Human Molecular Genetics

Genomic organization of murine Hic1

The comparison of the isolated mouse cDNA clones with the genomic sequence revealed that the murine Hic1 gene contains two exons, separated by a 1.5 kb intron. For the human HIC1 gene only a genomic clone was described and the exon-intron structure was predicted to have three exons (4). The presumptive first human 5[prime]-untranslated exon is not contained in the isolated murine cDNA and in a human HIC1 EST (Fig. 1a). The human second and third exon and the intron (500 bp) in between correspond to the second murine exon. The coding region of the second murine Hic1 exon exhibits 88% identity on DNA level to the corresponding region of the human HIC1 gene. Moreover, the murine Hic1 and human HIC1 nucleotide sequences exhibit 74% identity in the 3[prime]-UTR and 79% identity in ~650 bp of the 3[prime] region of the mouse intron directly upstream of the second murine exon. Interestingly, the exon-intron boundary of the first murine exon exhibited 96% (56 of 58 nt) identity to a human genomic fragment (accession no. M21147; ref. 33) (Fig. 1a). This genomic fragment is contained in a human HIC1 EST (Fig. 1a). It is thus conceivable that also the human HIC1 contains an additional exon in the 5[prime]-region. The reported genomic structure of human HIC1 (4) might represent an alternative transcript.

Chromosomal localization of Hic1

In this study Hic1 was mapped to mouse chromosome 11 at 48 cM employing the EUCIB. Its human orthologue was placed at 17p13.3 (4). Other genes that have been localized on the EUCIB, are Cryba1 at 45 cM, Cacna1g at 60 cM and Nfe2l1 at 61 cM. However, the human orthologues of these three genes map to 17q (Fig. 4b). Thus, Hic1 seems to be localized in a region that exhibits homology to a linkage group on human chromosme 17q. The order of loci that were placed on the Mouse Genome Database (MGD) map support this view (http://www.informatics.jax.org/map.html ). This suggests that chromosomal rearrangements in this region have occured between man and mouse. Similar discontinuities in the gene order within regions of conserved synteny were found among mouse chromosome 16 and the human DiGeorge syndrome region on 22q11.2 (34) and among human chromosome 5 and mouse chromosome 11 (35).

Sequences from the human MDS chromosomal region hybridized to mouse chromosome 11 (36). In addition, the gene order and relative distance of genes localized inside (LIS1/PAFAH1B1, MNT/ROX) and outside (14-3-3e/YWHAE) the MDS critical chromosomal region are conserved between man and mouse. The three genes reside within 1 Mb (37) and have been placed at 44 cM on the mouse chromosome 11 in the MGD map. In human, HIC1 is localized between MNT/ROX and 14-3-3e/YWHAE. For the establishment of an MDS mouse model it would be extremely important to determine the precise localization of Hic1 in relation to these genes.

Comparison of the expressions of Hic1 and [gamma]FBP during development

The POZ domain and the zinc-finger region of the murine Hic1, the human HIC1 and the chicken [gamma]FBP proteins are highly identical, thus suggesting that these proteins might exert similar functions. HIC1 is expressed in all adult tissues analysed (4), but its embryonic expression pattern is not known. Comparison of the murine Hic1 expression and that of a related gene in chicken embryos, [gamma]FBP, revealed some similarities, but also some differences. [gamma]FBP and Hic1 might share expression territories in the sclerotomes, head mesenchyme, developing extremities, pharynx, esophagus, tongue, urogenital ridge, intestine, vertebral arch and rib associated mesenchymes (32 and this study). Despite the absence of detailed information on the [gamma]FBP expression, these general similarities raise the possibility of common functions of these structurally related genes in differentiation of restricted mesenchymes. Moreover, [gamma]FBP was expressed during lens differentiation and functionally implicated in the regulation of the [gamma]F-Crystallin (Crygf) gene (32). However, we did not detect any expression of the murine Hic1 gene in lenses at embryonic, fetal, postnatal (p1, p7, p14, p21), and adult stages by means of ISHs of whole embryos and on paraffin sections (Fig. 8d, and data not shown). Thus, it is not likely that the murine Hic1 gene is implicated in lens differentiation.

