The association between retinal nerve fibre layer thickness and N-acetyl aspartate levels in multiple sclerosis brain normal-appearing white matter: a longitudinal study using magnetic resonance spectroscopy and optical coherence tomography.

BACKGROUND AND PURPOSE: N-acetyl aspartate (NAA) assessed using proton magnetic resonance spectroscopy (1 H MRS) has a high pathological specificity for axonal density. Retinal nerve fibre layer thickness (RNFLT) measured by using optical coherence tomography is increasingly used as a surrogate marker of neurodegeneration in multiple sclerosis (MS). Our aim was to investigate the relation between RNFLT and NAA/creatine in brain normal-appearing white matter (NAWM), their dynamics over time and the association with clinical outcome measures in relapsing MS. T2 WM lesions served as control tissue. METHODS: Forty-three MS patients underwent standardized neurological examination including the Expanded Disability Status Scale (EDSS), Multiple Sclerosis Functional Composite (MSFC) score, optical coherence tomography and magnetic resonance imaging including 1 H MRS at baseline and after 1 year. RESULTS: At baseline, NAA/creatine level was lower in T2 WM lesions than in NAWM (1.64 ± 0.16 vs. 1.88 ± 0.24, P < 0.001). Lowest levels were found in secondary progressive MS (SPMS). Mean RNFLT was higher in clinically isolated syndrome than in the combined group of relapsing-remitting MS and SPMS (99.8 ± 12.3 µm vs. 92.4 ± 12.8 µm, P = 0.038). In all patients, mean RNFLT decreased by 1.4% during follow-up. At baseline, MSFC z-scores correlated with NAA/creatine levels both in NAWM (r = 0.42; P = 0.008) and T2 WM lesions (r = 0.52, P = 0.004). NAWM NAA/creatine variation correlated with the RNFLT change over 1 year (ρ = 0.43, P = 0.046). CONCLUSIONS: N-acetyl aspartate/creatine level reduction correlated with RNFLT thinning over 1 year in an EDSS stable MS cohort suggesting that these techniques might be sensitive to detect subclinical disease progression.

[Prognostic effects of cardiac rehabilitation in patients with acute coronary syndrome]. / Impacto pronóstico de la realización de un programa de rehabilitación cardiaca tras un síndrome coronario agudo.

AIM: Reduction of major adverse cardiovascular events (MACE) at 5 years in patients with acute coronary syndrome (ACS) and percutaneous coronary intervention who completed a cardiac rehabilitation program. METHODS: We included 230 consecutive men with a diagnosis of ACS and percutaneous coronary intervention in the first half of 2015 according to the inclusion criteria. Two cohorts of 115 patients each were compared, one of them (rehabilitated group) with patients who completed the cardiac rehabilitation program and the other (control group) who received conventional outpatient care. Base-line characteristics and MACE at 5-years follow-up were compared and analyzed. MACE included re-infarction, heart failure, cerebrovascular accident and all-cause mortality. RESULTS: At 5 years of follow-up, a statistically significant reduction in MACE (27.19% vs 7.83%; OR 0.23 [IC 0.10-0.50]; P=.0001), all-cause mortality (OR 0.12 [IC 0.01-0.94]; P=0.01), re-infarction rate (OR 0.29 [IC 0.11-0.77]; P=.007) and cerebrovascular accident (P=.014) was found in the rehabilitated group (RG). MACE-free survival was significantly longer in the RG, regardless of risk (HR 0.25 [IC 0.12-0.53]; P=.000). CONCLUSION: In our sample, cardiac rehabilitation programs showed a prognostic benefit in patients with ACS and percutaneous coronary intervention, with a statistically significant reduction in MACE, re-infarction rate and CVA at five-year follow-up.

Prorhombomeric subdivision of the mammalian embryonic hindbrain: is it functionally meaningful?

