CNS-specific regulatory elements in brain-derived HIV-1 strains affect responses to latency-reversing agents with implications for cure strategies.

Latency-reversing agents (LRAs), including histone deacetylase inhibitors (HDACi), are being investigated as a strategy to eliminate latency in HIV-infected patients on suppressive antiretroviral therapy. The effectiveness of LRAs in activating latent infection in HIV strains derived from the central nervous system (CNS) is unknown. Here we show that CNS-derived HIV-1 strains possess polymorphisms within and surrounding the Sp transcription factor motifs in the long terminal repeat (LTR). These polymorphisms result in decreased ability of the transcription factor specificity protein 1 to bind CNS-derived LTRs, reducing the transcriptional activity of CNS-derived viruses. These mutations result in CNS-derived viruses being less responsive to activation by the HDACi panobinostat and romidepsin compared with lymphoid-derived viruses from the same subjects. Our findings suggest that HIV-1 strains residing in the CNS have unique transcriptional regulatory mechanisms, which impact the regulation of latency, the consideration of which is essential for the development of HIV-1 eradication strategies.

Heterozygous mutations in the tumor suppressor gene PATCHED provoke basal cell carcinoma-like features in human organotypic skin cultures.

Basal cell carcinoma of the skin is the most common type of cancer in humans. The majority of these tumors displays aberrant activation of the SONIC HEDGEHOG (SHH)/PATCHED pathway, triggered by mutations in the PATCHED tumor suppressor gene, which encodes a transmembrane receptor of SHH. In this study, we took advantage of the natural genotype (PATCHED(+/-)) of healthy keratinocytes expanded from patients with the nevoid basal cell carcinoma or Gorlin syndrome to mimic heterozygous somatic mutations thought to occur in the PATCHED gene early upon basal cell carcinoma development in the general population. PATCHED(+/-) epidermis developed on a dermal equivalent containing wild-type (WT) PATCHED(+/+) fibroblasts exhibited striking invasiveness and hyperproliferation, as well as marked differentiation impairment. Deciphering the phenotype of PATCHED(+/-) keratinocytes revealed slight increases of the transcriptional activators GLI1 and GLI2-the latter known to provoke basal cell carcinoma-like tumors when overexpressed in transgenic mice. PATCHED(+/-) keratinocytes also showed a substantial increase of the cell cycle regulator cyclin D1. These data show for the first time the physiological impact of constitutive heterozygous PATCHED mutations in primary human keratinocytes and strongly argue for a yet elusive mechanism of haploinsufficiency leading to cancer proneness.

Ultraviolet responses of Gorlin syndrome primary skin cells.

BACKGROUND: Gorlin syndrome, or naevoid basal cell carcinoma syndrome (NBCCS), is an autosomal dominant disorder associated with mutations in the PTCH1 gene, which encodes the receptor of SONIC HEDGEHOG. In addition to developmental abnormalities, patients with NBCCS are prone to basal cell carcinoma (BCC), the most frequent type of nonmelanoma skin cancer in humans. OBJECTIVES: As ultraviolet (UV) exposure plays a prominent role in the development of sporadic BCC, we aimed to determine whether primary NBCCS skin cells exhibit differential responses to UV exposure compared with wild-type (WT) skin cells. METHODS: Primary fibroblast and keratinocyte strains were isolated from nonlesional skin biopsies of 10 patients with characteristic NBCCS traits. After identification of PTCH1 mutations, capacities of NBCCS cells to repair UV-induced DNA lesions and to survive after UV irradiation, as well as p53 responses, were compared with those of WT skin cells. RESULTS: The c1763insG PTCH1 mutation is described for the first time. DNA repair and cell survival analyses following UV irradiation revealed no obvious differences between responses of NBCCS and WT fibroblasts and keratinocytes. However, p53 accumulation after UV irradiation was abnormally persistent in all NBCCS primary keratinocyte strains compared with WT keratinocytes. CONCLUSIONS: Our observations that NBCCS cells harbour normal DNA repair and survival capacities following UV irradiation better explain that BCC proneness of patients with NBCCS does not solely concern body areas exposed to sunlight and suggest rather that it might be due to cell cycle alterations.

