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.

Studies of human, mouse and yeast homologues indicate a mitochondrial function for frataxin.

Friedreich's ataxia is due to loss of function mutations in the gene encoding frataxin (FRDA). Frataxin is a protein of unknown function. In situ hybridization analyses revealed that mouse frataxin expression correlates well with the main site of neurodegeneration, but the expression pattern is broader than expected from the pathology of the disease. Frataxin mRNA is predominantly expressed in tissues with a high metabolic rate, including liver, kidney, brown fat and heart. We found that mouse and yeast frataxin homologues contain a potential mitochondrial targeting sequence in their N-terminal domains and that disruption of the yeast gene results in mitochondrial dysfunction. Finally, tagging experiments demonstrate that human frataxin co-localizes with a mitochondrial protein. Friedreich's ataxia is therefore a mitochondrial disease caused by a mutation in the nuclear genome.

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.

New reactor dedicated to in operando studies of model catalysts by means of surface x-ray diffraction and grazing incidence small angle x-ray scattering.

A new experimental setup has been developed to enable in situ studies of catalyst surfaces during chemical reactions by means of surface x-ray diffraction (SXRD) and grazing incidence small angle x-ray scattering. The x-ray reactor chamber was designed for both ultrahigh-vacuum (UHV) and reactive gas environments. A laser beam heating of the sample was implemented; the sample temperature reaches 1100 K in UHV and 600 K in the presence of reactive gases. The reactor equipment allows dynamical observations of the surface with various, perfectly mixed gases at controlled partial pressures. It can run in two modes: as a bath reactor in the pressure range of 1-1000 mbars and as a continuous flow cell for pressure lower than 10(-3) mbar. The reactor is connected to an UHV preparation chamber also equipped with low energy electron diffraction and Auger spectroscopy. This setup is thus perfectly well suited to extend in situ studies to more complex surfaces, such as epitaxial films or supported nanoparticles. It offers the possibility to follow the chemically induced changes of the morphology, the structure, the composition, and growth processes of the model catalyst surface during exposure to reactive gases. As an example the Pd(8)Ni(92)(110) surface structure was followed by SXRD under a few millibars of hydrogen and during butadiene hydrogenation while the reaction was monitored by quadrupole mass spectrometry. This experiment evidenced the great sensitivity of the diffracted intensity to the subtle interaction between the surface atoms and the gas molecules.

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.

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.

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.

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.

The Hox-4.8 gene is localized at the 5' extremity of the Hox-4 complex and is expressed in the most posterior parts of the body during development.

We report the isolation and expression pattern of a novel mouse homeobox gene, Hox-4.8. Hox-4.8 is the most 5'-located homeobox gene in the HOX-4 complex. Sequence analysis confirmed that Hox-4.8 is a member of the subfamily of AbdominalB-related Hox-4 genes and revealed strong interspecies conservation. As for the human locus, Hox-4.8 is probably the last Hox gene in this part of the HOX-4 complex. During development, Hox-4.8 transcripts are restricted to the extremities of the embryonic anteroposterior axis and limbs as well as in the developing tail bud and to the most posterior segment of the gut (the rectum). Within the limb mesenchyme, Hox-4.8 is expressed in more posterodistal regions than those of its neighbour Hox-4.7. Hence, Hox-4.8 expression appears to be related to the last significant phenotypic changes towards the extremities of the embryonic body and limb axes.

Restricted expression and retinoic acid-induced downregulation of the retinaldehyde dehydrogenase type 2 (RALDH-2) gene during mouse development.

Retinaldehyde dehydrogenase type 2 (RALDH-2) was identified as a major retinoic acid generating enzyme in the early embryo. Here we report the expression domains of the RALDH-2 gene during mouse embryogenesis, which are likely to indicate regions of endogenous retinoic acid (RA) synthesis. During early gastrulation, RALDH-2 is expressed in the mesoderm adjacent to the node and primitive streak. At the headfold stage, mesodermal expression is restricted to posterior regions up to the base of the headfolds. Later, RALDH-2 is transiently expressed in the undifferentiated somites and the optic vesicles, and more persistently along the lateral walls of the intraembryonic coelom and around the hindgut diverticulum. The RALDH-2 expression domains in differentiating limbs, which include presumptive interdigital regions, coincide with, but slightly precede, those of the RA-inducible RAR beta gene. The RALDH-2 gene is also expressed in specific regions of the developing head, including the tooth buds, inner ear, meninges and pituitary gland, and in several viscera. Administration of a teratogenic dose of RA at embryonic day 8.5 results in downregulation of RALDH-2 transcript levels in caudal regions of the embryo, and may reflect a mechanism of negative feedback regulation of RA synthesis.

Expression of the transcriptional intermediary factor TIF1alpha during mouse development and in the reproductive organs.

