Human Molecular Genetics, 2000, Vol. 9, No. 9 1443-1452
© 2000 Oxford University Press
Barhl1, a gene belonging to a new subfamily of mammalian homeobox genes, is expressed in migrating neurons of the CNS
1Telethon Institute of Genetics and Medicine (TIGEM), 2Department of Biological and Technological Research (DIBIT), 3Universita’ Vita-Salute San Raffaele, San Raffaele Biomedical Science Park, Via Olgettina 58, 20132 Milan, Italy and 4Department of Morphological Sciences, Faculty of Medicine, University of Murcia, Murcia, Spain
Received 7 February 2000; Revised and Accepted 27 March 2000.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The BarH1 and BarH2 (Bar) Drosophila genes are homeobox-containing genes, which are required for the fate determination of external sensory organs in the fly. By means of a bioinformatic approach, we have identified in mouse and human two homeobox genes highly related to the Bar Drosophila genes, Barhl1 and Barhl2. While Barhl1 represents a novel gene, Barhl2 turned out to correspond to the mBH1 cDNA recently described in rat. We isolated and sequenced the full-length mouse Barhl1 and mapped both the human BARHL1 and BARHL2 genes to chromosomes 9q34 and 1p22, respectively. Detailed analysis of the murine Barhl1 expression pattern by in situ hybridization revealed that this transcript is exclusively expressed in restricted domains of the developing CNS, which suggests that this gene, similar to its Drosophila counterparts BarH1 and BarH2, may play a crucial role in cell fate determination of neural structures. In particular, Barhl1 showed specific domains of expression in the diencephalon and in the rhombencephalon where it was found to be expressed in migrating cells giving rise to the cerebellar external granular layer and to specific populations of dorsal sensory interneurons of the spinal cord. Thus, Barhl1 function may be required for the generation of these specific subtypes of neuronal progenitors. Furthermore, the mapping assignment and the expression pattern make BARHL1 an attractive positional candidate gene for a form of Joubert syndrome, a rare developmental anomaly of the cerebellum in humans.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The development of the mammalian neural system is a very complex process that requires an ordered series of steps beginning with an initial growth phase of dividing precursor cells and the subsequent process of differentiation of post-mitotic cells. The molecular mechanisms controlling these events still remain largely elusive and involve many genes, most of which have not yet been identified and characterized. One way to gain insight into the genetic program of neural development in mammals is represented by the study of model organisms. In fact, many of the pathways involved in neurogenesis have been found to share similarities during evolution and numerous genes have been found to be conserved and to play similar roles even in distantly related species. In particular, the fruitfly Drosophila melanogaster is one of the most valuable model organisms to study development (1). Many genes playing a role in neurogenesis in the fly have been characterized at both the genetic and molecular levels (2,3). This has led to the understanding of some of the basic pathways underlying neural development in Drosophila, particularly of the sensory organs. The proneural genes such as the four members of the Achaete–scute complex, daughterless and atonal, act as positive regulators for sensory organ formation, as opposed to other genes like hairy, Enhancer of split and extramacrochetae, which act as negative regulators (for review see refs 4,5). A number of upstream regulators of proneural genes have also been identified and several of them are transcription factors which are required for the initial activation of proneural genes in sensory organs (6–9). Among these are the BarH1 and BarH2 genes. They are required for the fate determination of external sensory organs and for normal eye development in the fly (10–12). Recently, they have also been found to play a crucial role in regional pre-patterning of specific structures of the Drosophila nervous system, such as the notum. The BarH1 and BarH2 genes exert this function through an upstream regulation of the proneural achaete–scute genes (13).