[Clinical and genetic study in 22 patients with basal cell nevus syndrome]. / Etude clinique et recherche de mutations germinales du gène PTCH 1 dans le syndrome des hamartomes basocellulaires.

BACKGROUND: Nevoid basal cell carcinoma syndrome is an autosomal dominant disorder characterized by developmental abnormalities and cancer predisposition. The PTCH 1 gene, the human homolog of the Drosophila segment polarity gene patched, has been shown to be involved in the development of nevoid basal cell carcinoma syndrome. PTCH 1 is mapped to chromosome 9q22.3. The aim of the present study was to report on clinical and genetic characteristics in patients followed for nevoid basal cell carcinoma syndrome and to compare them to the data in the literature. PATIENTS AND METHODS: Screening for PTCH 1 mutations was done in 22 patients followed between 1981 and 2003 for clinical suspicion of nevoid basal cell carcinoma syndrome. Clinical and radiological data were reviewed retrospectively from records. Genetic analysis was performed using blood samples after patient informed consent was obtained. When possible, DNA was also analyzed from the parents of patients in whom PTCH 1 mutations were found. RESULTS: All patients had developed basal cell carcinomas: 45% palmar and plantar pitting, 62% jaw cysts and 66% calcification of falx cerebri. Medulloblastomas and meningiomas were the most common associated tumors. PTCH 1 mutations were identified in 13 patients: 6 familial cases, 3 sporadic cases and for 4 patients, it was not possible to conclude. Nine different new germ-line mutations were identified. DISCUSSION: Genetic analysis allows molecular confirmation of diagnosis in about half of all patients. Early diagnosis is essential for detection of clinical and radiological manifestations in young patients and for provision of advice concerning protection of the skin from the sunlight.

Gorlin syndrome presenting as prenatal chylothorax in a girl.

Gorlin syndrome (GS), also known as nevoid basal cell carcinoma syndrome, is a rare autosomal dominant condition with an estimated prevalence of 1:57 000. GS is associated with congenital malformations and predisposition to neoplasms. The main features observed in patients with GS are basal cell carcinomas, odontogenic keratocysts, skeletal anomalies including scoliosis and bifid ribs, palmar and plantar epidermal cysts, facial dysmorphism, and cerebral falx calcification. More than 100 other clinical manifestations have also been described in the literature including ovarian fibroma, enlarged cerebral ventricles, and lymphatic as well as chylous mesenteric cysts. The Patched (PTCH) gene is responsible for GS when mutated. Here, we report on a prenatal diagnosis of GS in a girl with a chylothorax, a previously unreported feature in GS. We discuss the clinical features observed in this family and we comment on the molecular studies that allowed us to describe a previously unreported Patched gene mutation.

Dynamic assembly of tight junction-associated proteins ZO-1, ZO-2, ZO-3 and occludin during mouse tooth development.

Tight junctions might play a role during tissue morphogenesis and cell differentiation. In order to address these questions, we have studied the distribution pattern of the tight junction-associated proteins ZO-1, ZO-2, ZO-3 and occludin in the developing mouse tooth as a model. A specific temporal and spatial distribution of tight junction-associated proteins during tooth development was observed. ZO-1 appeared discontinuously in the cell membrane of enamel organ and dental mesenchyme cells. However, endothelial cells of the dental mesenchyme capillaries displayed a continuous fluorescence at the cell membrane. Inner dental epithelium first showed an evident signal for ZO-1 at the basal pole of the cells at bud/cap stage, but ZO-1 was accumulated at the basal and apical pole of preameloblast/ameloblasts at late bell stage. Surprisingly, in the incisor ZO-1 decreased as the inner dental epithelium differentiated, and was re-expressed in secretory and mature ameloblasts. On the contrary, ZO-2 was confined to continuous cell-cell contacts of the enamel organ in both molars and incisors. The lateral cell membrane of inner dental epithelial cells was specifically ZO-2 labeled. However, ZO-3 was expressed in oral epithelium whereas dental embryo tissues were negative. In addition, occludin was hardly detected in dental tissues at the early stage of tooth development, but was distributed continuously at the cell membrane of endothelial cells of ED19.5 dental mesenchyme. In incisors, occludin was detected at the cell membrane of the secretory pole of ameloblasts. The occurrence and relation during tooth development of tight junction proteins ZO-1, ZO-2 and occludin, but not ZO-3, suggests a combinatory assembly in tooth morphogenesis and cell differentiation.

