G:T mismatches that result from the hydrolytic deamination of 5-methyl cytosines. TDG transcripts were uniformly and ubiquitously expressed from 7.5-13.5 days post-coitum, but were then markedly enriched in specific tissues of the developing fetus. At 14.5 gestational days, TDG was strongly expressed in the developing nervous system, thymus, lung, liver, kidney and intestine. At later stages, high levels of expression were detected in the thymus, brain, nasal epithelium and within proliferating regions of the intestine, skin, kidney, teeth and bone. This pattern of expression strongly correlated with those of the methyl transferase (MTase) gene, coding for the enzyme which specifically methylates CpG dinucleotides, and the p53 tumour suppressor gene. However, TDG and MTase were differentially expressed during maturation of the male and female germline. We also report that tumors occuring in mice which overexpress MMTV-v-Ha-ras or MMTV-c-myc transgenes or mice heterozygous for p53 gene disruption, all show elevated TDG and MTase expression specific to the transformed tissue.

Stra3/lefty, a retinoic acid-inducible novel member of the transforming growth factor-beta superfamily.

We report the structure, chromosomal localization and expression features of Stra3, a novel mouse gene whose expression is upregulated by retinoic acid in P19 embryonal carcinoma cells. The Stra3 cDNA sequence, which encodes a novel member of the TGF-beta superfamily, corresponds to, but extends more 3' than the recently published lefty sequence (Meno et al., 1996, Nature 381:151-155). The Stra3/lefty protein, which exhibits characteristics of secreted proteins, is synthesized as a precursor of 42 kDa and secreted after cleavage, suggesting that it may function as an intercellular signaling molecule. There are four exons in the Stra3/lefty gene and its 5' flanking region contains, among other putative regulatory elements, an unusual retinoid response element consisting of two half sites arranged as a palindrome, with an 8 base pairs spacer. We also show that Stra3/lefty is ectopically induced in the endodermal and ectodermal layers following in vivo administration of retinoic acid to gastrulating mouse embryos.

Defects of the chorioallantoic placenta in mouse RXRalpha null fetuses.

The active derivatives of vitamin A (the retinoids) play important and multiple roles in mammalian development and homeostasis. We have previously shown that specific retinoic acid receptors are expressed in the chorioallantoic placenta of the mouse and that among these, RXRalpha is strongly expressed in the developing labyrinthine zone (Sapin, V., Ward, S. J., Bronner, S., Chambon, P., Dollé, P., Dev. Dyn. 208, 199-210, 1997). Here, we show that mouse fetuses with a targeted disruption of the RXRalpha gene develop defects of the chorioallantoic placenta. Both morphological abnormalities and alterations in the expression of molecular markers were found, mostly confined to the labyrinthine zone of placentas from mid-late gestation mutants. This region exhibited edema, abnormal stasis of maternal blood, and signs of disruption of the endothelial layer of fetal vessels. We also detected a reduction in the number of lipid droplets in the trophoblastic layer and abnormal fibrin deposits in the junctional zone of the mutant placentas. These abnormalities most probably result in an impairment of the functional capacities of exchange between the maternal and fetal circulations in the mutant placentas. Thus, placental defects could represent an extraembryonic cause of lethality for RXRalpha null mutant fetuses, in addition to the previously described embryonic cardiac defects.

Meis2, a novel mouse Pbx-related homeobox gene induced by retinoic acid during differentiation of P19 embryonal carcinoma cells.

We report the cDNA cloning, partial genomic organization, and expression pattern of Stra10, a novel retinoic acid-inducible gene in P19 embryonal carcinoma cells. Four murine cDNA isoforms have been isolated, which are likely to result from alternative splicing. The predicted protein sequences exhibit approximately 85% identity with the Pbx-related Meis1 homeobox gene products, which are involved in myeloid leukemia in BXH-2 mice, and one of the Stra10 isoforms corresponds to the recently published Meis2 sequence (Nakamura et al. [1996] Oncogene 13:2235-2242). The Meis2 homeodomain is identical to that of Meis1, and is most closely related to those of the Pbx/TGIF homeobox gene products. By in situ hybridization analysis, we show that the Meis2 gene displays spatially restricted expression patterns in the developing nervous system, limbs, face, and in various viscera. In adult mice, Meis2 is mainly expressed in the brain and female genital tract, with a different distribution of the alternative splice forms in these organs.