We recently started a project aimed at the identification and characterization of novel human cDNAs similar to Drosophila mutant genes. By screening the Expressed Sequence Tag database (dbEST) using the BLAST software, we identified >200 such human cDNAs and we termed them Drosophila-related expressed sequences (DRES) (14,15). One of the DRES genes identified in the course of our dbEST screening, DRES115, was found to bear significant homology to the homeodomain of the Drosophila genes BarH1 and BarH2. Here, we report a detailed characterization of the Dres115 (Barhl1) gene including cDNA identification, sequence and mapping assignment in both human and mouse. In addition, we have isolated and mapped the human homolog of mBH1 (Barhl2), a gene highly related to Barhl1 which has been described previously in rat (16). A comprehensive study of Barhl1 expression by RNA in situ hybridization revealed that this gene is expressed in discrete domains of the CNS, particularly in migrating progenitor cells, giving rise to the cerebellar external granular layer and to specific populations of dorsal sensory interneurons of the spinal cord.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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cDNA identification and sequence analysis
By analyzing the DRES Search Engine (17), a collection of TBLASTN searches obtained using all Drosophila protein sequences as queries, we identified a mouse EST (GenBank accession no. W36375), significantly similar to the homeodomain of the Drosophila proteins BARH1 and BARH2 (GenBank accession nos B39369 and S27806, respectively). The EST W36375 was derived from the IMAGE mouse embryo cDNA clone 335997 and we named this clone DRES115. Due to its sequence similarity, the DRES115 cDNA was renamed BarH (Drosophila)-like 1 (Barhl1). Using the entire cDNA insert as a probe, we screened two different mouse cDNA libraries: an embryonic day 11.5 (E11.5) embryo and an embryonic carcinoma cDNA library. These screenings led to the identification of five cDNA clones, among which clone ME9 was found to span the entire Barhl1 transcript (1593 bp). Sequence analysis of Barhl1 revealed a single open reading frame of 984 bp (GenBank accession no. AJ237590). The putative initiation codon was identified at position 18 within a nucleotide sequence meeting Kozak’s criteria (18). The open reading frame ends with a TGA stop codon at nucleotide 999. The Barhl1 gene is therefore predicted to encode a protein product of 327 amino acids.
To identify the human BARHL1 gene, we screened a human lambda genomic library and identified one positive clone which, by sequence analysis, was found to contain the last two exons of the human gene. By joining together these two exons, we obtained a partial BARHL1 coding sequence of 518 bp containing the homeobox and the stop codon (GenBank accession no. AJ237816). The degree of sequence identity between the partial human BARHL1 coding sequence and the corresponding murine sequence was 94% at the nucleotide level and 99% at the amino acid level (with only one amino acid change in the two proteins; Fig. 1A).
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Sequence comparison analysis revealed that the Barhl1 gene is a new member of the bar subgroup of homeobox genes, characterized by the presence of a tyrosine residue at position 49 (within the third helix) of the homeodomain rather than the more common phenylalanine. This class includes a limited number of proteins, namely the Drosophila BARH1 and BARH2, the Caenorhabditis elegans CEH-30, the hydra CNOX3, the mammalian BARX1 and BARX2, and the rat mBH1 proteins (Fig. 1B). The BARHL1 predicted protein shows the highest level of sequence similarity to the rat mBH1 protein and, as expected, to the Drosophila proteins BARH1 and BARH2. In particular, BARHL1 was found to be closely related to the mBH1 protein, recently described by Saito et al. (16), as the two proteins show 71% similarity and 67% identity. The stretch of highest sequence conservation between the two proteins was found in a region of 107 amino acids encompassing the homeodomain: only seven amino acid changes were detected in this portion. The degree of similarity is much lower in the N- and the C-termini (Fig. 1A); in the N-terminus, high similarity between the two proteins is found in two regions that have been designated by Saito et al. (16) as ‘FIL’ domains (FIL1 and FIL2 domains; Fig. 1A) because their consensus sequence is composed of phenylalanine, isoleucine and leucine. The degree of sequence similarity between the murine BARHL1 and the rat mBH1 in the N- and C-termini (very low compared with the almost complete identity found in the human and murine Barhl1 genes in the C-terminal region of the proteins), as well as their different patterns of expression (see below), indicated that they are not orthologous genes but rather paralogous genes belonging to a new subclass of mammalian homeobox genes. We confirmed this hypothesis by PCR analysis on human and murine genomic DNA (Materials and Methods), by isolating a fragment of 208 bp corresponding to the human and murine homologs of the rat mBH1 gene, hereafter termed Barhl2 (HGNC-approved gene symbol). The human BARHL2 fragment (GenBank accession no. AJ251753) shared 92 and 74% nucleotide identity with the rat Barhl2 and the human BARHL1 cDNA sequences, respectively.