The technique of whole embryo culture has made significant contributions to understanding the mechanisms of morphogenesis in mammalian embryos, especially with respect to cranial neurulation and neural crest cell migration. This study traces the fate of two specifically mammalian structures, the preotic and otic sulci. Their formation at the 1/2- and 3-somite stages respectively, divides the hindbrain neuroepithelium into prorhombomeres A, B and C. The preotic sulcus is a deeply recessed structure that forms the rostral boundary of expression of both Hoxb-2 and the first domain of Krox-20. The otic sulcus is a shallow concavity in which the second Krox-20 domain is expressed. DiI labeling followed by whole embryo culture confirmed that the later fate of the preotic sulcus is the rhombomere 2/3 boundary, and the fate of the otic sulcus is the cranial part of rhombomere 5. Structurally, the preotic and otic sulci show no specialization with respect to actin, tubulin or proteoglycans, but their maintenance depends on contact with the subjacent mesenchyme. Their formation is inhibited by exposure of embryos to retinoic acid prior to the onset of somitic segmentation, indicating that the molecular events governing prorhombomeric subdivision of the hindbrain are retinoic acid-sensitive. The preotic sulcus may be essential for neuroepithelial cell movement towards and into the rapidly enlarging forebrain; the otic sulcus may simply delineate the caudal boundary of prorhombomere B, an area with a discrete neural crest cell population discontinuous with those rostral and caudal to it. Understanding the positional relationships of the preotic and otic sulci to later rhombomeric segments makes them useful landmarks for experimental purposes, but there is no evidence that prorhombomeres are functionally significant as the precursors of rhombomeric segments.

The colocalization of cholecystokinin and tyrosine hydroxylase mRNAs in mesencephalic dopaminergic neurons in the rat brain examined by in situ hybridization.

The colocalization of cholecystokinin and tyrosine hydroxylase mRNAs was studied with a cellular resolution in the mesencephalic dopaminergic neurons of the rat brain by in situ hybridization using synthetic oligonucleotides. An extensive colocalization of cholecystokinin-expressing cells, greater than that seen previously by immunohistochemistry, was found in the ventral tegmental area and in the substantia nigra pars compacta. We observed in these regions that cholecystokinin and tyrosine hydroxylase mRNAs coexisted in the same neurons but not all dopamine cells expressed cholecystokinin mRNA. 6-Hydroxydopamine-induced destruction of mesostriatal dopaminergic neurons resulted in a complete loss of cholecystokinin and tyrosine hydroxylase mRNA expression throughout the substantia nigra pars compacta, indicating that all cholecystokinin expressing cells are 6-hydroxydopamine-sensitive. While increased enkephalin mRNA expression in the striatum ipsilateral to the lesion was detected, no change of cholecystokinin mRNA expression was observed in any forebrain on the lesioned side, suggesting that cholecystokinin expression in the forebrain is not under dopaminergic control. These results show the usefulness of the in situ hybridization approach for the precise localization of cells in rat brain which express mRNAs for cholecystokinin and tyrosine hydroxylase and for the study of the effects of neurotoxic lesions on these cells.

Mammalian neural crest and neural crest derivatives.

In the mammalian embryonic trunk, neural crest cells emigrate from the closed neural tube in a cranio-caudal sequences and appear to have similar migration pathways and derivatives to those of avian embryos. In the cranial region, however, there are mammalian-specific features, which are related to the mammalian-specific pattern of cranial neurulation. Midbrain and rostral hindbrain neural crest cells emigrate from widely open neural folds; caudal hindbrain crest emigrates in a caudo-rostral sequence, following the sequence of neural tube closure in this region. The forebrain is also a source of neural crest cells at early stages of neurulation; both forebrain and midbrain crest cells contribute to the frontonasal mesenchyme, although their relative contributions have not been analysed. Few studies have provided direct information about mammalian neural crest cell derivatives. Studies on the effects of retinoid excess on craniofacial development provide indirect evidence that mammalian cranial neural crest, like that of avian embryos, includes two populations whose differentiated phenotype and morphological tissue structure are determined prior to emigration. Retinoid-induced shortening of the preotic hindbrain leads to abnormal migration pathways of the neural crest cells that normally migrate into the mandibular arch to form Meckel's cartilage, so that an ectopic Meckel's cartilage-like structure forms in the maxillary region of the face. Slow descent of the heart in retinoid-exposed embryos enables the "wrong" crest cell population to populate the wall of the truncus arteriosus. These observations correlate well with observations of retinoid-induced craniofacial and heart abnormalities in human infants.

Retinoid receptors in vertebrate limb development.

