| Human Molecular Genetics | Pages |
A novel mammalian Wnt gene, WNT8B, shows brain-restricted expression in early development, with sharply delimited expression boundaries in the developing forebrain
Introduction
Results
Structural characterization of the human WNT8B gene
WNT8B expression in early human embryogenesis
Discussion
Materials And Methods
Genomic clone isolation and characterization
cDNA structure
DNA sequence analysis
In situ hybridization on human and mouse embryo sections
Acknowledgements
References
A novel mammalian Wnt gene, WNT8B, shows brain-restricted expression in early development, with sharply delimited expression boundaries in the developing forebrain
DDBJ/EMBL/GenBank accession nos Y11094 and Y11108
Our current knowledge of mammalian forebrain development is meagre. The comparatively few relevant anatomical landmarks are, however, being supplemented by gene expression studies which are able to identify subsets of anatomical structures. We previously described cloning, subchromosomal localization and preliminary structural characterization of the human WNT8B gene, the first mammalian Wnt8b gene to be reported. Wnt genes encode intercellular signalling molecules which play a variety of critical roles in early development, including, in several cases, a presumed role in brain development. In the current report we present the full-length cDNA sequence and genomic organization of the human Wnt8b gene and report studies of expression of the Wnt8b gene in human and mouse embryos. The human and mouse expression patterns appeared identical and were restricted to the developing brain, with the great majority of expression being found in the developing forebrain. In the latter case expression was confined to the germinative neuroepithelium of three sharply delimited regions: the dorsomedial wall of the telencephalic ventricles (which includes the developing hippocampus), a discrete region of the dorsal thalamus and the mammillary and retromammillary regions of the posterior hypothalamus. Expression in the developing hippocampus may suggest a role for WNT8B in patterning of this region and subchromosomal localization of the human gene to 10q24 may suggest it as a candidate gene for partial epilepsy in families where the disease has been linked to markers in this region.
INTRODUCTION
The details of vertebrate brain pattern are gradually being understood and gene expression patterns in hindbrain have been tightly linked to transverse segmentation into rhombomeres and in midbrain to the activity of an isthmus signalling centre (1). Forebrain patterning is less well understood, but region-restricted gene expression patterns have also suggested the existence of transverse segmentation into neuromeres (prosomeres) plus longitudinal compartmentalization of the prosomeres (2). While good animal models of early development have been extremely valuable, the importance of the forebrain in defining unique human functions means that there is a need to supplement our rather meagre understanding of early human brain development, which has mostly been founded on anatomical studies of archived material (3).
Among the genes that are thought to have an important role in early brain development are various members of the Wnt gene family (4). Wnt genes encode intercellular signalling molecules which play important roles in key processes of embryonic development, including mesoderm induction, specification of the embryonic axis and patterning of the central nervous system and limb (5,6). Largely from studies in mouse, it has been established that mammalian genomes harbour over a dozen different Wnt genes, some of which have been classified into subfamilies, mostly on the basis of a comparatively high degree of sequence homology for paralogous genes (7). An analogous family of five genes is known in Drosophila, of which the archetypal member is the wingless (wg) gene (8,9 and references therein). Over the last few years the molecular details of the Wnt/wg signalling pathways have been clarified and the components of the pathway have been shown to be conserved from flies to mammals (for a review, see ref. 10). The functional importance of Wnt genes is endorsed by mutant phenotypes observed in the case of spontaneous mutations, such as the murine swaying (Wnt1) and vestigial tail (Wnt3a) mutants, and in a variety of artificially created mouse phenotypes, including various gene-targeted Wnt knock-outs (for a review, see ref. 11 and references therein) and also mutant phenotypes derived from antisense-mediated inhibition of Wnt gene expression in whole embryo culture (12).
We recently described cloning, subchromosomal localization and preliminary structural characterization of the human WNT8B gene, the first mammalian member of the Wnt8 subfamily (Wnt8, Wnt8b and Wnt8c) to be reported (13). Clearly identifiable orthologues of this novel mammalian gene demonstrate brain-restricted expression patterns in zebrafish (14), Xenopus (15) and the chick (16). We now present the full-length cDNA sequence and genomic organization of the WNT8B gene and report our studies on expression of WNT8B/Wnt8b in human and mouse embryos. The data obtained indicate that expression of the mammalian Wnt8b gene is brain-restricted, like that of non-mammalian vertebrate orthologues, but differs in being particularly confined to the forebrain, where there are highly delimited regions of expression which appear to be conserved in humans and mice. The data also indicate that WNT8B provides a very good molecular marker for the study of hippocampal differentiation as well as for subcortical structures (such as posterior hypothalamus and dorsal thalamus). The observed expression patterns also suggest that the WNT8B gene could be the locus for certain neurological disorders that arise as a result of abnormal development of the hippocampus in humans (see Discussion).