Macrophage tropism of human immunodeficiency virus type 1 isolates from brain and lymphoid tissues predicts neurotropism independent of coreceptor specificity.

The viral determinants that underlie human immunodeficiency virus type 1 (HIV-1) neurotropism are unknown, due in part to limited studies on viruses isolated from brain. Previous studies suggest that brain-derived viruses are macrophage tropic (M-tropic) and principally use CCR5 for virus entry. To better understand HIV-1 neurotropism, we isolated primary viruses from autopsy brain, cerebral spinal fluid, blood, spleen, and lymph node samples from AIDS patients with dementia and HIV-1 encephalitis. Isolates were characterized to determine coreceptor usage and replication capacity in peripheral blood mononuclear cells (PBMC), monocyte-derived macrophages (MDM), and microglia. Env V1/V2 and V3 heteroduplex tracking assay and sequence analyses were performed to characterize distinct variants in viral quasispecies. Viruses isolated from brain, which consisted of variants that were distinct from those in lymphoid tissues, used CCR5 (R5), CXCR4 (X4), or both coreceptors (R5X4). Minor usage of CCR2b, CCR3, CCR8, and Apj was also observed. Primary brain and lymphoid isolates that replicated to high levels in MDM showed a similar capacity to replicate in microglia. Six of 11 R5 isolates that replicated efficiently in PBMC could not replicate in MDM or microglia due to a block in virus entry. CD4 overexpression in microglia transduced with retroviral vectors had no effect on the restricted replication of these virus strains. Furthermore, infection of transfected cells expressing different amounts of CD4 or CCR5 with M-tropic and non-M-tropic R5 isolates revealed a similar dependence on CD4 and CCR5 levels for entry, suggesting that the entry block was not due to low levels of either receptor. Studies using TAK-779 and AMD3100 showed that two highly M-tropic isolates entered microglia primarily via CXCR4. These results suggest that HIV-1 tropism for macrophages and microglia is restricted at the entry level by a mechanism independent of coreceptor specificity. These findings provide evidence that M-tropism rather than CCR5 usage predicts HIV-1 neurotropism.

MLH1 and MSH2 protein immunohistochemistry is useful for detection of hereditary non-polyposis colorectal cancer in young patients.

AIMS: Hereditary non-polyposis colorectal cancer is related to germline mutations of DNA mismatch repair genes MLH1 and MSH2, which result in microsatellite instability and loss of protein expression of the corresponding mutated gene in the tumour tissue. METHODS AND RESULTS: MLH1 and MSH2 protein expression was studied by immunohistochemistry in paraffin-embedded surgical samples of 100 colorectal adenocarcinomas occurring before 50 years of age. Absence of tumour cell nuclear staining with positive internal control (normal mucosa, lymphoid follicles) was considered negative. Loss of MLH1 or MSH2 expression was found in 20 cases with microsatellite instability in 15 cases. Twelve of these patients had a family history of colorectal cancer. Compared with MLH1- and MSH2-positive cases, MLH1- or MSH2-deficient colorectal adenocarcinomas were significantly associated on multivariate analysis with a younger age (38 vs. 43 years, P;0.0224), a larger tumour size (60 +/- 6 vs. 46 +/- 2 mm, P=0.0291), an expanding margin (85% vs. 51%, P=0.0159), a higher number of tumour-infiltrating lymphocytes assessed by CD3 immunostaining (202 +/- 48 vs. 33 +/- 4 CD3+ lymphocytes/10 high-power fields, P=0.0039), and a grade 2 Crohn's like lymphoid reaction (70% vs. 9%, P=0.0037). The two groups were not different for tumour site, differentiation, pTNM stage, vascular and perineural invasion, peripheral adenomatous residue, and 5-year survival rates. CONCLUSIONS: MLH1- or MSH2-deficient colorectal carcinomas of young patients exhibit pathological and molecular features similar to hereditary non-polyposis colorectal cancer. This suggests that MLH1 and MSH2 immunohistochemistry is valuable for detecting hereditary non-polyposis colorectal cancer in young patients.