Nuclear receptors are important regulators of development and reproduction whose action can be modulated by transcriptional intermediary factors (TIFs). In situ hybridization was used to investigate the expression pattern of the putative nuclear receptor mediator TIF1alpha during mouse embryogenesis and adult life. TIF1alpha is ubiquitously expressed until midgestation. At 12.5 gestational days, TIF1alpha is preferentially expressed in the developing central and peripheral nervous system. Differential expression persists until perinatal stages, with high expression in the brain, nasal epithelium and within proliferating regions of the kidney and teeth. In the adult, TIF1alpha expression is predominant in both the male and female gonads. Immunogold electron microscopy revealed that TIF1alpha protein is most abundant in the nuclei of male germ cells at various stages of their maturation.

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.

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.

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.

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.

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.

Cloning of a novel retinoic-acid metabolizing cytochrome P450, Cyp26B1, and comparative expression analysis with Cyp26A1 during early murine development.

Tight regulation of retinoic acid (RA) distribution in the embryo is critical for normal morphogenesis. The RA-metabolizing enzymes Cyp26A1 and Cyp26B1 are believed to play important roles in protecting certain embryonic tissues from inappropriate RA signaling. We have cloned the murine Cyp26B1 cDNA and compared its expression pattern to that of Cyp26A1 from embryonic day (E) E7-E11.5 using in situ hybridization. Northern blot analysis shows the presence of two Cyp26B1 transcripts of approximately 2.3 and 3.5 kb in embryonic limb bud. Whereas Cyp26A1 is expressed in gastrulating embryos by E7, Cyp26B1 is first expressed at E8.0 in prospective rhombomeres 3 and 5. Cyp26B1 expression expands to specific dorso-ventral locations in rhombomeres 2-6 between E8.5 and E9.5, whereas Cyp26A1 hindbrain expression is limited to rhombomere 2 at E8.5. No (or very weak) Cyp26B1 expression is observed in the tail bud, a major site of Cyp26A1 expression. Differential expression is seen in branchial arches, with Cyp26A1 being mainly expressed in neural crest-derived mesenchyme, and Cyp26B1 in specific ectodermal and endodermal areas. Cyp26B1 is markedly expressed in the ectoderm and distal mesoderm of the limb buds from the beginning of their outgrowth. Cyp26A1 transcripts are seen later and at lower levels in limb ectoderm, and both transcripts are excluded from the apical ectodermal ridge.

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.

Differential expression of retinoic acid-inducible (Stra) genes during mouse placentation.

Several retinoid binding proteins and nuclear receptors are specifically expressed in murine placenta. However, little is known about molecular events and target genes regulated by retinoids during placentation. Here, we report that several retinoic acid-inducible (Stra) genes, originally isolated by a differential screening procedure, exhibit specific expression patterns in mouse placental tissues. Three Stra genes, including the ephrinB1 receptor tyrosine kinase ligand, are prominently expressed in the regions of exchanges between maternal and embryonic circulations, i.e. the yolk sac and/or the labyrinthine zone of the mature placenta. The Meis2 homeobox gene appears to be specifically expressed in maternally-derived cell populations. Three other Stra genes, including the AP-2-related gene AP-2gamma, are differentially expressed in the trophoblastic cell lineage. Thus, retinoids may regulate various signaling pathways in specific placental cell-types.

Tissue-specific expression of retinoic acid receptor isoform transcripts in the mouse embryo.

The three murine retinoic acid receptor (RAR) genes each contain two distinct promoters which give rise to protein isoforms differing in their N-terminal regions. This study used in situ hybridization to describe the expression patterns of RARalpha1, RARalpha2, RARbeta1/3, RARbeta2/4, RARgamma1 and RARgamma2 isoform transcripts during mouse embryogenesis. RARalpha1 transcripts are widely distributed, with the exception of the central nervous system. Highest expression is found in developing muscle, pituitary gland and various epithelia. On the other hand, RARalpha2 is essentially expressed along the spinal cord up to the hindbrain 7th rhombomere and in the 4th rhombomere, pons and developing basal ganglia (corpus striatum and pallidum). RARbeta2/4 transcripts account for most of the previously described RARbeta expression features being expressed specifically, or more prominently than RARbeta1/3, in foregut endoderm and its derivatives, olfactory and periocular mesenchyme, urogenital region, proximal limb bud mesenchyme and later within interdigital regions. RARbeta1/3 is more prominently expressed in the developing heart outflow tract mesenchyme, intervertebral disks, midgut loop mesenchyme and umbilical vessel walls. RARbeta1/3 and RARbeta2/4 are coexpressed in the developing corpus striatum. They exhibit, however, distinct dorsoventral distributions along the spinal cord and caudal hindbrain. RARgamma2 is the RARgamma isoform expressed at high levels in the caudal neural groove at embryonic day 8.5. At later stages, both RARgamma isoforms are essentially coexpressed, although the progressive restriction of RARgamma1 transcripts to craniofacial or limb precartilaginous condensations appears to precede that of RARgamma2.