The similarity of BARHL1 to the Drosophila BARH1 and BARH2, on the other hand, is mostly restricted to a region of 80 amino acids (Fig. 1A) including the homeodomain, and 20 amino acids immediately downstream (81% identity with BARH1 and 85% identity with BARH2). Also, the nematode protein CEH-30 shows a striking homology to BARHL1 in the same portion (70% identity). The degree of similarity between the murine BARHL1 and the murine BARX1 and BARX2 proteins is less significant (63 and 65% identity, respectively) and is restricted to the homeodomain. This indicates that the BARX proteins, previously reported to be very closely related to the Bar Drosophila genes, belong to a different subfamily of homeodomain-containing proteins which is also suggested by the different expression pattern (19,20).
Genomic mapping of Barhl1 and Barhl2
To establish the mapping assignment of the human BARHL1 gene, we performed a PCR analysis on the GeneBridge4 radiation hybrid panel (21) using a sequence tagged site generated in the genomic region downstream of the third coding exon of this gene. As a result, BARHL1 was assigned to chromosome 9q34, between markers WI-9685 and WI-6494. Interestingly, Saar et al. (22) recently reported on the genetic mapping of a locus for Joubert syndrome (a rare developmental disorder characterized by aplasia/hypoplasia of the cerebellar vermis) to 9q34, between the markers D9S114 and D9S1826. The latter region overlaps with the BARHL1 radiation hybrid (RH) mapping interval suggesting that BARHL1 is a positional candidate gene for this form of Joubert syndrome.
We also decided to establish the localization of Barhl1 in the mouse genome. By haplotype and linkage analysis of the BSS backcross DNA panel generated and maintained at The Jackson Laboratory (Bar Harbor, ME), we mapped the Barhl1 mouse gene to murine chromosome 2 (Fig. 2). Mapping of Barhl1 was made possible by the existence of an EcoRI polymorphism between the parental DNAs (C57BL/6JEi and SPRET/Ei). Such polymorphism allowed us to distinguish BS heterozygotes from SS homozygotes in the N2 progeny of the cross. Barhl1 maps between D2Ertd217e (q = 1.09; LOD = 25.3) distally and D2Ucl22 (q = 9.78; LOD = 14.9) proximally and cosegregates with Notch1. This region of murine chromosome 2 has been found to be syntenic to human chromosome 9q34 because the human homolog of Notch1 has been mapped to 9q34.3.
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We also decided to establish the mapping assignment of BARHL2, the human homolog of the rat mBH1 gene. For this purpose, we designed oligonucleotide primers on the BARHL2 fragment and performed a PCR analysis on the GeneBridge4 RH panel. This analysis revealed that the BARHL2 gene maps to chromosome 1p22, between the markers AFMA205XD5 and WI-7719.
Expression of the murine Barhl1 gene during embryonic development
To determine the temporal and spatial expression of Barhl1 during mouse development, we used in situ hybridization to histological sections and whole-mount preparations at several developmental stages. Barhl1 expression was highly dynamic over the course of development but exclusively restricted to the CNS. The earliest embryonic day examined was E7.5 and the first expression was detected at 9.5 days’ gestation in the caudal part of the diencephalic anlage. At later stages other domains of expression appeared in the mesencephalon, rhombencephalon and spinal cord. To interpret Barhl1 expression, we used the terminology of the prosomeric model which suggests that the forebrain (prosencephalon) can be divided into two preneuromeric domains: the diencephalon and the secondary prosencephalon. These two structures can be regionally subdivided into three diencephalic (p1–p3) and three secondary prosencephalic (p4–p6) molecular compartments called prosomeres (23,24). In its alar components, the diencephalon consists of the pretectum (p1), dorsal thalamus and epithalamus (p2), and ventral thalamus (p3) (Fig. 3O). The secondary prosencephalon includes the telencephalic and optic vesicles, the preoptic area and the hypothalamus (Fig. 3O).