Overexpression of Stra13, a novel retinoic acid-inducible gene of the basic helix-loop-helix family, inhibits mesodermal and promotes neuronal differentiation of P19 cells.

We report the cDNA cloning of Stra13, a novel retinoic acid (RA)-inducible gene from P19 embryonal carcinoma cells that encodes a basic helix-loop-helix (bHLH) protein that shows the highest sequence similarities to the Drosophila Hairy and Enhancer of split and mouse Hes proteins. Stra13 does not bind to the known consensus motifs (E-box and N-box) for bHLH proteins, but can repress activated transcription (through an alpha-helix rich domain) in part by interaction with general factors of the basal transcription machinery. During mouse embryogenesis, Stra13 RNA is expressed in the neuroectoderm, and also in a number of mesodermal and endodermal derivatives. Remarkably, overexpression of Stra13 in P19 cells results in neuronal differentiation in monolayer culture, under conditions where wild-type P19 cells only undergo mesodermal/endodermal differentiation. This neuronal differentiation is accompanied by an altered expression of mesodermal and neuronal markers, indicating that Stra13 could be one of the earliest RA target genes whose expression is required for repression of mesodermal/endodermal differentiation and/or induction of neuronal differentiation when P19 cell aggregates are exposed to RA. Our results raise the possibility that Stra13 could be involved as a repressor in a number of decision events occurring during differentiation of various cell lineages.

Developmental expression pattern of Stra6, a retinoic acid-responsive gene encoding a new type of membrane protein.

Retinoic acid plays important roles in development, growth and differentiation by regulating the expression of target genes. A new retinoic acid-inducible gene, Stra6, has been identified in P19 embryonal carcinoma cells using a subtractive hybridization cDNA cloning technique. Stra6 codes for a very hydrophobic membrane protein of a new type, which does not display similarities with previously characterized integral membrane proteins. Stra6, which exhibits a specific pattern of expression during development and in the adult, is strongly expressed at the level of blood-organ barriers. Interestingly, in testis Sertoli cells, Stra6 has a spermatogenic cycle-dependent expression which is lost in testes of RAR alpha null mutants where Stra6 is expressed in all tubules. We suggest that the Stra6 protein may be a component of an as yet unidentified transport machinery.

Genomic structure and developmental expression of the mouse cell cycle regulatory transcription factor DP1.

The E2F/DP family of transcription factors play an important role in the control of cell cycle progression. By direct regulatory interactions with the retinoblastoma family of proteins, they integrate extracellular growth promoting signals impinging on the cyclin and cyclin dependent kinase complex during the G1 phase, with cell cycle progression. This is accomplished by direct transcriptional activation of genes required for nucleotide biosynthesis and DNA replication in the S phase. In addition, these transcription factors also play a role in the control of genes involved in regulating G1 and S phase progression including, autoregulatory control, as in the case of E2F1 itself. In this report, we describe the characterisation of the genomic locus encoding DP1, a member of this family. The DP1 gene has a TATA-less promoter and transcription initiates at multiple sites. Using transient transfection assays we have delineated sequences in the upstream region which have promoter or enhancer activity. The DP1 gene was localised to mouse chromosome 8 by metaphase chromosome analysis. We describe a dynamic pattern of DP1 expression using in situ hybridisation on cryostat sections of mouse embryos at various stages of development and a variable level of expression by Northern blot analysis of RNA from various adult tissues.

Characterization of a premeiotic germ cell-specific cytoplasmic protein encoded by Stra8, a novel retinoic acid-responsive gene.

The full-length cDNA corresponding to Stra8, a novel gene inducible by retinoic acid (RA) in P19 embryonal carcinoma cells, has been isolated and shown to encode a 45-kD protein. Both Stra8 mRNA and protein were induced in cells treated by all-trans and 9-cis retinoic acids. Two-dimensional gel analysis and dephosphorylation experiments revealed that the two stereoisomers of RA differentially regulate the phosphorylation status of the Stra8 protein, which was shown to exist in differently phosphorylated forms. Subcellular fractionation and immunocytochemistry studies showed that the Stra8 protein is cytoplasmic. During mouse embryogenesis, Stra8 expression was restricted to the male developing gonads, and in adult mice, the expression of Stra8 was restricted to the premeiotic germ cells. Thus, Stra8 protein may play a role in the premeiotic phase of spermatogenesis.