RESULTS
Structural characterization of the human WNT8B gene
We previously reported isolation of an ~900 bp human WNT8B cDNA clone which contained a partial coding sequence (13). We have subsequently defined sequences at the 5[prime]- and 3[prime]-ends to complete the full-length 2.1 kb cDNA sequence (Fig. 1). The coding sequence is interpreted to specify a 351 amino acid polypeptide containing 24 cysteine residues, 20 of which are found at identical positions in all the other Wnt8 proteins characterized so far (13,14,16,17). A highly conserved Wnt family `signature sequence' present in all other Wnt proteins is found in the inferred WNT8B protein (amino acids 180-189; Fig. 1), as well as two asparagine-linked glycosylation sites (amino acid 103 and 259; Fig. 1), which are present in other Wnt8 proteins. The predicted amino acid sequence of the human WNT8B gene is very similar to that of the inferred products of its assumed orthologous genes, including mouse Wnt8b (95.1% identity; the mouse Wnt8b sequence was compiled from our own unpublished results and data provided by Dr J. Mason and Prof. A. McMahon and the sequence of EST X91941), Xenopus wnt8b (86.6%) and zebrafish wnt8b (80.7%). The equivalent sequence comparisons for the products of other members of the Wnt8 subfamily, such as the zebrafish Wnt8 gene (65% identity) and the chick Wnt8c gene (68% identity), indicate that these genes, although paralogues, are clearly closely evolutionarily related.
Figure 1. WNT8B gene organization. The figure shows the full-length cDNA sequence and inferred amino acid sequence of the polypeptide product. The previously published sequence (13) extends from nucleotide 207 to 1088. Exon boundaries are indicated by small arrows and the polyadenylation signal is highlighted by bold type and underlining. Conserved cysteine residues are shown in bold, N-linked glycosylation sites are underlined and a highly conserved Wnt family `signature sequence' is boxed. The termination codon indicated by an asterisk is at the start of the 3[prime]-untranslated region. Intron sizes: intron 1, several kilobases (not fully sequenced); intron 2, 790 bp; intron 3, 986 bp; intron 4, 787 bp; intron 5, 224 bp. The cDNA and genomic sequences have been deposited in the EMBL database under accession nos Y11094 and Y11108 respectively. The predicted WNT8B transcript size of ~2.1 kb conforms well with the results obtained from northern blots, where the size of the main transcript observed is ~2.1 kb, with minority transcripts of greater size possibly reflecting the occasional usage of alternative polyadenylation sites (data not shown). The WNT8B cDNA sequence is specified by six exons which are separated by rather small introns, with the exception of intron 1 (Fig. 1). We found no evidence, however, for an extra exon of the type that has been reported to be spliced out in the Xenopus Wnt8b gene (15). The location of this exon corresponds to the exon 3-exon 4 boundary of the human WNT8B gene, but after sequencing the interval separating these two exons in genomic DNA we were unable to find any sequences showing significant homology to the extra exon in the Xenopus Wnt8b gene or any indication of additional exons in the other introns.
WNT8B expression in early human embryogenesis
We studied expression of the WNT8B/Wnt8b gene by in situ hybridization to sections from human embryos (see below) and also from mouse embryos [10-13 days post-coitum (d.p.c.)]. Unlike equivalent human/mouse comparisons for some other Wnt genes (see Discussion), we observed no significant differences in the embryonic expression patterns of the human and mouse Wnt8b orthologues (data not shown). As a result, we report here only the human embryonic expression patterns. In all we studied sections from eight human embryos, spanning Carnegie stages 15-21 (33-52 post-ovulatory days; see ref. 3) and these comprised six embryos cut in saggital section (CS15, CS16, CS17, CS18, CS19 and CS21) and two embryos cut in transverse section (CS15 and CS17). During all these stages expression was seen to be confined to the brain, principally in three regions of the forebrain as detailed below. Throughout the stages studied WNT8B transcripts were only detected in the germinative neuroepithelium (also called the ventricular layer and described as the proliferative cell layer nearest the ventricle in each brain region) of these forebrain regions.