Hic1 is expressed in perineural mesenchymes

Embryonic expression of Hic1 was detected in subterritories of the sclerotomes, first intersomitically, and shortly later, in the rostral sclerotome halves. Formation of the segmental array of the components of the peripheral nervous system (PNS), the peripheral ganglia, peripheral nerves and their branches, depends on the permissive properties of the rostral sclerotome halves and the repulsive properties of the caudal sclerotome halves (38-41). Fetal expression of the Hic1 gene was observed in the mesenchymes embedding elongating peripheral nerves in the head, trunk and limbs. Moreover, the spatio-temporal dynamics of Hic1 expression appeared to reflect the course of branching in the peripheral nerves whose target regions are situated more and more peripherally (42,43). Thus, Hic1 expression might play a role in the specification of the sclerotome- and lateral plate-derived as well as cranial mesenchymes implicated in the spatial organization of the PNS. Finally, Hic1 was expressed in mesenchymes at many sites where peripheral nerves probably make contact with the developing muscles and the visceral organs. Interestingly, also the Drosophila POZ-C2-H2 zinc-finger protein lola (longitudinals lacking) plays essential roles in PNS development; lola is required for axon growth and guidance of the intersegmental nerves (44). Moreover, the Drosophila POZ-C2-H2 zinc-finger BR-C (broad complex) regulates muscle attachment in Drosophila (45). The possible contribution of the HIC1 gene to the development of peripheral nerves remains to be elaborated, because also the LIS1/PAFAH1B1 and the MNT/ROX genes may be implicated in peripheral nerve development (12,46). Moreover, in syndromes encompassing lissencephaly, it is difficult to decide if the primary reasons of functional deficiencies of the PNS reside solely in the brain or also the PNS itself.

Hic1 is activated in embryonic anlagen of tissues that are affected in MDS patients

Besides lissencephaly, MDS patients exhibit facial dysmorphism and frequently additional birth defects, e.g. anomalies of the heart, kidney, gastrointestinal tract and the limbs (7-11). Genes deleted in MDS patients are from the centromere to the telomere: LIS1/PAFAH1B1, MNT/ROX, HIC1, OVCA1/DPH2L and OVCA2. In the ILS, mutations and deletions of the LIS1/PAFAH1B1 gene were observed (12-14), and in several cases, also the locus D17S379 residing in the 3[prime]-UTR of the MNT/ROX gene is deleted (10,13). However, the deletions in MDS patients always extend more distally than those in the deleted ILS patients (13). ILS patients sometimes exhibit subtle cranio-facial anomalies reminiscent of those in MDS patients, like bi-temporal hollowing and small jaw, but they rarely have other consistent anomalies (10), strongly suggesting that loss of function in the LIS1/PAFAH1B1 and MNT/ROX genes does not cause the defects in cranio-facial and limb development in MDS patients.

This raises the possibility that other candidate genes in the MDS critical region may be implicated in the defects other than lissencephaly. OVCA1/DPH2L and OVCA2 contain no known functional motifs and are ubiquitously expressed in adult tissues (21) and their developmental expression is not known. In this study, we present the mouse gene Hic1, the orthologue of HIC1, and the striking correlation of its expression with additional sites of defective development in MDS patients.

First, MDS patients exhibit a characteristic facial appearence consisting of a prominent forehead, small jaw (micrognathia), bi-temporal hollowing, short nose with upturned nares, a broad nasal bridge and a protuberant upper lip. Frequently, wrinkling of the forehead was also observed (9). During development, Hic1 is expressed in the first branchial arch that gives rise to the maxillar and mandibular components of the jaw and the lips. In addition, Hic1 expression was observed in the mesenchyme surrounding the infolding nasal epithelium in embryonic and fetal development. Thus, the developmental expression of Hic1 in spatially restricted cranio-facial mesenchymes whose derivatives are affected in MDS patients suggests that HIC1 may be implicated in the morphogenesis of the nose, jaw and lips.