Although the precise role of retinoids in limb development remains obscure, the finding that retinoic acid can produce major alterations in limb patterning suggests that this ligand might be involved in the process of limb morphogenesis. Here we describe the patterns of expression of retinoic acid receptors and cytosolic retinoid binding proteins during the course of limb morphogenesis. Examining the distribution of these molecules in the limb and correlating their presence with important processes in limb development could help elucidate their possible functions.

Differential distribution patterns of CRABP I and CRABP II transcripts during mouse embryogenesis.

We have compared the transcript distribution of cellular retinoic acid binding protein (CRABP) I and II genes in mouse embryos at various stages of development. Both CRABP transcripts are present in embryonic structures from the earliest stages studied and exhibit specific patterns of distribution, suggesting that the two retinoic acid (RA) binding proteins perform different functions during mouse embryogenesis. The CRABP I transcript distribution correlates well with structures known to be targets of excess retinoid-induced teratogenesis (e.g. neural crest cells and hindbrain), suggesting that cells expressing CRABP I are those that cannot tolerate high levels of RA for their normal developmental function. The embryonic structures expressing CRABP II transcripts include those structures that have been shown to be adversely affected by excess of retinoids, such as limbs and hindbrain, but CRABP II transcripts are also found in structures not known to be specifically vulnerable to raised RA levels. The CRABP II gene is coexpressed with retinoic acid receptor (RAR)-beta and cellular retinol binding protein (CRBP) I genes in a number of tissues such as the gut endoderm, hypophysis and interdigital mesenchyme, all of which are devoid of CRABP I transcripts. Interestingly, the expression of the three genes, RAR-beta, CRABP II and CRBP I, is induced by retinoic acid, which suggests a link between the synthesis of RA from retinol and the control of expression of subsets of RA-responsive genes. The transcript distribution of CRABP I and II is discussed in relation to the teratogenic effects of RA, and compared to the RA-sensitive pattern of expression of other important developmental genes.

Retinoic acid receptors and cellular retinoid binding proteins. II. Their differential pattern of transcription during early morphogenesis in mouse embryos.

In situ hybridization with 35S-labelled RNA probes was used to study the distribution of transcripts of genes coding for the retinoic acid receptors, RAR-alpha, -beta and -gamma, and the cellular binding proteins for retinoic acid (CRABP I) and retinol (CRBP I), in mouse embryos during the period of early morphogenesis. Primary mesenchyme formation was associated with CRBP I labelling of both epiblast and mesenchyme of the primitive streak, while the CRABP probe labelled the migrating primary mesenchyme cells. Neural crest cell emigration and migration were associated with CRABP labelling of both neural epithelium (excluding the floor plate) and neural crest cells, while CRBP I expression was restricted to basal and apical regions of the epithelium (excluding the floor plate). The strongest neuroepithelial signal for CRABP was in the preoptic hindbrain. RAR-beta was present in presomitic stage embryos, being expressed at highest levels in the lateral regions. RAR-alpha was associated with crest cell emigration and migration, while RAR-gamma was present in the primitive streak region throughout the period of neurulation. There was a change from RAR-beta to RAR-gamma expression at the junction between closed and open neural epithelium at the caudal neuropore. RAR-alpha and RAR-beta were expressed at specific levels of the hindbrain and in the spinal cord. These distribution patterns are discussed in relation to segmental expression patterns of other genes, and to maturational changes in the caudal neuropore region. The CRABP transcript distribution patterns correlated well with known target tissues of excess retinoid-induced teratogenesis (migrating primary mesenchyme and neural crest cells, preoptic hindbrain), providing further support for our hypothesis that cells expressing CRABP are those that cannot tolerate high levels of RA for their normal developmental function.

Retinoic acid receptors and cellular retinoid binding proteins. III. Their differential transcript distribution during mouse nervous system development.