A new mouse member of the Wnt gene family, mWnt-8, is expressed during early embryogenesis and is ectopically induced by retinoic acid.

We have identified a novel mouse Wnt genc using a cDNA differential screening procedure for retinoic-acid-induced transcripts in P19 embryonal carcinoma cells. Sequence analysis showed that this gene represents the first murine Wnt-8 (mWnt-8) gene reported to date. The expression of the mWnt-8 gene, which is rapidly induced by retinoic acid in P19 and embryonic stem cells, appears to be restricted to early stages of mouse embryogenesis. mWnt-8 transcripts are first detected in the posterior region of the epiblast of early primitive streak-stage embryos. As gastrulation proceeds, mWnt-8 expression spreads into the embryonic ectoderm up to a sharp rostral boundary at the base of the developing headfolds. mWnt-8 is also transiently expressed in the newly formed mesoderm. mWnt-8 expression is rapidly down-regulated during early somitogenesis, the latest detectable expression domains corresponding to the presumptive fourth rhombomere and the caudal region of the neural plate. The expression pattern of mWnt-8 is clearly distinct from those of other murine Wnt genes expressed during gastrulation, but shows striking similarities with that of the chicken Cwnt-8C gene. We also show that mWnt-8 expression is ectopically induced in the rostral neural plate in response to RA exposure of presumitic (7-7.5 days post coitum) cultured mouse embryos.

AP-2.2: a novel AP-2-related transcription factor induced by retinoic acid during differentiation of P19 embryonal carcinoma cells.

A 2.8-kb cDNA encoding a new transcription factor (AP-2.2) has been cloned from mouse P19 embryonal carcinoma cells, in which the corresponding mRNA begins to accumulate 30 min after retinoic acid (RA) addition. The predicted protein is 449 amino acids long and exhibits approximately 65% overall identity with other AP-2-related proteins (human AP-2, mouse AP-2alpha and beta). A 96-amino-acid-long sequence, which is almost fully conserved between all these proteins, corresponds to the previously characterized human AP-2 DNA binding domain. Expression of AP-2.2 in Escherichia coli generated a protein that formed a specific complex with the AP-2 recognition site GCCN3GGC. AP-2.2 activated transcription from a reporter gene containing an AP-2 DNA binding site and acted synergistically with RARalpha to activate transcription from the CRABPII gene promoter. Transcriptional activation required the AP-2.2 amino-terminal region that contains a domain rich in proline and glutamine residues. The pattern of AP-2.2 expression in adult tissues, which is distinct from that of AP-2alpha, is essentially restricted to male and female gonads, to most if not all the squamous epithelia, and to several exocrine glands.

Restricted expression of a novel retinoic acid responsive gene during limb bud dorsoventral patterning and endochondral ossification.

Using a differential substractive hybridization cloning procedure we have recently identified Stra6 as a novel retinoic acid-induced gene in murine P19 embryonal carcinoma cells. The putative amino acid sequence of Stra6 shows no similarity with previously characterised proteins. We report here the pattern of expression of Stra6 transcripts during mouse limb development as revealed by in situ hybridization. In 8.5-9.0 days post-coitum (dpc) embryos, Stra6 was expressed in the lateral plate mesenchyme prior to limb bud outgrowth. By 9.5 dpc, expression was restricted to the proximal and dorsal forelimb bud mesoderm. Over the next 2 gestational days, Stra6 expression was specific of the dorsal mesoderm of the undifferentiated forelimb and hindlimb buds with the exception of their distal-most region or progress zone. A novel proximal-ventral expression domain appeared, however, by 11.0-11.5 dpc. Stra6 also remained expressed in the flank mesoderm. From 11.5-13.5 dpc, Stra6 expression was restricted to the superficial mesenchyme surrounding the chondrogenic blastemas, and progressively extended until the distal extremities of the limbs upon disappearance of the progress zone. Progressive restriction of Stra6 expression to perichondrium and developing muscles was seen at 13.5-14.5 dpc. Upon the initiation of endochondral ossification (15.5-16.5 dpc), Stra6 expression was limited to the area of perichondrium opposing cells of high metabolic and proliferative activity (the elongation zone). We suggest that Stra6 may play a role in early dorsoventral limb patterning and later in the control of endochondral ossification.

