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Human Molecular Genetics Pages 727-737

Sequence analysis, identification of evolutionary conserved motifs and expression analysis of murine tcof1 provide further evidence for a potential function for the gene and its human homologue, TCOF1
Introduction
Results
   Elucidation of the murine cDNA sequence
   Mouse/human homology
   Chromosomal localization
   Whole-mount in situ hybridization
Discussion
Materials And Methods
   Zooblot analysis
   Isolation and characterization of cDNA clones
   RNA extraction and RT-PCR analysis
   Rapid amplification of cDNA ends (RACE)
   PCR conditions
   Chromosomal localization
   Whole-mount in situ hybridization
Acknowledgements
References
Table

Sequence analysis, identification of evolutionary conserved motifs and expression analysis of murine tcof1 provide further evidence for a potential function for the gene and its human homologue, TCOF1

Sequence analysis, identification of evolutionary conserved motifs and expression analysis of murine tcof1 provide further evidence for a potential function for the gene and its human homologue, TCOF1 Jill Dixon, Karine Hovanes1, Rita Shiang1 and Michael J. Dixon*

School of Biological Sciences and Departments of Dental Medicine and Surgery, 3.239, Stopford Building University of Manchester, Oxford Road, Manchester M13 9PT, UK and 1Department of Biological Chemistry, College of Medicine, University of California, Irvine, CA 92717, USA

Received December 11, 1996; Revised and Accepted February 5, 1997

The gene mutated in Treacher Collins syndrome, an autosomal dominant disorder of facial development, has recently been cloned. While the function of the predicted protein, Treacle, is unknown, it has been shown to share a number of features with the highly phosphorylated nucleolar phosphoproteins, which play a role in nucleolar-cytoplasmic transport. In the current study, the murine homologue of the Treacher Collins syndrome gene has been isolated and shown to encode a low complexity, serine/alanine-rich protein of 133 kDa. Interspecies comparison indicates that the proteins display 61.5% identity, with the level of conservation being greatest in the regions of acidic/basic amino acid repeats and nuclear localization signals. These features are shared with the nucleolar phosphoproteins. Confirmation that the gene isolated in the current study is orthologous with the Treacher Collins syndrome gene was provided by the demonstration that it mapped to central mouse chromosome 18 in a conserved syntenic region with human chromosome 5q21-q33. Expression analysis in the mouse indicated that the gene was expressed in a wide variety of embryonic and adult tissues. Peak levels of expression in the developing embryo were observed at the edges of the neural folds immediately prior to fusion, and also in the developing branchial arches at the times of critical morphogenetic events. These observations support a role for the gene in the development of the craniofacial complex and provide further evidence that the gene encodes a protein which may be involved in nucleolar-cytoplasmic transport.

INTRODUCTION

Treacher Collins syndrome (TCS) is a congenital disorder of craniofacial development which occurs with an incidence of ~1/50 000 live births (1 ,2 ). The clinical features of TCS, which are usually bilaterally symmetrical in nature (3 ), include: (i) abnormalities of the external ears which are frequently associated with atresia of the ear canals and anomalies of the middle ear ossicles. Bilateral conductive hearing loss is therefore a common feature of TCS (4 ); (ii) hypoplasia of the facial bones, particularly the mandible and zygomatic complex; (iii) lateral downward slanting of the palpebral fissures with colobomas of the lower eyelids and a lack of eyelashes medial to the defect; (iv) cleft palate (1 ,5 ). However, a high degree of inter- and intra-familial phenotypic variability is observed in TCS (6 ,7 ). Moreover, while TCS is inherited in an autosomal dominant fashion, 60% of cases arise without a previous family history, presumably as the result of a de novo mutation (8 ). These combined facts create diagnostic and genetic counselling difficulties.

The TCS locus was initially mapped to human chromosome 5q31-q34 (9 ) and the gene, TCOF1, was subsequently identified using positional cloning strategies (10 ). TCOF1 contains an open reading frame of 4233 bp, which encodes the low complexity, serine/alanine-rich, 144 kDa protein, Treacle. The identification of all 26 exons of TCOF1 has permitted the identification of >50 largely family-specific mutations which result in the introduction of a premature termination codon into Treacle (10 -12 ; unpublished data). The identification of the gene, and delineation of the wide mutational spectrum, have failed to reveal the exact biochemical nature of the disorder, as, to date, database comparisons have only revealed weak, but significant, homology to a family of highly phosphorylated proteins, the nucleolar phosphoproteins (13 ). Nevertheless, a series of repeated units have been identified within the gene and these have been shown to map onto individual exons. The function of the repeating units is unclear, however, each unit contains a number of potential sites for casein kinase II phosphorylation, suggesting that phosphorylation is important for the correct function of the protein. The homology between the nucleolar phosphoproteins and TCOF1 also appears to be greatest at these motifs (13 ). Moreover, a number of potential nuclear localization signals have been noted towards the 3' end of the coding sequence of both the nucleolar phosphoproteins and Treacle.