Diminished production of human immunodeficiency virus type 1 in astrocytes results from inefficient translation of gag, env, and nef mRNAs despite efficient expression of Tat and Rev.

Astrocytes infected with human immunodeficiency virus type 1 (HIV-1) produce only minimal quantities of virus. The molecular events that limit acute-phase HIV-1 infection of astrocytes were examined after inducing acute-phase replication by transfection with the pNL4-3 proviral plasmid. The levels of HIV-1 mRNA were similarly high in both astrocytes and HeLa cells, but astrocytes produced approximately 50-fold less supernatant p24 than HeLa cells. We found that diminished HIV-1 production in astrocytes resulted from inefficient translation of gag, env, and nef mRNAs that were efficiently transported to the cytoplasm. Tat- or Rev-dependent reporter constructs showed no defect in Tat or Rev function in astrocytes compared with HeLa cells. HIV-1 mRNAs were correctly spliced, but only Rev and Tat proteins were efficiently translated from their native mRNAs. Pulse-chase labelling and immunoblot experiments revealed no defect in protein processing, but levels of Gag, Env, or Nef protein expressed were dramatically reduced in astrocytes compared to HeLa cells. These results demonstrate that inefficient translation of HIV-1 structural proteins underlies the restricted infection of astrocytes. The efficient expression of functional Tat and Rev by astrocytes may contribute to HIV-1 neuropathogenesis.

Restricted HIV-1 infection of human astrocytes: potential role of nef in the regulation of virus replication.

A small percentage of astrocytes are consistently infected in vivo by HIV-1 and may contribute to neuropathogenesis despite a non-productive infection. Overexpression of the nef gene product has been associated with their infection both in vivo and in vitro. We examined the role of the nef gene during HIV replication in astrocytes (U251MG cells) following transfection with pNL4-3 proviral plasmid or isogenic strains containing a deletion or point mutation in the nef gene (pNL4-3deltaNef; pNL4-3-nef-stop). We were able to initiate virus replication which peaked at 5 days post-transfection and became non-productive after 21 days. Nef protein expression by wild type pNL4-3 was observed at low levels compared to control HeLa cells at peak virus replication. At later time points after development of a non-productive infection, viral antigen and Nef protein was not detectable however virus was readily recovered by co-culture with CD4+T-cells. Interestingly, virus production was significantly enhanced by a 222 base pair deletion in the nef reading frame. This was not observed with a frame shifting point mutation in nef, indicating a suppressive effect of nef on virus production in astrocytes. The enhanced virus production from nef-deleted pNL4-3 in U251MG cells was not reversed by co-expression of Nef from a second Nef-expressing plasmid, and in fact Nef expression in trans had a further positive effect on virus production. This suggested opposing effects of the Nef protein and elements contained within the nef sequence on virus production in astrocytes. Despite the low expression of Nef by U251MG astrocytes, relatively high amounts of multiply spliced 2 kb mRNA were present compared to HeLa cells. These data demonstrate that an acute low-level infection of astrocytes rapidly becomes a non-productive infection and this process is assisted by sequences in nef. The low level Nef protein expression, despite high levels of mRNA, suggests a block in translation of multiply spliced HIV mRNA in astrocytes, or a translational control mechanism not yet characterised.