We have studied the transcript distribution of the retinoic acid receptors (RARs) and the cytoplasmic retinoid binding proteins during embryonic development of the mouse nervous system. Of the three retinoic acid receptors, only RAR-gamma was not expressed in developing neural structures. RAR-beta and RAR-alpha both showed rostral limits of expression in the medulla oblongata equivalent to their patterns of expression in the neuroepithelium of the early hindbrain neural tube. Within their expression domains in the spinal cord and brain, RAR-alpha was ubiquitously expressed, whereas RAR-beta transcripts showed very specific patterns of expression, suggesting that this receptor is involved in mediating retinoic acid-induced gene expression in relation to the development of specific neural structures or pathways. The cytoplasmic binding proteins, cellular retinoic acid binding proteins type I and II (CRABP I and CRABP II) and cellular retinol binding protein type I (CRBP I), were widely distributed in developing neural structures. Their differential spatiotemporal patterns of expression suggest that fine regional control of availability of retinoic acid (RA) to the nuclear receptors plays an important role in organization and differentiation of the nervous system. For instance, expression of CRABP I in the migrating cells that give rise to the olivary and pontine nuclei, which develop abnormally in conditions of retinoid excess, is consistent with observations from a variety of other systems indicating that CRABP I limits the access of RA to the nuclear receptors in normal physiological conditions. Similarly, expression of CRBP I in the choroid plexuses, which develop abnormally in conditions of vitamin A deficiency, is consistent with observations indicating that this binding protein mediates the synthesis of RA in tissues requiring high levels of RA for their normal developmental programme. RAR-beta and CRABP II, which are both RA-inducible, were coexpressed with CRBP I in the choroid plexus and in many other sites, perhaps reflecting the fact that all three genes are RA-inducible. The function of CRABP II is not well understood; its domains of expression showed overlaps with both CRABP I and CRBP I.

Retinoic acid receptors and cellular retinoid binding proteins. I. A systematic study of their differential pattern of transcription during mouse organogenesis.

We report here the gene expression patterns, as revealed by in situ hybridisation, of the retinoic acid receptors alpha, beta and gamma (RAR-alpha, -beta and -gamma), and the cellular binding proteins for retinol and retinoic acid (CRBP, CRABP) in non-neural tissues of mouse embryos during the period of organogenesis. At all stages, RAR-alpha transcripts were almost ubiquitous, whereas the distribution of transcripts of the other four genes was distinctive in all systems. At early stages in the formation of an organ, the expression patterns were different in the epithelium, the adjacent mesenchyme, and in mesenchyme more distant from the epithelium, suggesting a role for RA and RA receptors in epithelial-mesenchymal tissue interactions. In the developing face, limb bud and genital tubercle, where large expanses of mesenchyme are present, differential patterns of expression were established before the onset of overt tissue differentiation, suggesting some significance for pattern formation in these regions. The distribution of RAR-beta transcripts in tracheobronchial, intestinal and genital tract epithelial is consistent with the possibility that RAR-beta plays a role in mediating retinoid effects on the differentiated stage of these epithelia. Possible developmental roles of RARs in relation to the expression patterns of other genes are discussed. CRBP expression domains showed a high degree of overlap with RAR-beta and RAR-gamma, and a mutual exclusivity with CRABP expression domains. Correlation of these expression patterns with the morphogenetic effects of vitamin A deficiency and retinoid excess lead us to propose that the function of CRBP is to store and release retinol where high levels of RA are required for specific morphogenetic processes, while CRABP serves to sequester RA in regions where normal developmental functions require RA levels to be low. Where both binding protein genes are expressed in a non-overlapping pattern within a large area of mesenchyme, a gradient of free RA may be created between them by release of retinol-derived RA from CRBP-expressing cells, with binding to CRABP enhancing the steepness of the decline in concentration distant to the source.

Specific spatial and temporal distribution of retinoic acid receptor gamma transcripts during mouse embryogenesis.

Retinoic acid (RA), a putative morphogen in vertebrates, has profound effects on development during embryogenesis, chondrogenesis and differentiation of squamous epithelia. The distribution of the transcripts of the retinoic acid receptor gamma (RAR-gamma) gene has been studied here by in situ hybridization during mouse development from days 6.5 to 15.5 post-coïtum (p.c.). RAR-gamma transcripts are detected as early as day 8 p.c. in the presomitic posterior region. Between days 9.5 and 11.5 p.c., the transcripts are uniformly distributed in the mesenchyme of the frontonasal region, pharyngeal arches, limb buds and sclerotomes. At day 12.5 p.c., RAR-gamma transcripts are found in all precartilaginous mesenchymal condensations. From day 13.5 p.c., the transcripts are specifically localized in all cartilages and differentiating squamous keratinizing epithelia, irrespective of their embryological origin. RAR-gamma transcripts are also found in the developing teeth and whisker follicles. The developmental pattern of expression of the RAR-gamma gene suggests that RAR-gamma plays a crucial role for transducing RA signals at the level of gene expression during morphogenesis, chondrogenesis and differentiation of squamous epithelia.

