The DNA damage dependence of poly(ADP-ribose) polymerase-2 (PARP-2) activity is suggestive of its implication in genome surveillance and protection. Here we show that the PARP-2 gene, mainly expressed in actively dividing tissues follows, but to a smaller extent, that of PARP-1 during mouse development. We found that PARP-2 and PARP-1 homo-and heterodimerize; the interacting interfaces, sites of reciprocal modification, have been mapped. PARP-2 was also found to interact with three other proteins involved in the base excision repair pathway: x-ray cross complementing factor 1 (XRCC1), DNA polymerase ␤, and DNA ligase III, already known as partners of PARP-1. XRCC1 negatively regulates PARP-2 activity, as it does for PARP-1, while being a polymer acceptor for both PARP-1 and PARP-2. To gain insight into the physiological role of PARP-2 in response to genotoxic stress, we developed by gene disruption mice deficient in PARP-2. Following treatment by the alkylating agent N-nitroso-N-methylurea (MNU), PARP-2-deficient cells displayed an important delay in DNA strand breaks resealing, similar to that observed in PARP-1 deficient cells, thus confirming that PARP-2 is also an active player in base excision repair despite its low capacity to synthesize ADP-ribose polymers.In response to DNA interruptions, PARP-1, 1 the founding member of the poly(ADP-ribose) polymerase superfamily, catalyzes the successive covalent addition of ADP-ribose units from NAD to a limited number of nuclear acceptors to form a branched anionic polymer. PARP-1 is a nuclear enzyme involved in the detection and signaling of DNA strand breaks introduced either directly by ionizing radiation or indirectly following enzymatic incision of a DNA lesion (abasic sites or oxidized or alkylated bases) repaired by the base excision repair (BER) pathway (see for review Ref. 1). The discovery of numerous PARP-1 protein partners and/or poly(ADP-ribose) acceptors involved in DNA architecture (histones H1 and H2B, lamin B, and high mobility group proteins) or in DNA metabolism (DNA replication factors, DNA repair proteins, i.e. XRCC1, transcription factors, topoisomerases, and PARP-1 itself) has shed light onto the implication of PARP-1 in these processes (see for review Ref. 1).The function of PARP-1 in BER has long been assumed, until direct evidence demonstrated the presence of PARP-1 in the BER complex, associated to XRCC1 (2, 3) and DNA polymerase (pol) ␤ (4). The polymer produced by PARP-1 upon activation by DNA breaks triggers the recruitment of XRCC1, which shows high affinity for oligo(ADP-ribosyl)ated PARP-1 (3-5). The requirement of PARP-1 in BER was established in vivo, because PARP-1 knock-out cells displayed a severe defect in strand breaks resealing following genotoxic treatment (6, 7). The preferential role of PARP-1 in long patch BER was observed using extracts from these PARP-1 knock-out cells (4). Photoaffinity labeling experiments revealed that PARP-1 binds to BER intermediates (8). In reconstituted in vitro systems containing purified human B...

Three retinaldehyde dehydrogenases (RALDH1, RALDH2 and RALDH3), which catalyze the oxidation of retinaldehyde into retinoic acid, have been shown to be differentially expressed during early embryogenesis. Here, we report their differential expression patterns throughout later mouse organogenesis. Raldh1 is prominently expressed in developing lung (notably in bronchial and tracheal epithelia), and shows stage-specific expression in stomach and intestine epithelial and mesenchymal layers. Raldh3 expression is specific to the differentiating intestinal lamina propria. Raldh2 is expressed throughout the kidney nephrogenic zone, whereas Raldh1 and Raldh3 are mostly expressed in collecting duct epithelia. Raldh3 expression is more restricted than that of Raldh1 in the urogenital tract and sex gland epithelia, whereas Raldh2 expression is mesenchymal. Raldh1 is coexpressed with Raldh2 in the early heart epicardium, and is later specifically expressed in developing heart valves. All three genes exhibit distinct expression patterns in respiratory and olfactory epithelia and/or mesenchymes, and in developing teeth. Only Raldh1 expression is seen after birth in specific brain structures. These data indicate a requirement for regulated RA synthesis in various differentiating organs.