AP-2.2, a novel gene related to AP-2, is expressed in the forebrain, limbs and face during mouse embryogenesis.

Using a differential subtractive hybridization cloning procedure we have recently identified the AP-2.2 gene as a novel early retinoic acid-induced gene in murine P19 embryonal carcinoma cells. We have also shown that the AP-2.2 protein, which is highly related to the AP-2 transcription factor, can activate transcription when bound to an AP-2 consensus binding site [Oulad-Abdelghani et al. (1995) Mol. Cell. Biol., submitted]. We report here the in situ hybridization pattern of expression of AP-2.2 transcripts during mouse embryogenesis. At 7.5 days post-coitum, AP-2.2 transcripts were detected in the boundary region between neural plate and surface ectoderm, as well as in extra-embryonic tissues. By 8.0-8.5 gestational days, AP-2.2 transcripts appeared to be expressed in premigratory and migrating neural crest cells. Over the following days, the AP-2.2 gene displayed region-restricted expression in the facial mesenchyme, especially around the embryonic mouth cavity and the nasal cavities, as well as in the surface ectoderm, nasal and oral epithelia. AP-2.2 RNA was also specifically expressed in the presumptive cortical region of the forebrain vesicles. AP-2.2 transcripts were restricted to the distal mitotic area (the 'progress zone') of the limb buds and of the genital bud. AP-2.2 expression also appeared to be specific for primordial germ cells in the genital ridges. Thus, the AP-2.2 gene is expressed in several embryonic areas whose development can be affected by retinoids, such as the forebrain, face and limb buds.

Sequence and expression pattern of the Stra7 (Gbx-2) homeobox-containing gene induced by retinoic acid in P19 embryonal carcinoma cells.

The cDNA sequence of Stra7, a retinoic acid (RA)-inducible gene in P19 embryonal carcinoma (EC) cells, was determined. The deduced Stra7 protein contains a homeodomain highly similar to that of the previously described chicken CHox7 gene product, and is highly conserved during evolution, from hemichordates to vertebrates. The mouse Stra7 cDNA corresponds to the full-length form of the 77 bp homeodomain-encoding cDNA fragment which was previously cloned and termed MMoxA or Gbx-2. Reverse-transcriptase-PCR analysis revealed the presence of Stra7/Gbx-2 transcripts in the adult brain, spleen, and female genital tract, whereas no expression could be observed in heart, liver, lung, kidney, or testes. In situ hybridization analysis showed a restricted expression pattern of Stra7/Gbx-2 in the three primitive germ layers during gastrulation. Restricted expression was also detected in the pharyngeal arches. Subsequently, there were specific expression domains in the developing central nervous system, at the midbrain/hindbrain boundary and later in the cerebellum anlage, in certain rhombomeres, in dorsal regions of the spinal cord, and in the developing dorsal thalamus and corpus striatum.

Mouse Lbx1 and human LBX1 define a novel mammalian homeobox gene family related to the Drosophila lady bird genes.

We have cloned two novel homeobox genes which are the mouse (Lbx1) and human (LBX1) homologs of the Drosophila lady bird genes. They are highly related not only within the coding region but also in 5' and 3' untranslated regions. Several amino acid residues inside and around the homeodomain, have been conserved between the mammalian Lbx genes and their Drosophila counterparts. The mouse Lbx1 gene is located on chromosome 19 (region D) and the human LBX1 gene maps to the related q24 region of chromosome 10, known as a breakpoint region in translocations t(7;10) and t(10;14) involved in T-cell leukemias. Thus, LBX1 and the protooncogene HOX11 map to a common chromosomal region, as do their Drosophila counterparts, the lady bird and 93Bal genes. The mouse Lbx1 gene is specifically expressed during embryogenesis. From 10.5 days of gestation, Lbx1 expression is detected in the central nervous system and some developing muscles. In the CNS, Lbx1 transcripts are expressed in the dorsal part of the mantle layer of the spinal cord and hindbrain, up to a sharp boundary within the developing metencephalon. Thus, Lbx1 may be inolved in spinal cord and hindbrain differentiation and/or patterning, and its restricted expression pattern could depend upon evolutionarily conserved inductive signals involving some mammalian Wnt and Pax genes, as is the case for Drosophila lady bird genes and wingless or gooseberry.