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In the forebrain, Barhl1 is expressed exclusively in a restricted domain of the caudal diencephalon at early neural tube stage (E9.5), and more specifically in the ventral area of p1 and p2 (Fig. 3A). By E10.5, Barhl1 expression is detected along the neural tube in two separate rostral and caudal domains (Figs 3B, and 4A, D and G). The rostral diencephalic domain extends: (i) anteriorly, into the basal plate of prosomeres 3 and 4 (p3, p4), anlage of the mammillary area; (ii) dorsally, along the p2–p3 boundary corresponding to the zona limitans (ZL), which will separate the dorsal from the ventral thalamus; and (iii) posteriorly to the pretectum (p1) (Fig. 3B, E, F, G and M). The mammillary area corresponds to the only region where the mouse Barhl1 gene is expressed in the ventral midline (p4 in Fig. 3B and E). In the pretectum, two distinct domains of expression can be recognized, one in the basal plate and the other in the most alar and caudal region (Fig. 3B and M). This latter expression, corresponding to the commissural area, will extend basally by E11.5 (Fig. 3C, K, N and O). At E10.5, the caudal domain extends from the midbrain/isthmic boundary, through the rhombencephalon (cerebellar plates and rhombic lips) to the entire extent of the spinal cord. In these structures, this gene is expressed as a longitudinal stripe along the most dorsal area of the alar plate, but not in the roof plate (Figs 3B and H, and 4A, D and G).
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By E11.5 the expression pattern of Barhl1 changes dynamically: a positive band appears in the mesencephalic basal plate (Fig. 3C, L, N and O), and in the spinal cord the expression expands laterally, following the ventral migrating cells from the dorsal alar plate to the boundary between the basal and alar plate (Fig. 4E and H). At E12.5, the expression shows another significant change of pattern: in the diencephalon, expression becomes mainly restricted to the mammillary region (basal p4) (Fig. 3D, I and J), whereas mesencephalic expression is now localized in the rostral alar plate in the presumptive area of the superior colliculus (Figs 3D and 4C). In the caudal rhombencephalon, a large lateral band of expression is observed and probably corresponds to the cells migrating ventrally, which will form the pontine and inferior olivary nuclear complex (25,26) (Figs 3D and 4C). At this stage, most of the expression in the spinal cord is confined to the mantle layer in a lateral band between the basal and alar plate (Figs 3D, and 4F and I) and this pattern will not change during later stages of development (data not shown).
The sites of expression at late embryonic stages (E16.5), besides the spinal cord, are the mantle layer of the inferior colliculus anlage, with a clear caudo–rostral gradient, the pontine nucleus and the cerebellar external granular layer (Fig. 5A and B). Another site of expression, only evident at this stage of development, is the sensory epithelium of the inner ear, where Barhl1 expression is possibly restricted to the sensory hair cells (Fig. 5C).
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The expression pattern of Barhl1 was also examined in postnatal brain. Expression was observed in brain regions that derived from sites of earlier expression. In particular, Barhl1 is expressed at postnatal days 4 and 7 (P4 and P7) in the superficial layers of the superior colliculus, in the entire inferior colliculus, in the external cerebellar granular layer, in the pontine nucleus and in a restricted hypothalamic area (Fig. 5D and E, and data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In this paper, we describe the isolation and characterization of a novel mammalian gene, Barhl1, which represents a new member of the Bar subgroup of homeobox genes, characterized by the presence of a tyrosine residue at position 49 of the homeodomain instead of the more common phenylalanine (Fig. 1B). In particular, Barhl1 is highly related in terms of sequence similarity to another homeobox-containing gene, mBH1, recently described in rat by Saito et al. (16). The predicted proteins encoded by the mouse Barhl1 and the rat mBH1 cDNAs show very significant sequence similarity particularly in a central portion of 107 amino acids spanning the homeodomain, while the N- and C-termini show much lower homology (Fig. 1A). To rule out the possibility that the mBH1 gene could be the rat homolog of Barhl1, we isolated two partial cDNA fragments belonging to the murine and human counterparts of mBH1 and verified that they are different from Barhl1. Also, the expression patterns of mBH1 (Barhl2) and Barhl1 are different, since mBH1 has been reported to be expressed in retina and in the dorsal thalamus (16), sites where Barhl1 was never detected in our experiments. Therefore, Barhl1 and mBH1 (now renamed Barhl2, HGNC-approved symbol) represent two members of a new subfamily of Bar homeobox genes. Barhl1 and Barhl2 are the mammalian genes showing the highest sequence similarity to the two Bar Drosophila genes BarH1 and BarH2. In fact, the murine proteins BARX1 and BARX2, which are also characterized by the presence of a Bar-like homeodomain and have previously been reported to be closely related to the Bar Drosophila proteins (19,20), show a much lower sequence similarity (restricted to the homeodomain) to the BARH1 and BARH2 proteins (Fig. 1B). These data indicate that the Barhl genes represent the mammalian transcripts most closely related to the Bar Drosophila genes.