A striking characteristic of vertebrate embryos is their bilaterally symmetric body plan, which is particularly obvious at the level of the somites and their derivatives such as the vertebral column. Segmentation of the presomitic mesoderm must therefore be tightly coordinated along the left and right embryonic sides. We show that mutant mice defective for retinoic acid synthesis exhibit delayed somite formation on the right side. Asymmetric somite formation correlates with a left-right desynchronization of the segmentation clock oscillations. These data implicate retinoic acid as an endogenous signal that maintains the bilateral synchrony of mesoderm segmentation, and therefore controls bilateral symmetry, in vertebrate embryos.

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Chicken ovalbumin upstream promoter transcription factor-interacting proteins 1 and 2 (CTIP1 and CTIP2) are related transcriptional regulatory proteins. While overexpression of both of these proteins has been linked to the development of several lymphoid malignancies, lack of CTIP1 and CTIP2 expression results in defective lymphopoiesis and abnormal thymocyte development, respectively. Here, we describe the expression patterns of CTIP1 and CTIP2 during mouse embryogenesis and in the post-natal brain. Both CTIP1 and CTIP2 were expressed diffusely in the embryo at 10.5 days post-coitum (d.p.c.). However, the expression of both genes became increasingly restricted to the central nervous system (CNS) during the course of fetal development, culminating with high, but differential, expression levels throughout the hippocampal subregions, olfactory bulb and cortex, limbic system, basal ganglia and frontal cortex of the developing brain, and in dorsal cells of the spinal cord. The brain expression domains of CTIP1 and CTIP2 were maintained into adulthood. Outside the CNS, both genes exhibited differential expression within the facial mesenchyme at 12.5 d.p.c., and CTIP2 was selectively expressed from day 12.5 onwards in the olfactory epithelium and developing thymus, and to a lesser extent in oral and gut epithelia. Strong CTIP2 expression was maintained in the thymus at 18.5 d.p.c. These results support the selective contributions of both CTIP1 and CTIP2 in the development and function of both the central nervous and immune systems and the importance of future investigations to define the function(s) of both proteins. et al., 2000). Recruitment of either CTIP1 (Avram et al., 2000) or CTIP2 (Avram, unpublished studies) to the template by a COUP-TF family member was found to result in transcriptional repression of a reporter gene harboring a COUP-TF binding site. Additionally, CTIP1 and CTIP2 are sequence-specific DNA binding proteins that repress transcription via direct, COUP-TF-independent binding to a motif that is related to the canonical GC box (Avram et al., 2002). Thus, CTIPs may either be recruited to the template by a COUP-TF family member or bind directly to the template in a sequence-specific manner. In both cases, CTIP1 and CTIP2 appear to repress transcription in a manner that does not involve trichostatin A-sensitive histone deacetylation (Avram et al., 2000;Senawong et al., 2003). Although, the mechanistic basis of CTIP-mediated transcriptional repression has not been defined, overexpressed CTIP2 ( Expression of both CTIP1 and CTIP2 has recently been linked to the etiology of disease. Overexpression of CTIP1 following proviral integration led to the transformation of NIH3T3 cells and the development of myeloid leukemia in mice . This transformation event may be facilitated by a physical interaction of CTIP1 with BCL6, a known human B cell proto-oncogene product. Recently, an association between p53 and CTIP2 in mice has been implicated in the development of thymic lymphomas (Wakabayashi et al., ...