On the basis that the tissues affected by TCS are derived from the first and second branchial arches, which in turn have a significant contribution from the neural crest, it has been proposed that the disorder may be the result of a defect in neural crest cell migration, improper cellular differentiation during development (14 ,15 ) or an abnormality of the extracellular matrix (16 ). Additionally, phenocopies of TCS have been produced in mice following acute maternal exposure to 13-cis-retinoic acid at 9.0-9.5 days post-fertilization (17 ), suggesting that the disorder may result from abnormal development of the first and second branchial arch ectodermal placodes. Whatever the underlying mechanism of the disorder, it is evident that this gene plays a crucial role in the formation of the craniofacial complex during early embryonic development.

In the current investigation, we have isolated the murine homologue of TCOF1 which has allowed us to make a preliminary study of the spatio-temporal distribution of the gene during embryonic development. Comparison of the human and mouse cDNA sequences has allowed us to identify evolutionary conserved regions of the gene which are likely to be important for its function. The results of these analyses provide further support for the hypothesis that Treacle may play a role in nucleolar-cytoplasmic transport.

RESULTS

Elucidation of the murine cDNA sequence

Zooblot analysis performed using a human TCOF1 cDNA clone (10 ) showed that the gene is highly conserved in genomic DNA extracted from dog, pig, sheep, cow and monkey. The intensity of the bands observed in murine and chicken DNA was weaker, suggesting a lower level of evolutionary conservation in the genomes of these animals (Fig. 1 ). In order to isolate a full-length murine tcof1 cDNA clone, the combined techniques of cDNA library screening and rapid amplification of cDNA ends (RACE) were employed. Initially, a mouse embryonic day 10 (E10) cDNA library was screened at reduced stringency with a human TCOF1 cDNA clone (10 ). Fourteen clones were identified, of which eight were purified. The longest of these clones, designated E10-1C, was sequenced in its entirety and found to contain a single open reading frame of 1991 bp encompassing the entire clone. As this clone did not contain a translation initiation signal, a polyadenylation signal or a poly A tail, the ends of the remaining clones were sequenced, but were found not to extend the sequence in either a 5' or 3' direction. The E10-1C clone was therefore used to screen a mouse embryonic day 15 (E15) cDNA library resulting in the identification of three further clones, none of which extended the existing sequence.


Figure 1. Conservation of TCOF1 across species as assessed by zooblot analysis. The blot was probed with a partial length cDNA clone (10), washed with 0.5* SSC/0.1% SDS at 65oC and exposed for 24 h.

To identify additional cDNA libraries which could be screened, and to provide preliminary information on the expression of tcof1, RT-PCR analysis was performed using a variety of murine adult and embryonic tissues. Using the primers 5'-TGA ACA CCA CAA AGA AGG CC-3' and 5'-TAG ACT GCT CAC TCT TGC TG-3', which were designed corresponding to nucleotide positions 2546-2656 and 2706-2725, respectively, of the murine cDNA (Fig. 2 ), and are predicted to flank intron 17 based on the relative positions in the human coding sequence, a single PCR product of 180 bp was detected in RNA extracted from placenta, adult heart, spleen, brain, tongue and also in RNA extracted from E9, E10, E11 and E15 mouse embryos (Fig. 3 ). A product of ~1.3 kb was obtained when the same primers were used to amplify mouse genomic DNA (Fig. 3 ). Radiolabelled fragments of the original E10-1C clone were therefore used to screen an adult mouse spleen library, a further eight clones being identified. Two of these clones, [lambda]9 and [lambda]4, were restriction mapped and sequenced. Clone [lambda]9 generated an additional 1433 bp of sequence; [lambda]4 extended further 5' to [lambda]9 producing 254 bp of additional sequence which included a strong Kozak consensus initiation sequence (18 ,19 ). Concurrently, the sequence generated from cDNA clone E10-1C was used to design primers for use in RACE. Both 5' and 3' RACE were employed using RNA extracted from adult mouse heart as the substrate. 3' RACE resulted in the identification of two PCR products of ~500 bp and 1 kb. The smaller product extended the sequence of E10-1C by 165 bp, but did not contain a polyadenylation signal or a poly A tail. The larger product extended further 3' and contained both a polyadenylation signal and a poly A tail. 5' RACE produced a 500 bp fragment which extended the sequence of E10-1C by 355 bp which was subsequently found to be contained entirely within cDNA clones [lambda]4 and [lambda]9.