Retinoic acid-induced glandular metaplasia in mouse skin is linked to the dermal expression of retinoic acid receptor beta mRNA.

The distribution of transcripts of nuclear (RAR alpha, RAR beta, and RAR gamma) and cytosolic (CRABP) retinoic acid receptors was analyzed in 13.5-day mouse embryo upper-lip skin, cultured in vitro for 48 hr with or without added retinoic acid. The results show a significant up-regulation of the transcription of the RAR beta gene concomitant with the initiation of an alteration of hair vibrissae follicle development, leading, after transfer for 8 days to the chick embryo chorioallantoic membrane, to an exocrine-type gland morphogenesis.

Developmental analysis of the retinoic acid-inducible RAR-beta 2 promoter in transgenic animals.

Retinoic acid (RA) is a signalling molecule important for pattern formation during development. There are three known types of nuclear receptors for RA in mammals, RAR-alpha, RAR-beta and RAR-gamma, which transduce the RA signal by inducing or repressing the transcription of target genes. Here we describe the developmental expression pattern of the mouse RAR-beta 2 promoter. Independent lines of transgenic animals expressing RAR-beta 2 promoter sequences fused to the E. coli beta-galactosidase gene were examined throughout the course of embryogenesis and found to exhibit reproducible and specific patterns of beta-galactosidase expression in a majority of sites that have been shown previously to contain mRAR-beta transcripts. In the limbs, mRAR-beta 2 promoter activity and mRAR-beta transcripts were both excluded from precartilagenous condensations; interestingly, mRAR-beta 2 promoter activity was observed in the apical ectodermal ridge (AER) where mRAR-beta transcripts could not be detected, while no mRAR-beta 2 promoter activity or mRAR-beta transcripts were associated with the limb region that contains the zone of polarizing activity (ZPA). Analysis of the lacZ expression pattern in embryos from mothers treated with teratogenic doses of RA, indicated that mRAR-beta 2 promoter is selectively induced in a manner suggesting that overexpression of the mRAR-beta 2 isoform is involved in RA-generated malformations. The normal and induced expression pattern of the mRAR-beta 2 promoter suggests several possible roles for mRAR-beta 2 in development of the limbs, as an inhibitor of cartilage formation, in programmed cell death and in the formation of loose connective tissue.

Involvement of cellular retinoic acid-binding proteins I and II (CRABPI and CRABPII) and of the cellular retinol-binding protein I (CRBPI) in odontogenesis in the mouse.

The coordination of the activities of individual cells during development is regulated in part by epigenetic signals either encoded in the insoluble extracellular matrix or provided by small diffusible factors such as growth factors peptides and retinoids. Odontogenesis offers a suitable model to correlate the temporospatial distributions of such molecules, and of their cell receptors and ligands, with particular developmental processes. We have analyzed, by in situ hybridization, the distribution patterns of CRABPI, CRABPII and CRBPI transcripts during odontogenesis in the mouse. CRABPI transcripts were restricted to the mitogenic regions of the dental mesenchyme during late bell stages and were absent from post-mitotic odontoblasts. The only epithelial site of CRABPI transcription was the labial epithelial loop of the continuously growing incisor. CRABPII transcription was preponderant in the mitogenic zones of the dental epithelium: differential labeling of the dental epithelium occurred as early as the dental bud stage and during subsequent molar morphogenesis, this labeling became confined in the epithelial loops. The graded distribution of CRABPII transcripts along the anteroposterior axis of the continuously growing incisor was superimposed with the gradient of mitoses. CRABPII transcripts were absent from post-mitotic ameloblasts. It is concluded that during odontogenesis the expressions of the CRABPI and CRABPII genes are confined to regions exhibiting the highest rate of cell proliferation whenever differential mitotic activity is required. Moreover, the putative effects of retinoic acid on the regulation of cell proliferation kinetics in the dental epithelium and in the dental mesenchyme imply distinct CRABPs. CRBPI transcripts were restricted to the dental mesenchyme prior to the onset of CRABPI transcription. This observation supports the hypothesis that the two proteins might perform antagonistic functions in some retinoic acid-mediated developmental processes.