MDM2 expression during mouse embryogenesis and the requirement of p53.

We compared mouse embryonic expression of the MDM2 proto-oncogene, p21WAF1/CIP1 and their transcriptional regulator, p53. MDM2 expression is ubiquitous from 7.5 to 11.5 days post coitum (dpc) and more restricted from 12.5 dpc, with the highest levels in the testes and neural tube. From 14.5 to 18.5 dpc, the nasal respiratory epithelium expresses high levels of MDM2 RNA and protein and p21WAF1/CIP1 RNA, in both wild type and p53 null embryos. MDM2 expression during development is tissue-specific and, like p21WAF1/CIP1, is independent of p53. MDM2 may have a developmental role after 6.5 dpc, when MDM2 null mice die (Jones, S.N., Roe, A.E., Donehower, L.A., Bradley, A., 1995. Rescue of embryonic lethality in Mdm2-deficient mice by absence of p53. Nature 378, 206-208; Montes de Oca Luna, R., Wagner, D.S., Lozano, G., 1995. Rescue of early embryonic lethality in mdm2-deficient mice by deletion of p53. Nature 378, 203-206).

Expression of p21(WAF1/CIP1) during mouse odontogenesis.

p21(WAF1/CIP1) is a cyclin-dependent kinase (Cdk) inhibitor. This protein may function during development as an inducible growth inhibitor that contributes to cell cycle exit and differentiation. The expression pattern of p21 during mouse embryogenesis was correlated with terminal differentiation of multiple cell lineages including skeletal muscles, cartilage, skin and nasal epithelium. p21 expression was analyzed by in situ hybridization during odontogenesis as well as during in vitro tooth development in chemically defined medium with or without retinoic acid. p21 transcripts were detected in the restricted area of the inner dental epithelium during late cap and initial bell stages and then confined to the post-mitotic odontoblasts and ameloblasts. The replicating cells were devoid of any signal. The distribution of p21 mRNA in vitro, whatever the culture conditions, was similar to the in vivo pattern. p21 protein immunolocalization was superimposed on the transcripts distribution but more restricted in ameloblasts. TGFbeta1 is known to induce p21 expression. During dental cytodifferentiations, TGFbeta1 and p21 expressions overlap. Growth inhibition by TGFbeta1 may be associated with p21 induction.

Limited specificity and large overlap of the functions of the mouse RAR gamma 1 and RAR gamma 2 isoforms.

The RAR gamma gene generates two major isoforms, RAR gamma 1 and RAR gamma 2, which originate from two distinct promoters. We report here the engineering of mice lacking RAR gamma 1, but in which RAR gamma 2 is normally expressed. The effect of this null mutation has been compared with those previously described for RAR gamma 2 and all RAR gamma isoforms (total RAR gamma gene inactivation), both in single mutants and in double mutants bearing additional null mutations in their RAR alpha, RAR beta or RXR alpha genes. RAR gamma 1 mutants, but not RAR gamma 2 mutants, displayed a subset of the abnormalities exhibited by total RAR gamma null mutants (growth deficiency, abnormal cricoid cartilage and occasional cervical vertebra defects), suggesting that RAR gamma 1 is the main isoform mediating the corresponding RAR gamma functions. Interestingly, cricoid cartilage defects were also found in a fraction of heterozygote animals for the RAR gamma 1, RAR gamma or RAR alpha mutations, indicating that wild type levels of RARs are required for the normal morphogenesis of this structure. Compound RAR alpha/RAR gamma 1 and RAR alpha/RAR gamma 2 double null mutants exhibited only a small fraction of the defects found in RAR alpha/RAR gamma double null mutants. Moreover, these defects were often partially penetrant, or corresponded to a less severe form. However, they occurred preferentially in certain compound mutants, demonstrating that given isoforms mediate specific functions of RAR gamma in the context of a RAR alpha null background. In a RXR alpha null background, both RAR gamma 1 and gamma 2 isoform mutations resulted in increased severity of the RXR alpha null ocular phenotype. Together, the present observations indicate that the functions of the two RAR gamma isoforms overlap to a large extent, but also that each of these isoforms exhibits a limited functional specificity. Furthermore, the occurrence of morphological defects in heterozygote mutants for a single RAR isoform provides a basis for explaining the strong conservation of these isoforms during vertebrate evolution.