The composite sequence of mouse tcof1 is presented in Figure 2 . It contains an open reading frame of 3906 nucleotides (nt), followed by a termination codon and a 3' UTR of 450 bp, which contains a single polyadenylation signal. A 5' UTR of 48 bp was also identified. While the 5' UTR does not contain an in-frame termination codon, unlike its human counterpart (13 ), no additional potential upstream initiation codons were identified. The sequence of tcof1 predicts a serine/alanine-rich protein of 1302 amino acids with a predicted molecular weight of 133 kDa. The sequence is of low complexity with five amino acids (serine, alanine, lysine, proline and glutamic acid) accounting for almost 60% of the residues (Table 1 ). In addition to extensive homology with TCOF1, database sequence comparisons revealed only very weak similarity to Xenopus laevis nucleolar phosphoprotein (S57757; P = 4.2e-8) (20 ). Bioinformatics analysis of the conceptual protein using dotplots showed that there were repeating units within tcof1 (data not shown). These consist of clusters of acidic amino acids separated by stretches with few or no acidic residues, but a relatively large number of basic amino acids such as lysine (Fig. 2 ). The protein sequence was also compared against pattern databases in an attempt to identify functional motifs within the sequence. These comparisons resulted in the identification of multiple motifs for casein kinase II phosphorylation (55 in total), many of which fall within the clusters of acidic residues that occur within the repeat units, although other acidic clusters lie outside this region. As the acceptor serine residues in the casein kinase II phosphorylation motifs fall in acidic clusters their phosphorylation rate is likely to be increased. Interestingly, if a number of the serine residues in this region are phosphorylated, many of the remainder will themselves become acceptor sites for casein kinase II phosphorylation (21 ). In addition, a number of motifs of the type K-R/K-X-R/K, which represents the minimal nuclear localization signal consensus sequence (22 ) were predicted, particularly towards the 3' end of the coding sequence. Nevertheless, as these motifs tend to be overpredicted by the programs, their significance was unclear. Comparison of the mouse and human sequences allowed those motifs which were conserved, and therefore likely to be of functional significance, to be identified.

Mouse/human homology

Comparison of the human and mouse sequences revealed that the two genes display 74.3% identity at the nucleotide level, with 72% identity in the 3' UTR. Despite the fact that the predicted protein produced from the mouse gene is 109 amino acids shorter than its human counterpart, the orthologues display 61.5% identity and 71% similarity (Fig. 4 ). Alignment of the sequences indicated that the size discrepancy was predominantly due to three large gaps in the mouse sequence, the largest of which (37 amino acids) corresponds to the region surrounding the position of intron 18 in the human gene (Fig. 4 ). The fact that this was not artefactual was confirmed by analysis of clones from different cDNA libraries. The homology between the two genes is greatest in the 5' and 3' regions of the coding sequence. The area corresponding to the repeated region in the human gene is less homologous with the exception of the potential casein kinase II phosphorylation sites which are well-conserved (Fig. 4 ). The predicted nuclear localization signals at the 3' end of the coding sequence are also conserved (Fig. 4 ).

Chromosomal localization

In order to confirm the orthologous nature of the human and mouse sequences, and to assess whether tcof1 mapped in close proximity to any of the known mouse mutants, genotyping of the BSS Jackson Laboratory Backcross panel was undertaken. HindIII digestion of mouse genomic DNA generated tcof1 fragments of 16 and 2 kb in C57BL/6J mice and 18 kb in SPRET/Ei mice. Genotyping of this polymorphism indicated that tcof1 maps to central mouse chromosome 18 between the markers Lmnb1, proximal, and D18Hun11/D18Bir6, distal. This is a conserved syntenic region with human chromosome 5q21-q33. The local gene order and inferred distances are depicted in Figure 5 . Genotyping of a polymorphic tract of cytosine residues (SPRET/Ei = 5 residues, C57BL/6J = 7 residues) located in intron 17 of tcof1 across the European Collaborative Interspecific Backcross panel produced a similar result (data not shown).AB


Figure 2.(A) Composite sequence of the tcof1 cDNA (GenBank accession number U81030) and the derived amino acid sequence. The region of alternating acidic and more basic amino acid residues is indicated (acidic regions are double-underlined, more basic regions are underlined). The single polyadenylation signal is in bold face. (B) Diagrammatic representation of the cDNA clones and RACE products isolated and the riboprobes used in the whole-mount in situ hybridization.