Barhl1 expression and neural development
Barhl1 is exclusively expressed in restricted domains of the developing CNS. The regionalization of gene expression in the embryonic CNS is believed to provide positional information that regulates the generation of defined populations of neurons and appropriate guidance of their axons. On the basis of the analysis of its expression pattern, we argue that Barhl1 can be involved in such processes at different levels of the neural axis.
Barhl1 is first detected at E9.5 in the basal and caudal regions of the prosencephalon (p1 and p2) that correspond to the anlage of the diencephalon, a structure that will differentiate into the pretectum and the thalamus. From E10.5, expression is detected in the basal zone of the secondary prosencephalon and diencephalon. In the pretectum, Barhl1 is expressed in the most ventral part of the basal zone (basal p1) and, separately, in the alar domain (alar p1). In the latter region, three other homeobox genes are expressed: Pax6, Lim1 and Gsh1 (27). Unlike Pax6, which begins expression at an earlier stage, Barhl1, Lim1 and Gsh1 expression in p1 initiate at E10.5. It is therefore reasonable to hypothesize that these genes are involved in establishing the identity of p1 cells as different from mesencephalic cells, and that the timing of Lim1, Gsh1 and Barhl1 expression suggests that these genes may be downstream of Pax6.
At E11.5, Barhl1 expression extends into the basal plate of the caudal hypothalamus, stopping abruptly at the borders between the mammillary and infundibulary region, the limit between the prosomeres 4 and 5 (Fig. 3). More caudally, basal expression in P2 and P3 extends dorsally into the ZL between the ventral and dorsal thalamus. Expression in the hypothalamus also persists postnatally: Barh1l is clearly detected at P4 in the anterior and lateral mammillary nuclei and, at lower levels, in the retromammillary domains (Fig. 5). The extensive expression in the above mentioned embryonic structures, such as the thalamus and the mammillary region, suggests that Barhl1 may have a role in the development of these central processing stations. In adult brain, the thalamus connects rostral brain centers with the brainstem and the spinal cord, while the mammillary region is selectively involved in some aspects of learning and memory.
In the mesencephalon, Barhl1 starts to be expressed in the basal plate in a region corresponding to the periaqueductal grey at E11.5. By E12.5, strong expression is detected in the mantle of the tectum. At this stage, Barhl1 is first expressed in the entire superior colliculi, whereas by E16.5, its expression in the tectum is confined to the caudal colliculus anlage, with a clear caudo–rostral gradient. The inferior colliculi, functionally related to the auditory system, and the superior colliculi, layered structures which are part of the visual system, are generated in the alar plate region of the mesencephalon through a complex neuroblast migration process.
Barhl1 is a novel marker of cerebellar migrating neurons
The post-mitotic neurons generated in the alar rhombencephalon and, in particular, in the region of incomplete closure of the dorsal neural tube named rhombic lip, migrate to various destinations, giving rise to cerebellar granule cells and the precerebellar nuclei. Here, we show that Barhl1 is strongly expressed in the rhombic lip as well as in the structures deriving from it. The cerebellar granule cell precursors originate from the rostral lip, while the caudal lip will give rise to the ventrally migrating neurons generating the precerebellar nuclei of the medulla and of the pons. Within this migrating stream from the lower lip, Barhl1 is expressed only in neurons migrating to the pontine nuclei and not in the neurons migrating to the medulla. Interestingly, the formation of these nuclei is selectively disrupted in several knock-out mice, such as the mouse mutants for Pax6 (26), Math1 (28), Netrin1 (25,29) and its receptor Deleted in colorectal cancer (Dcc) (30). Cells at the dorsal margin of the rostral rhombic lip give rise to granule neuron progenitors (31,32). These progenitors, specified to become granule cell progenitors, express the transcription factor genes Zic1, Zic2 (33,34), Zipro1 (35) and the basic HLH gene Math1 (36,37). Recently, it has been shown that Math1 function is required for the generation of granule cell progenitors (38). Nevertheless, the molecular steps involved in the specification of granule cell fate remain largely unknown.