Developmental roles of the retinoic acid receptors.

Retinoic acid, one of the principle active metabolites of vitamin A (retinol), is believed to be essential for numerous developmental and physiological processes. Vitamin A deprivation (VAD) during development leads to numerous congenital defects. Previous studies of retinoic acid receptor (RAR) deficient mice failed to reveal any of these VAD-induced defects. This finding suggested that either the RARs are functionally redundant or that they are not critically required during development. In order to address these possibilities, we derived a number of RAR compound mutants. Unlike RAR single mutants, these compound null mutants died either in utero or shortly following birth. Histological analysis revealed essentially all of the defects characteristic of fetal VAD. A number of additional malformations, not described in previous VAD studies, were also observed. These included defects of the ocular and salivary glands and their ducts, the skeletal elements of the fore- and hindlimbs, and the cervical region of the axial skeleton. In addition, with the exception of derivatives forming within the first pharyngeal arch, most of the elements derived from mesectoderm emanating from cranial and hindbrain levels were affected. A number of these mutants also exhibited supernumerary cranial skeletal elements characteristics of the reptilian skull. A summary of the defects found in these RAR double mutants is presented.

Mice deficient in cellular retinoic acid binding protein II (CRABPII) or in both CRABPI and CRABPII are essentially normal.

We have disrupted the CRABPII gene using homologous recombination in embryonic stem cells, and shown that this disruption results in a null mutation. CRABPII null mutant mice are essentially indistinguishable from wild-type mice as judged by their normal development, fertility, life span and general behaviour, with the exception of a minor limb malformation. Moreover, CRABPI-/-/CRABPII-/- double mutant mice also appear to be essentially normal, and both CRABPII-/- single mutant and CRABPI-/-/CRABPII-/- double mutant embryos are not more sensitive than wild-type embryos to retinoic acid excess treatment in utero. Thus, CRABPI and CRABPII are dispensable both during mouse development and adult life. Our present results demonstrate that CRABPs are not critically involved in the retinoic acid signaling pathway, and that none of the functions previously proposed for CRABPs are important enough to account for their evolutionary conservation.

Function of the retinoic acid receptors (RARs) during development (I). Craniofacial and skeletal abnormalities in RAR double mutants.

Numerous congenital malformations have been observed in fetuses of vitamin A-deficient (VAD) dams [Wilson, J. G., Roth, C. B., Warkany, J., (1953), Am. J. Anat. 92, 189-217]. Previous studies of retinoic acid receptor (RAR) mutant mice have not revealed any of these malformations [Li, E., Sucov, H. M., Lee, K.-F., Evans, R. M., Jaenisch, R. (1993) Proc. Natl. Acad. Sci. USA 90, 1590-1594; Lohnes, D., Kastner, P., Dierich, A., Mark, M., LeMeur, M., Chambon, P. (1993) Cell 73, 643-658; Lufkin, T., Lohnes, D., Mark, M., Dierich, A., Gorry, P., Gaub, M. P., Lemeur, M., Chambon, P. (1993) Proc. Natl. Acad. Sci. USA 90, 7225-7229; Mendelsohn, C., Mark, M., Dollé, P., Dierich, A., Gaub, M.P., Krust, A., Lampron, C., Chambon, P. (1994a) Dev. Biol. in press], suggesting either that there is a considerable functional redundancy among members of the RAR family during ontogenesis or that the RARs are not essential transducers of the retinoid signal in vivo. In order to discriminate between these possibilities, we have generated a series of RAR compound null mutants. These RAR double mutants invariably died either in utero or shortly after birth and presented a number of congenital abnormalities, which are reported in this and in the accompanying study. We describe here multiple eye abnormalities which are found in various RAR double mutant fetuses and are similar to those previously seen in VAD fetuses. Interestingly, we found further abnormalities not previously reported in VAD fetuses.(ABSTRACT TRUNCATED AT 250 WORDS)

The cellular retinoic acid binding protein I is dispensable.