Figure 3. RT-PCR analysis of RNA extracted from a range of murine adult tissues and embryos as indicated above each lane. The size marker (M) is [Phi]X174 DNA digested with HaeIII. The primer set amplifies a 180 bp product from cDNA and a product of ~1.3 kb from genomic DNA. The negative control (-ve) is a reverse transcription of spleen RNA performed in the absence of reverse transcriptase.

Whole-mount in situ hybridization

To investigate the expression of tcof1 during embryonic development, whole-mount in situ hybridization was used to screen E8-E15 mouse embryos. A variety of probes corresponding to different portions of the gene were tested (Fig. 2 ), all of which produced identical staining patterns. Whole-mount studies revealed that tcof1 was expressed throughout all of the embryonic ages examined, albeit at apparently different levels (Fig. 6 ). The general pattern was of a high level of expression at E8 which decreased with advancing embryonic age. At E8, a high level of general expression was evident in embryos stained with the antisense probe compared with the sense control (Fig. 6 A). At E8.5, staining was particularly strong in the forebrain, hindbrain rhombomeres, notochord and the developing first branchial arch (Fig. 6 B). The intense staining at the edges of the neural folds in the region immediately rostral to neural tube closure in the cervical region is of particular interest as this appears to be confined to a region about to undergo fusion (Fig. 6 C). At E9, the expression of tcof1 remained generally high throughout the embryo, but tended to be concentrated in the developing branchial arches, particularly the first, at the inter-somitic junctions and in the notochord (Fig. 6 D). Areas of increased staining were noted on the superior aspect of the mandibular processes of the first arch and the lateral aspects of the first and second arches (Fig. 6 E and F). By E10, expression of tcof1 appeared to be greatly reduced, the only evidence of significant staining being in the leading edges of the developing limbs, the branchial arches and in the somites of the most caudal portion of the tail (data not shown). From this stage onwards, the staining patterns were almost indistinguishable from the controls, although the antisense embryos were always slightly darker than the sense controls in identical staining regimes (Fig. 6 G). Parallel positive control experiments using a sonic hedgehog probe produced identical results to those detailed in Bitgood and McMahon (23 ) (Fig. 6 H).

Table 1 Comparison of the amino acid compositions of the predicted proteins produced from mouse tcof1 and human TCOF1
Amino

 Percentage

 Percentage

 Amino acid

 Percentage

 Percentage
acid

 in tcof1

 in TCOF1

 acid

 in tcof1

 in TCOF1
Ser

 14.88

 13.59

 Asp

 4.22

 3.96
Ala

 14.42

 14.86

 Arg

 3.07

 3.40
Lys

 11.66

 11.18

 Asn

 1.30

 1.42
Pro

 9.74

 9.06

 Ile

 1.00

 1.06
Glu

 8.28

 9.13

 Met

 1.00

 0.99
Gly

 6.90

 7.57

 His

 0.84

 0.50
Thr

 6.83

 6.79

 Tyr

 0.46

 0.28
Gln

 5.21

 5.59

 Cys

 0.15

 0.28
Val

 4.91

 5.52

 Trp

 0.15

 0.28
Leu

 4.91

 4.32

 Phe

 0.07

 0.22



DISCUSSION

In the current paper we report the isolation of the murine homologue of the Treacher Collins syndrome gene and present preliminary expression data. Comparison of the human and mouse cDNA sequences indicates that the two genes display 74.3% identity at the nucleotide level. This level of conservation is consistent with the data generated by zooblot analysis in which the intensity of bands observed in DNA extracted from mouse was less than that observed in DNA extracted from dog, pig, sheep, cow and monkey, suggesting a lower level of evolutionary conservation. While this level of homology is much less than many other recently identified genes, such as the neurofibromatosis type 2 (NF2) gene (24 ), in which the mouse and human genes are 98% identical suggesting strong evolutionary constraints on the sequence of the entire gene, this lower level of conservation highlights a number of regions which are potentially important for protein function (see below). Interestingly, although the level of conservation observed in the coding sequence is less than the average value calculated by Makalowski et al. (25 ), that observed in the 3' UTR is relatively high (72%) suggesting a potential regulatory role for this region.