Recent studies in the development of the cerebellar primordium suggest the involvement of patterning signals, such as FGF and Wnt proteins, that control regional identity at the boundary between the mesencephalon and metencephalon (mes/met boundary) (39), and inductive factors, such as the BMP proteins, which have an important role in the specification of the granule cell precursors (40). The first specified granule neuron progenitors appear in the rhombic lip laterally to the roof plate, a source of several BMPs, at around E10 and they seem to express both the Math1 (36,37,41) and Barhl1 genes. The ability of several BMPs (BMP6 and 7, and GDF7) to induce the expression of Math1 and other dorsal markers (Zic1, Zic2 and Wnt3a) has been proved recently (40), suggesting that the induction of the granule precursors may depend on BMP signaling with a mechanism similar to the induction of dorsal interneurons of the spinal cord (42). Once the specification of the granule progenitors has occurred, the putative coexpression of the Math1 and Barhl1 genes is maintained. The co-expression is also maintained in the subsequent events characterized by migration to the external granular layer (EGL), expansion of the progenitor population and final differentiation and migration into the internal granular layer (IGL) (36,37; data not shown). Therefore, it is reasonable to hypothesize that the generation of the EGL may depend on a BMP-triggered transduction pathway involving not only Math1 (38) but also Barhl1 which has a similar spatial and temporal pattern of expression.
Barhl1 expression and development of dorsal sensory interneurons of the spinal cord
The spinal cord is composed of different cell types which are generated and occupy different positions along the dorso–ventral axis of the neural tube (43). The motor neurons are generated laterally to the floor plate, which represents the ventral midline of the neural tube. Dorsally, after the migration of the neural crest cells and the closure of the neural tube, the roof plate is formed along the dorsal midline and cells lateral to it differentiate into different types of dorsal sensory interneurons. The specification of ventral and dorsal cell types, as well as of individual neuronal subtypes, seems to be regulated by inductive signaling factors, provided by the notochord and floor plate ventrally, and by the epidermal ectoderm and roof plate dorsally. Recent evidence provides strong support for a role of the BMP subclass of TGFß-related proteins in the control of the differentiation of dorsal neurons (42,44). In particular, it has been demonstrated that GDF7, a BMP family member expressed selectively by roof plate cells, is able to induce the differentiation of dorsal commissural interneurons (42). These neurons are generated close to the roof plate, and can be divided in D1A and D1B neurons (45). The Math1 gene is specifically expressed by the progenitor cells adjacent to the roof plate which generate both subclasses of the D1 neurons (36,37,41,42,44). At E10.5, the Barhl1 and Math1 domains of expression seem to overlap entirely in this region. However, at E12.5, Barhl1 expression becomes restricted to a more ventral domain corresponding to the region where the D1B subclass of neurons migrate (45), while Math1 expression is confined to the D1A neurons which, for the most part, remain close to the roof plate (42). Therefore, both D1A and D1B neurons seem to originate from Math1+ and Barhl1+ progenitors, but they subsequently reside in different sites of the dorsal spinal cord with D1A neurons maintaining the Math1 expression and D1B neurons maintaining the expression of Barhl1. It is worth noting that in GDF7 mutants, only the late Math1+ cells, corresponding to the D1A neurons, are lost, whereas the D1B differentiate normally (42). Thus, there might be different TGFß-related proteins from the roof plate regulating the formation and identity of the specific subtypes of dorsal interneurons.
The expression pattern of Barhl1 in the developing CNS strongly suggests that this gene, similar to its Drosophila counterparts BarH1 and BarH2, may play a crucial role in cell fate determination of neural structures and may therefore be involved in the pathogenesis of CNS developmental anomalies. Interestingly, the human BARHL1 gene maps to chromosome 9q34, in a region overlapping the critical interval for one form of Joubert syndrome described recently by Saar et al. (22). Joubert syndrome, is a rare syndrome with autosomal recessive inheritance characterized by aplasia/hypoplasia of the cerebellar vermis with abnormal eye movements, ataxia and mental retardation. Both the mapping assignment and its remarkable expression in the primordia of the cerebellum and the brainstem, render BARHL1 an attractive candidate gene for Joubert syndrome. Mutation analysis in patients as well as the generation of a Barhl1 knockout mouse will be necessary to determine whether the Barhl1 gene plays a causative role in Joubert syndrome or in other developmental defects of the mammalian nervous system.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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cDNA identification
To identify the murine Barhl1 full-length transcript, two murine cDNA libraries were used: an E11 embryo (Clontech ML3003a) and an embryonic carcinoma (P19 cell line; Stratagene 937317) cDNA library. For the isolation of the BARHL1 gene, we used a fibroblast genomic library (Stratagene 946204). Plating, hybridization and washing conditions were as described previously (46).