Secondly, frequently, clinodactyly and sometimes syndactyly were observed in MDS patients (9). Hic1 is expressed in restricted domains of the limb mesenchyme, surrounding the skeletal condensations, as well as in rostral and caudal mesenchymes of each digit anlagen. Thus, loss of HIC1 function may be involved in the limb anomalies observed in MDS.

Thirdly, less frequently, inguinal hernia/omphalocoele was associated with MDS, leading to the suggestion that a gene involved in the closure of the lateral folds or in the return of the midgut from the body stalk to the abdomen during weeks 5-11 of gestation resides at 17p13.3 (11). Hic1 is expressed in the lateral body wall and in the gut. This raises the possibility that its human homologue might play a role in the omphalocoely observed in MDS patients. Finally, two patients with kidney defects were reported and one case with a duodenal atresia (7,8). Hic1 is expressed during development of the gut and the kidney.

In conclusion, the mouse homologue of HIC1, during embryonic development, is expressed in restricted territories of facial mesenchymes (in the branchial arches, surrounding the infolding nasal epithelium and the facial nerves), in the lateral body wall, kidney, gut and limb anlagen. All these expression territories correspond to sites where developmental defects are observed in MDS patients. Therefore, alterations in the gene dosage of the putative transcription factor HIC1 might be involved in the anomalies observed in MDS patients, that are hemizygous for HIC1. An example for gene dosage effects of transcription factors is the Pax family of paired domain proteins. All analyzed heterozygous Pax null mutants exhibit severe developmental defects due to haploinsuffiency (reviewed in ref. 47).

The generation and investigation of heterozygous and homozygous Hic1 knock-out mutant mice will help to reveal if the gene dosage of Hic1 is critical for development and if its human orthologue might be involved in the MDS.

Animals and embryos

Mice were obtained from the GSF animal facilities. C3H/El and (102/El × C3H/El)F₁ hybrid mice were used for the supply of all tissues and the preparation of embryos; for the latter also C57BL/6 mice were used. Staging of embryos was performed according to Kaufman (48). Individual somites and early vertebral condensations were identified according to Spörle and Schughart (49).

Expressed sequence tags

The murine EST of the IMAGE Consortium [CloneID 337300, partial sequence under accession no. W20732; (22)] was obtained from the Human Genome Mapping Project-Resource Centre (HGMP-RC, Cambridge, UK).

The zebrafish EST (ICRFp524N033, partial sequence under accession nos AA497316 and AA497240) was kindly provided by Dr M. Clark (Resource Centre of the German Human Genome Project at the Max-Planck-Institute for Molecular Genetics, Berlin, Germany).

Isolation of the murine Hic1

The 0.6 kb EcoRI-NotI insert of the IMAGE Consortium CloneID 337300 was labelled with [[alpha]-³²P]dCTP (Amersham, Braunschweig, Germany) by random priming using the Megaprime DNA labelling system (Amersham) to screen a mouse embryonic day 11 cDNA library of the mouse strain SwissWebster (Novagen, Madison, WI) using stringent hybridization conditions (50% formamide, 5× SSC, 5× Denhardt’s, 0.1% SDS, 100 µg/ml herring sperm DNA, 42°C). Filters (Qiabrane Nylon; Qiagen, Hilden, Germany) were washed in 0.1× SSC/0.1% SDS at 50°C. Sixteen positive clones were isolated and subcloned into pSHlox-1 by in vivo excision. Both strands of the largest clone (7-3-3, 2.3 kb) were sequenced. To obtain the 5[prime] region, a second mouse embryonic day 11 cDNA library of the mouse strain SwissWebster (kindly provided by Dr G. Borsani, TIGEM, Milano, Italy) was screened using a 935 bp fragment (EcoRI-NdeI) of the 5[prime] end of clone 7-3-3 as a probe. One of the isolated clones (6-1), revealing a 1.9 kb insert, exhibited further 5[prime]-coding region of Hic1 and was subcloned into the EcoRI sites of pBluescript KS and sequenced (Sequiserve, Vaterstetten, Germany).