Like its human homologue, tcof1 encodes a serine/alanine-rich protein of low complexity. Indeed, five amino acids (serine, alanine, lysine, proline and glutamic acid) account for almost 60% of the protein. At present, the precise function of the protein remains unclear as database sequence comparisons using either the human or murine sequence have shown only weak similarity with a class of nucleolar phosphoproteins (20 ,26 ). On closer inspection, however, the nucleolar phosphoproteins have a number of interesting features in common with both the human and murine homologues of TCOF1. Firstly, the nucleolar phosphoproteins are also low complexity proteins in which the same five amino acids make up the majority of the protein. Secondly, the genes all possess nuclear localization signals towards the 3' end of the coding sequence. Thirdly, the genes show a series of repeating units, consisting of clusters of acidic repeats, containing numerous consensus sites for casein kinase II phosphorylation, separated by basic amino acid stretches comprising a majority of lysine, alanine and proline residues. Overall, the mouse and human sequences display 61.5% identity/71.7% similarity at the amino acid level. Nevertheless, the regions that appear to be most highly conserved between the human and murine genes are those elements which also appear to be in common with the nucleolar phosphoproteins. The nucleolar phosphoproteins are a family of highly phosphorylated proteins that shuttle between the nucleus and cytoplasm along a limited number of curvilinear tracks from the dense fibrillar component of the nucleolus, across the nucleoplasm to certain nuclear pore complexes. While the precise role of the nucleolar phosphoproteins has not been elucidated, their shuttling between the nucleolus and the cytoplasm suggests a function in transport, for instance they might play a role in protein import/export (26 ). Rat nucleolar phosphoprotein, Nopp140, has also been shown to be associated with a highly conserved 57 kDa protein which has putative homologs in yeast and even prokaryotes (27 ). This suggests a fundamental and highly conserved function in both prokaryotic and eukaryotic cells. Whether Treacle shows similar characteristics and interactions remains to be shown, however, experiments to address these issues are in progress.

While the identity of the proteins predicted from the sequence of TCOF1 and its murine homologue, tcof1, is significantly less than the average value of 85% calculated by Makalowski et al. (25 ) in their comparison of 1196 mouse and human cDNAs, the orthologous nature of the two genes has been confirmed by the data generated from a comparison of the map locations in the respective genomes. tcof1 has been localized to mouse chromosome 18 in a conserved syntenic region with human chromosome 5, between the markers Lmnb1, proximal, and D18Hun11/D18Bir6, distal. tcof1 shows no recombination with the Ia-associated invariant chain locus, Ii. Analysis of cosmid contigs containing TCOF1 and DHLAG (the human homologue of Ii) has shown that these loci map within a small physical interval of <40 kb (10 ). The mouse mutant shaker-with-syndactylism, sy, has been mapped to this broad region of the genome, however, the sy locus has previously been mapped only in two and three point crosses in relation to the visible markers bouncy, plucked and balding, each more than 10 cM proximal of sy, giving only an approximate location (28 ,29 ). It is therefore not clear whether sy maps to a conserved syntenic region with human chromosome 5q31-q33 or 18q21. In any event this mutant would not appear to be a good model for TCS, as despite the fact that deafness is a feature of both TCS and sy, in the latter case this results from malformation of the inner ear (30 ), whilst in the case of TCS the deafness is of a conductive nature, resulting from middle ear anomalies (4 ). Moreover, whilst malformations of the limbs are seen in a number of the acrofacial dysostoses such as Miller and Nager syndromes, they are not seen in TCS. In the apparent absence of an existing mouse mutant, the elucidation of the genomic organization of tcof1 will be central to the creation of an animal model of TCS via gene targeting. In this regard, the genomic organization of TCOF1 is already known, which should facilitate this process. Interestingly, the predicted mouse protein is 109 amino acids smaller than its human counterpart. Whilst this size discrepancy is partially accounted for by a number of small gaps, the programs BESTFIT and GAP both predict three large intervals in the mouse sequence. Based on extrapolations of the genomic organization of the human gene, and assuming that the positions of the intron/exon boundaries in the mouse are similar to those in the human gene, these gaps are predicted to encompass the intron/exon boundary of exons 4/5, 10/11 and 18/19. When the genomic organization of tcof1 is elucidated it will be interesting to see if this represents merely smaller exons either side of the gap, or if any of the intron/exon boundaries are not conserved; experiments to address this issue are in progress.