To isolate the human and murine BARHL2 genes, we carried out PCR analysis on human genomic DNA using the oligonucleotide primers TO-9557 and TO-9558 designed on the sequence of the rat mBH1 cDNA (16). Oligonucleotide sequences were as follows: TO-9557, 5"-GTGGAAGCGGCAGACTGCAG-3" and TO-9558, 5"-CAGGCCGTGGATGAGCACGC-3". PCR conditions were 35 cycles of 94°C for 45 s, 50°C for 45 s and 72°C for 45 s.
cDNA sequence analysis
cDNA sequence analysis, and nucleotide and protein database searches were performed as described previously (14). Data on similarity/identity were obtained using the Bestfit program of the GCG software package, version 8.1. The multiple alignment analyses were generated using the PileUp program of the Wisconsin GCG software package, version 8.1.
Genomic mapping in human and mouse
For the RH mapping assignment of the human BARHL1 and BARHL2, we used a previously described procedure (14). PCR analysis on the GeneBridge4 radiation hybrid panel (21) was performed using the following two primers designed in the BARHL1 genomic region, 2 kb downstream of the beginning of the third exon: TO-5571, 5"-TTCTCAACTTGCCCCAGTCC-3" and TO-5572, 5"-CCTCATAAACGCCGACTCAAC-3". For the mapping of the BARHL2 gene, we used oligonucleotide primers TO-9840, 5"-CCGAGGCAGGGAACTACTCGG-3" and TO-9841, 5"-CCGCTGCAGCTGGGGATGG-3". PCR conditions were 35 cycles of 94°C for 45 s, 52°C for 45 s and 72°C for 45 s for both BARHL1 and BARHL2.
Genetic mapping was achieved using a (C57BL/6jxSPRET/Ei)F1xSPRET/Ei (BSS) backcross generated and distributed by the Jackson Laboratory (47). An EcoRI RFLP was identified by hybridization of C57BL/6j and SPRET/Ei parental DNAs (cut with each of the following six restriction enzymes: EcoRI, EcoRV, KpnI, MspI, TaqI and XbaI) with a Barhl1 cDNA probe corresponding to the insert of the IMAGE clone 335997. The polymorphism consisted of a single SPRET/Ei-specific EcoRI band, and of two C57BL/6JEi-specific EcoRI fragments. Four Southern panels containing EcoRI-cut parental DNAs and N2 progeny (n = 94) DNAs were hybridized with a Barhl1 cDNA probe. The analysis of the strain distribution pattern observed in the N2 progeny (n = 92) of the BSS backcross (47) was analyzed with the Map Manager 2.6 program (48) and permitted the localization of Barhl1 to chromosome 2.
Expression studies
Mouse embryo tissue sections were prepared and RNA in situ hybridization experiments were performed as described (49). The IMAGE EST clone 335997 containing part of the Barhl1 transcript was linearized with appropriate restriction enzymes to transcribe either sense or antisense 35S-labeled riboprobes. Slides were exposed for 10 days. Micrographs are double exposures: red color represents the in situ hybridization signal, and the blue shows the nuclei stained with Hoechst 33258 dye.
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ACKNOWLEDGEMENTS |
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We thank E. Roncoroni and the TIGEM sequencing staff for technical support, and M. Smith for preparation of the manuscript. This work was supported by the Italian Telethon Foundation, by the Merck Genome Research Institute (grant no. 37), and by the Armenise Harvard Foundation. S.M. is supported by grants from the EC (BIO4-CT98-0303, DGICYT PM98-0056) and from Fundacion la Caixa 97/101.
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FOOTNOTES |
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+ These authors contributed equally to this work
§ Present address: Telethon Institute of Gene Therapy (TIGET), San Raffaele Biomedical Science Park, Milan, Italy
¶ To whom correspondence should be addressed. Tel: +39 02 215601; Fax: +39 02 21560220; Email: banfi@tigem.it
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REFERENCES |
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