Antibodies specific to the retinoic acid human nuclear receptors alpha and beta.

Two cDNAs encoding two human receptors for retinoic acid (RA), RAR-alpha and RAR-beta, have been characterized recently. Synthetic peptides corresponding to the cDNA-deduced amino acid sequences unique to RAR-alpha and RAR-beta were used to generate anti-RAR-alpha antiserum (SP171) and anti-RAR-beta antisera (SP172 and SP248). The specificity of these antisera was confirmed both by immunocytochemical detection of these receptors in COS-1 cells transfected with RAR-alpha and RAR-beta expression vectors and by immunoblot analyses performed with whole extracts of these cells. We also demonstrate that these antisera recognize RAR-alpha and RAR-beta endogenously expressed in the RA-responsive human promyelocytic leukemia cell line HL-60.

An absolute requirement for both the type II and type I receptors, punt and thick veins, for dpp signaling in vivo.

TGF beta elicits diverse cellular responses by signaling through receptor complexes formed by two distantly related transmembrane serine/threonine kinases called type II and type I receptors. Previous studies have indicated that the product of the Drosophila thick veins (tkv) gene is a type I receptor for decapentaplegic (dpp). Here, we show that the Drosophila gene punt encodes a homolog of a vertebrate type II receptor, and we demonstrate that punt, like tkv, is essential in vivo for dpp-dependent patterning processes. Because no dpp-related signalling is apparent in the absence of either the punt or tkv receptor, we infer that both receptors act in concert to transduce the dpp signal and that their functions cannot be replaced by the other extant type II and I receptors.

Differential expression of genes encoding alpha, beta and gamma retinoic acid receptors and CRABP in the developing limbs of the mouse.

Retinoic acid has profound effects on vertebrate limb morphogenesis (refs 1-6, reviewed in refs 7-9), including in the mouse, where it can act as a teratogen generating phocomelia and bone defects. A retinoic acid gradient, possibly amplified by a graded distribution of cellular retinoic acid-binding protein (CRABP), could provide positional information across the antero-posterior axis of the chick limb bud. The discovery of nuclear retinoic acid receptors (RARs) acting as retinoic acid-inducible enhancer factors provided a basis for understanding how retinoic acid signals could be transduced at the level of gene expression. We have now used in situ hybridization to study the distribution of messenger RNA transcripts of the three murine receptors (mRARs) and CRABP during mouse limb development. Both mRAR alpha and mRAR gamma transcripts, but not those for mRAR beta, are present and uniformly distributed in the limb bud at day 10 post-coitum, whereas CRABP transcripts have a graded proximo-distal distribution, indicating that differential expression of CRABP, but not of mRAR alpha or mRAR gamma, could participate in the establishment of the morphogenetic field. At later stages, mRAR gamma transcripts become specific to the cartilage cell lineage and to the differentiating skin and mRAR beta transcripts are mostly restricted to the interdigital mesenchyme. CRABP transcripts, however, are excluded from regions expressing mRAR gamma and mRAR beta. These results indicate that all three RARs and CRABP have specific functions during morphogenesis and differentiation of the mouse limb.

DPP controls tracheal cell migration along the dorsoventral body axis of the Drosophila embryo.

We report that DPP signaling is required for directed tracheal cell migration during Drosophila embryogenesis. The failure of tracheal cells to receive the DPP signal from adjacent dorsal and ventral cells results in the absence of dorsal and ventral migrations. Ectopic DPP signaling can reprogram cells in the center of the placode to adopt a dorsoventral migration behavior. The effects observed in response to ectopic DPP signaling are also observed upon the tracheal-specific expression of a constitutive active DPP type I receptor (TKV(Q253D)), indicating that the DPP signal is received and transmitted in tracheal cells to control their migration behavior. DPP signaling determines localized gene expression patterns in the developing tracheal placode, and is also required for the dorsal expression of the recently identified BRANCHLESS (BNL) guidance molecule, the ligand of the BREATHLESS (BTL) receptor. Thus, DPP plays a dual role during tracheal cell migration. It is required to control the dorsal expression of the BNL ligand; in addition, the DPP signal recruits groups of dorsal and ventral tracheal cells and programs them to migrate in dorsal and ventral directions.

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.