The cellular retinoic acid binding proteins I and II (CRABPI and CRABPII) bind retinoic acid with high affinity, exhibit distinct patterns of expression during embryonic development, and are thought to play important roles in the RA signaling pathway. We have generated a targeted mutation of the CRABPI gene using the "hit-and-run" strategy and shown that it prevents the production of a functional CRABPI protein. Homozygous mutant mice were normal, indicating that CRABPI does not play a crucial role in the RA signaling pathway.

Two rhombomeres are altered in Hoxa-1 mutant mice.

This study provides a detailed description of the anatomical defects in the Hoxa-1-/- mutant mice previously generated in our laboratory (T. Lufkin, A. Dierich, M. LeMeur, M. Mark and P. Chambon, 1991; Cell 66, 1105-1119). Three-dimensional reconstructions of the Hoxa-1-/- rhombencephalon reveals that it bears only five rhombomeric structures (ie. morphological segments) instead of the normal seven. The first three of these rhombomeres appear normal as judged from the distribution pattern of CRABPI transcripts in the neurectoderm and from the histological analysis of the cranial nerve components derived from these structures. In contrast, the neural-crest-cell-free region normally located opposite rhombomere 5 is lacking in Hoxa-1-/- embryos, and motor neurons of the facial and abducens nerves, which normally differentiate within rhombomeres 4, 5 and 6, are missing in Hoxa-1-/- fetuses. These morphological data, combined with the determination of the molecular positional identities of the rhombomeres 4 and 5 (P. Dollé, T. Lufkin, R. Krumlauf, M. Mark, D. Duboule and P. Chambon, 1993; Proc. Natl. Acad. Sci. USA, in press), suggest that rhombomere 4 is markedly reduced, whereas rhombomere 5 is almost absent. Thus, the remnants of rhombomeres 4 and 5 appear to be fused caudally with rhombomere 6 to form a single fourth rhombomeric structure. Moreover, the migration of neural crest cells contributing to the glossopharyngeal and vagus nerves occurs in a more rostral position, resulting in abnormalities of these cranial nerves, which were visualized by whole-mount anti-neurofilament immunostaining. The mutual relationship along the rostrocaudal axis between the otic pit and the neuroepithelial site of int-2 protein secretion (a putative otogenic cue) is not significantly changed in Hoxa-1-/- embryos. However, the abnormal relationship between the rhombencephalon and the epithelial inner ear may account for the aplasia and faulty differentiation of the membranous labyrinth, the disruption of the cartilaginous otic capsule and the disorganisation of some middle ear structures. This phenotype is compared with that of the Hoxa-1-/- mutants generated by O. Chisaka, T. S. Musci and M. R. Capecchi, 1992 (Nature 335, 516-520) and with that of the mice homozygous for the kreisler mutation.

High postnatal lethality and testis degeneration in retinoic acid receptor alpha mutant mice.

Retinoic acid (RA) plays a critical role in normal development, growth, and maintenance of certain tissues. The action of RA is thought to be mediated in part by the three nuclear receptors (RAR alpha, -beta, and -gamma), each of which is expressed as multiple isoforms. To investigate the function of the RAR alpha gene, we have disrupted, in the mouse, the whole gene or the isoform RAR alpha 1. Although RAR alpha 1 is the predominant isoform and is highly conserved among vertebrates, RAR alpha 1-null mice appeared normal. However, targeted disruption of the whole RAR alpha gene resulted in early postnatal lethality and testis degeneration. These results, showing that RAR alpha is indeed involved in the transduction of the RA signal, also suggest an unexpected genetic redundancy.