We recently cloned the murine homologue of Cyp26B1, a novel retinoic acid (RA)-metabolizing enzyme and showed that its gene expression pattern is unique from that of Cyp26A1 during early embryogenesis. Here, we complete this comparative expression analysis from embryonic day (E) 12 to postnatal stages. Cyp26B1 expression was found in developing tendons and precartilaginous elements and in perichondrium by E14.5, while Cyp26A1 expression was restricted to extremities of rib and vertebral cartilage. Cyp26A1 and Cyp26B1 were expressed, in the distal epithelium and mesenchyme of the limbs and genital tubercle, respectively. High Cyp26B1 expression was found in craniofacial areas undergoing morphogenetic growth, whereas Cyp26A1 message was restricted to the mouth and dental epithelium. Cyp26A1 alone was expressed in the developing neural retina, while both genes were co-expressed in the retinal pigment epithelium. Cyp26B1 was specifically expressed in the developing hindbrain (pons, cerebellum) and forebrain (striatum, hippocampus), with forebrain expression persisting postnatally. In addition, Cyp26B1 was expressed at specific levels of the differentiating upper and lower thoracic spinal cord, adjacent to the cervical and lumbar regions that express the RA-synthesizing enzyme RALDH-2. In viscera, Cyp26B1 transcripts were detected in the developing lung, kidney, spleen, thymus and testis, whereas Cyp26A1 transcripts were found in the diaphragm and outer stomach mesenchyme. Cyp26B1 was also specifically expressed in dermis surrounding the developing hair follicles. Regulated RA metabolism may therefore be required in many developing systems.

Knockout of the murine retinoic acid (RA)-synthesizing enzyme retinaldehyde dehydrogenase 2 (RALDH2) gene leads to early morphogenetic defects and embryonic lethality. Using a RAresponsive reporter transgene, we have looked for RA-generating activities in Raldh2-null mouse embryos and investigated whether these activities could be ascribed to the other known RALDH enzymes (RALDH1 and RALDH3). To this end, the early defects of Raldh2 ؊/؊ embryos were rescued through maternal dietary RA supplementation under conditions that do not interfere with the activity of the reporter transgene in WT embryos. We show that RALDH2 is responsible for most of the patterns of reporter transgene activity in the spinal cord and trunk mesodermal derivatives. However, reporter transgene activity was selectively detected in Raldh2 ؊/؊ embryos within the mesonephric area that expresses RALDH3 and in medial-ventral cells of the spinal cord and posterior hindbrain, up to the level of the fifth rhombomere. The craniofacial patterns of RA-reporter activity were unaltered in Raldh2 ؊/؊ mutants. Although these patterns correlated with the presence of Raldh1 and͞or Raldh3 transcripts in eye, nasal, and inner ear epithelia, no such correlation was found within forebrain neuroepithelium. These data suggest the existence of additional RAgenerating activities in the differentiating forebrain, hindbrain, and spinal cord, which, along with RALDH1 and RALDH3, may account for the development of Raldh2 ؊/؊ mutants once these have been rescued for early lethality.

Retinoic acid signaling plays essential roles in morphogenesis and neural development through transcriptional regulation of downstream target genes. It is believed that the balance between the activities of synthesizing and metabolizing enzymes determines the amount of active retinoic acid to which a developing tissue is exposed. In this study, we investigated spatiotemporal expression patterns of four synthesizing enzymes, the retinaldehyde dehydrogenases 1, 2, 3, and 4 (Raldh1, Raldh2, Raldh3, and Raldh4) and two metabolizing enzymes (Cyp26A1 and Cyp26B1) in the embryonic and postnatal mouse inner ear by using quantitative reverse transcriptase polymerase chain reaction (RT-PCR), in situ hybridization, and Western blot analysis. Quantitative RT-PCR analysis and Western blot data revealed that the expression of CYP26s was much higher than that of Raldhs at early embryonic ages but that Cyp26 expression was downregulated during embryonic development. Conversely, the expression levels of Raldh2 and -3 increased during development and were significantly higher than the Cyp26 levels at postnatal day 20. At this age, Raldh3 was expressed predominantly in the cochlea, whereas Raldh2 was present in the vestibular end organ. At early embryonic stages, as observed by in situ hybridization, the synthesizing enzymes were expressed only in the dorsoventral epithelium of the otocyst, whereas the metabolizing enzymes were present mainly in mesenchymal cells surrounding the otic epithelium. At later stages, Raldh2, Raldh3, and Cyp26B1 were confined to the stria vascularis, spiral ganglion, and supporting cells in the cochlear and vestibular epithelia, respectively. The downregulation of Cyp26s and the upregulation of Raldhs after birth during inner ear maturation suggest tissue changes in the sensitivity to retinoic acid concentrations.