A genomic Hic1 clone was isolated by screening a genomic [lambda]DASH DNA library of the mouse strain 129/SV (50) using the 935 bp EcoRI-NdeI fragment of the 5[prime] region of clone 7-3-3 as a probe. One positive clone (G6-2), containing an ~16 kb insert, was further analyzed. An ~6 kb BamHI fragment of this clone was subcloned into pBluescript KS and sequenced.

Sequence analyses were performed with Gene works 2.45N, clustalW 1.7, MatInspector V2.2 (25) (http://www.gsf.de/biodv/ ) and the ‘BLAST’ network service (51).

Northern analysis

Total RNA from frozen tissues was isolated with RNeasy (Qiagen). Twenty micrograms of total RNA were separated on a 1% agarose gel according to Sambrook et al. (52), transferred onto nylon membranes (Qiabrane Nylon Plus; Qiagen) and UV crosslinked (Stratalinker; Stratagene, Heidelberg, Germany). The 0.6 kb EcoRI-NotI insert of the IMAGE Consortium CloneID 337300 was labelled with [[alpha]-³²P]dCTP (Amersham). Labelling and hybridization was performed as described above. Filters were exposed to X-ray film (Kodak XAR; Eastman Kodak Company, New York, NY) at -80°C with two intensifying screens for 3 days.

Chromosomal localization

FISH was performed on metaphase chromosome preparations obtained from mouse bone marrow cells according to standard procedures. The ~16 kb genomic Hic1 clone G6-2 in [lambda]DASH was labelled with biotin-dUTP using the Bio-Prime DNA Labelling System (Gibco BRL, Eggenstein, Germany). The specific chromosome 11 paint probe was obtained from Appligene Oncor; (Heidelberg, Germany). Hybridizations were performed according to Pinkel et al. (53). Metaphase chromosomes were analyzed under an Axiophot microscope (Zeiss, Germany), and pictures were taken by digitizing the microscopic image with the computer program ISIS3 (Metasystems, Altlußheim, Germany) and adapted in Adobe Photoshop 3.0.

Genomic DNA samples from the EUCIB were obtained from the HGMP-RC (28). A polymorphism between M.spretus and C57BL/6J was detected in the 3[prime]-UTR of Hic1 using the primer pair 5[prime]-aaccctgcctctccctgtggc-3[prime]/5[prime]-gtagggctgggagcgtcc-3[prime]. To confine the amplified products, the PCR fragments of M.spretus and C57BL/6J were cloned into the pCR[trade]2.1 vector (Invitrogen, Leek, The Netherlands) and sequenced. A 99 bp fragment was amplified from C57BL/6J and a 95 bp fragment from M.spretus. PCR was run in a GeneAmp PCR System 9600 (Perkin Elmer, Weiterstadt, Germany) using the following conditions: 6 min 94°C, 30 cycles of 94, 60 and 72°C each step lasted for 30 s and a final extension step for 10 min at 72°C. For each PCR reaction, 20 ng genomic DNA, 2 U Taq polymerase (Gibco BRL) 1× Taq reaction buffer (Boehringer Mannheim, Germany), 200 µM of each dNTP (Boehringer Mannheim), 1.5 mM MgCl₂, 0.25 µM of each oligonucleotide in a total volume of 20 µl were used. Bands were separated on an 8% polyacrylamide/1× TBE gel. A total of 130 backcross animals were typed for the Hic1 locus. Genetic linkage to EUCIB anchor loci was analysed using the Mbx program (28).