Figure 4. BESTFIT alignment of the predicted mouse and human Treacle sequences with the mouse (M) sequence above and human (H) sequence below. The conserved casein kinase II phosphorylation sites are in bold face and the conserved nuclear localization signals situated towards the C-terminus are underlined.



Figure 5. (A) A partial recombination map of mouse chromosome 18 is shown with marker flanking the tcof1 locus (code 443) which is in bold face. Numbers to the left of the chromosome indicate centiMorgans and loci mapped on the panel are shown to the right. (B) Key recombinant animals are shown with markers flanking the tcof1 locus. Animal numbers are shown to the left of the recombinant chromosomes and markers are listed above. The solid squares indicate alleles derived from the C57BL/6J parent and unfilled squares are alleles derived from the SPRET/Ei parent. The shaded square represents a recombination event between flanking markers, but its exact location is unknown. The map and recombinant data were obtained from the Jackson Laboratory (http://www.informatics.jax.org).

RT-PCR analysis has indicated that tcof1 is expressed in a range of adult and embryonic tissues. This is entirely consistent with those data generated for the human gene using northern analysis (10 ). Nevertheless, the expression patterns observed when using whole-mount in situ hybridization experiments are consistent with a fundamental role for tcof1 in the development of the craniofacial complex during embryonic development. The earliest expression observed in the current study is in early E8 embryos. At this stage, while staining is seen throughout the embryo, it is more intense in the developing first branchial arch, which will ultimately give rise to the mandible and maxilla. Expression also appears to be strong in the developing brain, in a pattern which reflects the rhombomere boundaries. Of particular interest is the region of increased expression in the neural folds immediately rostral to their point of fusion in E8 embryos. This area of expression appears to occur on the crests of the neural folds, and may coincide with a signalling event connected with fusion of the neural folds or migration of neural crest cells in the unfused region. Unfortunately, although the technique of whole-mount in situ hybridization provides an excellent three-dimensional overview of gene expression, it does not provide precise information regarding expression in specific cell layers. In situ hybridization to tissue sections is currently in progress to address this issue. As development progresses, the level of expression of tcof1 appears to be down-regulated. It is still evident at E9.0-9.5, although expression now appears to be restricted to specific areas of the brain, notochord, branchial arches and the inter-somitic junctions. The expression remaining in the branchial arches appears to be restricted to the superior aspect of the first branchial arch and the lateral aspects of the first and second arches. Our results suggest that peak expression of the gene occurs during the times of critical morphogenetic events in the craniofacial complex, including the formation and fusion of the branchial arches with the rest of the developing face. This peak of expression decreases once the facial complex has formed, and by E10 expression has reduced to near background levels. Nevertheless, expression of tcof1 can still be detected in E15 embryos by RT-PCR. It seems highly unlikely that the low levels of expression observed after E10, as assessed by whole-mount in situ hybridization, were due to problems with probe penetration as control experiments performed using a sonic hedgehog probe produced identical results to those detailed by Bitgood and McMahon (23 ). Nevertheless, the developmental gradient of tcof1 expression revealed by whole-mount in situ hybridization was not observed in RT-PCR experiments. In the latter case, however, no attempt was made to quantify the levels of tcof1 expression, saturation being achieved in all cases.


Figure 6. Expression patterns of tcof1 as assessed by whole-mount in situ hybridization. (A) Lateral view of E8.0 embryos. A high level of expression is evident throughout the embryo hybridized with the antisense probe (left) compared to that hybridized with the sense probe (right). (B) Dorso-lateral view of E8.5 embryos. The antisense probe (left) results reveal a continued high level of expression, which is particularly noticeable in the first branchial arch (arrowed). The corresponding region is not stained in the sense embryo (right). (C) Staining is present throughout the developing cranial and caudal regions. An area of strong staining is evident rostral to the region of neural tube fusion in the cranial region (arrowed). Both embryos were hybridized with the antisense probe and are viewed from the dorsal aspect. (D) Lateral view of E9.0 embryos. Expression of tcof1 remains high throughout the antisense embryo (left), particularly in the first branchial arch (arrowed). A sense embryo (right) exhibits only non-specific staining of the forebrain and heart. (E) At E9.5, the antisense embryo shows high tcof1 expression, staining appears to be particularly strong on the superior aspect of the first branchial arch (1) and the lateral aspects of the first (1) and second (2) arches. (F) The corresponding sense embryo. (G) At later stages of development the expression of tcof1 decreases to near-background levels. The antisense embryos (right) appear slightly darker than sense embryos in identical staining regimes, indicating a lower, but constant, level of expression. (H) Positive control using sonic hedgehog. Staining is evident in the rugae (r) and developing incisor tooth germs (tg).