In situ hybridization

RNA probes labelled with Digoxigenin-rUTP (Boehringer Mannheim) were synthesized from linearized cDNA templates according to the manufacturer’s instructions. The IMAGE Consortium CloneID 337300 was linearized with EcoRI and NotI, and synthesized with T3 and T7 RNA polymerases for antisense and sense probes, respectively. The clone 7-3-3 was linearized with SacI and transcribed with SP6 RNA polymerase for the antisense probe. The Myod1 and the Col2a1 probes were previously described (54,55).

ISHs of whole mount embryos (WISHs) were performed according to Wilkinson (56) and Rosen and Beddington (57) with modifications described by Spörle and Schughart (58). WISH embryos were cut transversely with a scalpel, and section planes were documented. Staining of WISH embryos by precipitating BM Purple AP Substrate (Boehringer Mannheim) with some riboprobes yielded two different colours in the same reaction. In WISHs, bright blue instead of purple precipitates were observed in the expression territories of Hic1 that were situated medially to the hypaxial somitic buds (Fig. 6d[prime] and h, white arrows).

ISHs on paraffin sections were performed as previously described (59,60) with the following modifications. After fixation, embryos were dehydrated in isopropanol, transferred to Histowax saturated with isopropanol and embedded in Histowax (Cambridge Instruments, Nußloch, Germany). Slides were fixed for 30 min at room temperature in both 4% PFA/PBS prehybridization fixation steps, and treated for 3 min with 2 µg/ml proteinase K in 1 mM EDTA, 20 mM Tris-HCl (pH 7.0). Before hybridization, the slides were washed twice for 2 min in 2× SSC, and incubated for at least 30 min in 0.1 M Tris, 0.1 M glycine. Hybridization solution (60 µl/slide) contained 50% formamide, 5× SSC, 5% dextran sulfate, 2 mg/ml heparin, 100 µg/ml tRNA, 0.1% Tween-20 (pH adjusted with citric acid to 6.0) and a 1:100 or 1:50 dilution of the riboprobes. Riboprobes were the same as for WISH. Hybridization occurred overnight at 65°C under a siliconized coverslip or a parafilm strip. Following hybridization, slides were washed for 1-2 h in 0.5× SSC, 20% formamide at 65°C. Sections were treated with 10 µg/ml RNaseA for 30 min at 37°C in NTE (0.5 M NaCl, 5 mM EDTA, 10 mM Tris-HCl, pH 7.0-7.5), then washed for 4 h in 0.5× SSC, 20% formamide at 65°C and for 30 min in 2× SSC, and blocked for 1 h at room temperature in 1% blocking reagent (Boehringer Mannheim)in MAB-T (0.1 M maleic acid, 0.15 M NaCl, 0.1% Tween-20,pH 7.5). A 1:5000 dilution of anti-digoxigenin-AP conjugate (Boehringer Mannheim) was preincubated for at least 1 h in 1% blocking reagent in MAB-T at 4°C. Slides were incubated with the antibody overnight at 4°C, washed for 6 h in TBS-T, for 30 min in NTM-T (0.1 M NaCl, 50 mM MgCl₂, 0.1 M Tris-HCl, pH 9.5, 0.1% Tween-20), and stained using centrifuged BM purple AP substrate (Boehringer Mannheim) in 0.3% Tween-20 for 1-7 days at 4°C and/or room temperature. Slides were washed in NTM-T, then in distilled water, and embedded in KAISER’S glycerol gelatin (Merck, Darmstadt, Germany). Sections were analysed under a Zeiss Axioplan microscope and documented with a high resolution CCD colour camera (Fujix HC-2000; Fuji) connected to an Apple PowerMacintosh. Pictures were adjusted for brightness, contrast and colour balance in Adobe Photoshop3.0.