While the exact mechanism underlying TCS is still unknown, the expression patterns observed in the current study are consistent with the hypothesis that this disorder results from haploinsufficiency. Given that the vast majority of the 50 plus mutations observed in TCOF1 to date result in the creation of a premature termination codon and occur prior to the penultimate exon (12 ), they are likely to be associated with reduced cytoplasmic levels of mRNA (31 ,32 ). As we have observed generalized expression of tcof1, with increased levels in the developing craniofacial complex, rather than `all-or-none' expression, it may be that dosage is important for facial development. In the event of a certain threshold of expression not being reached, for example as a result of a mutation in TCOF1, abnormality of the craniofacial complex may result. If this is the case, it is interesting to speculate that the phenotypic variation may result from the total level of transcript produced from the wild-type and mutant alleles with lower levels of transcript resulting in more severe abnormality.

MATERIALS AND METHODS

Zooblot analysis

Genomic DNA (10 [mu]g) extracted from chick, mouse, dog, pig, sheep, cow, monkey and human was digested with EcoRI, the restriction fragments separated by agarose gel electrophoresis and transferred to Biodyne A membrane (Pall) using standard methods (33 ). The membranes were hybridized with a radiolabelled TCOF1 cDNA probe at 65oC (34 ). The membranes were washed to either 1.0* or 0.5* SSC/0.1% SDS at 65oC for 30 min. Autoradiography was performed at -80oC with double intensifying screens for 1-2 days using Fuji RX film.

Isolation and characterization of cDNA clones

Bacteriophage from mouse embryonic day 8.5, day 10 and day 15 (E8.5, E10 and E15) (Dr B. Hogan, Vanderbilt University and Novagen Inc.) and mouse spleen (Stratagene Cloning Systems) cDNA libraries were plated at 5 * 104 p.f.u./140 mm petri dish. Approximately 6 * 105 plaques were screened with restriction fragments of the original TCOF1 cDNA (10 ) and subsequently with fragments of the murine clone E10-1C using standard procedures. Positive primary clones were purified by two additional rounds of screening and were subcloned into pBluescript. The resulting plasmids were restriction mapped, suitable restriction fragments subcloned into M13mp18/19 and sequenced via the dideoxy chain termination method (35 ) using the Sequenase version 2.0 kit (US Biochemical Corp.).

RNA extraction and RT-PCR analysis

Tissue dissected from young adult mice was snap-frozen in liquid nitrogen and total RNA extracted according to the method of Chomczynski and Saachi (36 ) for use in RT-PCR assays and RACE strategies. In the RT-PCR assay, 1 [mu]g of RNA was incubated with 100 ng of random primer at 70oC for 10 min. The samples were chilled on ice and MMLV reverse transcriptase buffer, 10 mM DTT, 1 mM dNTPs (all BRL) and 0.5 U RNAsin (Promega) were added. The reactions were equilibrated at 37oC for 2 min, 200 U MMLV reverse transcriptase added and the samples incubated at 37oC for 1 h. Controls included reactions performed in the absence of RNA or in the absence of reverse transcriptase. The samples were heated to 95oC and 3 [mu]l of cDNA was used in the PCR using the primers 5'-TGA ACA CCA CAA AGA AGG CC-3' and 5'-TAG ACT GCT CAC TCT TGC TG-3' as detailed below.

Rapid amplification of cDNA ends (RACE)

First-strand cDNA synthesis was performed on 1 [mu]g total RNA isolated from spleen and heart, using the 5' or 3' RACE kit (BRL) according to the manufacturer's instructions. In the case of 5' RACE, cDNA synthesis was initiated from the gene-specific primer 5'-TCC TTC TGA CTC AGA CTC-3'. The original mRNA template was then removed by treatment with RNase H. In the case of 5' RACE products a homopolymeric tail was added to the 3' end of the cDNA using TdT and dCTP. PCR amplification of the target cDNA was performed using the universal amplification primer and a 5' RACE gene-specific primer 5'-TTG AAT TCG GAA GAG TCC ACA TCT GAC C-3' or a 3' RACE gene-specific primer 5'-AAC AGC ATC ACC CAG CGC C-3'. After electrophoretic separation on a 2% agarose gel, the PCR product was diluted and subjected to a second round of PCR using the abridged universal amplification primer and a nested 5' RACE gene-specific primer 5'-CCG AAT TCC ACA TCT GAC CTC TGG GTG C-3' or nested 3' RACE gene-specific primer 5'-TTG AAT TCT GGT GAA GGT CCT GAC AGA G-3'. The resulting PCR products were gel-purified, digested with EcoR1/SalI, cloned into M13mp18/19 and sequenced as above.

PCR conditions

PCR assays were performed in 25 [mu]l volumes containing 50 pmol each primer, 200 [mu]M dNTPs, 10 mM Tris-HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl2 and 0.01% gelatin. The samples were overlaid with mineral oil, heated to 96oC for 10 min and cooled to 55oC. After addition of 0.75 U Taq DNA polymerase, the samples were processed through 35 amplification cycles of 92oC for 30 s, 55oC for 30 s, 72oC for 30 s using a Hybaid thermal cycler. The final extension step was lengthened to 10 min. Positive and negative controls were established for all reactions. The PCR products were analysed on 2-3% agarose gels.

Chromosomal localization

Interspecific backcross mapping was carried out using the BSS panel generated at the Jackson Laboratory (37 ). A probe corresponding to nt 2828-3795 of the mouse cDNA (Fig. 2 ) was used to screen Southern blots of HindIII digested DNA using standard methods (33 ). This probe hybridized to fragments of 16 and 2 kb in C57BL/6, and 18 kb in SPRET/Ei. The 94 progeny screened were scored and a map position was obtained by comparison with other markers previously typed on the panel. Recombinational distances and standard errors were calculated according to Green (38 ), and loci were ordered by minimizing the number of recombinants. Alternatively, the primers 5'-GTG ATC TGG TAA TGC CTT GC-3' and 5'-TAG ACT GCT CAC TCT TGC TG-3', which amplify a species-specific polymorphic tract of cytosine residues in intron 17 of tcof1, were used to genotype the EUCIB panel (39 ) using the PCR according to previously published methods (40 ).

Whole-mount in situ hybridization

Embryos derived from time-mated ICR mice were dissected into sterile 1* phosphate buffered saline and fixed in 4% paraformaldehyde overnight. The ages of the embryos were initially determined relative to the detection of a vaginal plug, midday of that day being taken as E0.5, prior to staging according to external morphological criteria (41 ). A number of probes representing different portions of the gene were used in initial whole-mount studies and all were found to produce identical staining patterns. One probe was therefore chosen to screen a range of developmental ages. A portion of the mouse cDNA clone corresponding to nt 2910-3630 cloned into pBluescript was linearized with BssHII and sense and antisense digoxygenin-labelled riboprobes generated using the T3 and T7 promoters. Whole-mount in situ hybridizations were performed essentially as described previously (42 ) and detection was achieved by incubating the hybridized embryos with an alkaline phosphatase-coupled, anti-digoxygenin antibody (Boehringer Mannheim, UK). Positive controls were established using a sonic hedgehog probe (23 ).

ACKNOWLEDGEMENTS

We should like to thank Julie Yang, Julie Vargas and Deanna Church for technical support; Professor M.W.J. Ferguson and Drs C. Byrne, S. Kimber and S. Winokur for advice; Dr A. McMahon for providing the sonic hedgehog plasmid and Dr B. Hogan for the E8.5 cDNA library. We should also like to thank Dr L. Rowe and the Human Genome Mapping Resource Centre, UK for access to the Jackson Laboratory and European Collaborative Interspecific Backcrosses, respectively. This work benefitted from the use of the SEQNET and Human Genome Mapping Resource Centre, UK computing facilities. The financial support of the Wellcome Trust, grant numbers 044684/Z/95/Z (MJD) and 044327/Z/95/Z (MJD), and NIH grant AR-42377-03 (RS) are acknowledged.

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*To whom correspondence should be addressed. Tel: +44 161 275 5620; Fax: +44 161 275 5620; Email: mdixon@fs2.scg.man.ac.uk

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