Dorsomedial wall of the telencephalon. At all stages WNT8B transcripts were detected in a highly regionalized manner in the dorsomedial wall of each telencephalic vesicle, continuing in the midline with the roof plate of the third ventricle (Figs 2. a, d and e and 3. a). Overall the expression domain resembles a triangular shape expanding at the top of the cerebral vesicle and narrowing down towards the midline until the level of the commisural plate. During Carnegie stage 15 (CS15, 33 post-ovulatory days) the forebrain consists of two telencephalic vesicles, each enclosing a lateral ventricle, and the diencephalon, whose cavity is the third ventricle. At this stage WNT8B expression in the telencephalon is observed in the germinative neuroepithelium of the dorsomedial wall, a region which includes the primordial hippocampus (18; Fig. 4a). During Carnegie stage 17 (41 post-ovulatory days) the primordial hippocampus is thought to subdivide into two regions, one corresponding to the primordium gyrus dentatus and the other the primordium cornu Ammonis, which is going to give rise to the hippocampus itself (19). WNT8B transcripts are detected in both areas, probably indicating a role in differentiation of the hippocampal primordia from other cortical formations, rather than hippocampus specialization itself (Fig. 2b, e and f). During the following stages (CS18-CS21) WNT8B continues to be expressed in the ventricular layer of the primordial hippocampus. While at the earlier stages of development (C15-CS16) we cannot be sure that the cells that express WNT8B will only give rise to the primordial hippocampus, by Carnegie stage 17 it is clear that while there is expression in the primordial hippocampus, no expression is detected in the adjacent structures, such as the commisural plate (Figs 2. a and 3. a). We observed an identical expression pattern in mouse embryos at 10-13 d.p.c. (data not shown). A schematic view of the expression domain in the dorsomedial wall of the telencephalon is presented in Figure 5. a and b.Figure 2. Expression of WNT8B in the brain of a Carnegie stage 17 embryo (41 post-ovulatory days). The figure shows in situ hybridization of an antisense human WNT8B probe to sections of a CS17 embryo as revealed by dark field microscopy. Individual plates show sections as follows: (a and b) medial and lateral sagittal sections respectively; (c-f) transverse sections, obtained by cutting in the approximate planes indicated in (a). Bar 500 µm. Indicated regions are as follows: AC, archicortex (the region which includes the primordial hippocampus); cp, commisural plate; chp, chiasmatic plate; D, diencephalon; DT, dorsal thalamus; ET, epithalamus; HT, hypothalamus; IVF, intraventricular foramen; LV, lateral ventricle; M, midbrain; mr, mammillary recess; NC, neocortex; OG, optical groove; PHC, primordial hippocampus; phd, posterior hypothalamus, dorsal; phv, posterior hypothalamus, ventral; RF, roof plate of third ventricle; SR, striatal ridge; tdd, telencephalon diencephalon dorsal fissure; VT, ventral thalamus; 3rd V, third ventricle; 4th V, fourth ventricle. Figure 3. WNT8B expression in the forebrain of a Carnegie stage 19 embryo (48 post-ovulatory days). (a) Dark field microscopy of in situ hybridization of an antisense WNT8B probe to a medial sagittal section; (b) bright field microscopy of a sagittal section adjacent to that in Figure 4a following staining with haematoxylin and eosin. The germinative neuroepithelium shows a darker colour than the marginal layer. Bar 500 µm. Indicated regions are as follows: AC, archicortex (the region which includes the primordial hippocampus); cp, commisural plate; ET, epithalamus; LV, lateral ventricle; mr, mammillary area; phd, posterior hypothalamus, dorsal; phv, posterior hypothalamus, ventral; 3rd V, third ventricle. Figure 4. WNT8B expression in the brain of a Carnegie stage 15 embryo (33 post-ovulatory days). The figure shows in situ hybridization of antisense human WNT8B probe to sections of a CS15 embryo as revealed by dark field microscopy. Individual plates represent sections as follows: (a and b) medial and lateral sagittal sections respectively; (c-f) transverse sections, obtained by cutting in the approximate planes indicated in (a). Bar 500 µm. Indicated regions are as follows: D, diencephalon; DT, dorsal thalamus; DTJ, diencephalon-telencephalon junction; ECV, edge of cerebral ventricles; H, hindbrain; HT, hypothalamus; LT, lamina terminalis; LV, lateral ventricle; M, midbrain; mr, mammillary recess; rmr, retromammillary area; phv, posterior hypothalamus, ventral; ST, subthalamus; T, telencephalon; 3rd V, third ventricle. Figure 5. Schematic presentation of human WNT8B expression in embryonic forebrain. (a) Dorsal view of the telencephalon; (b) medial view of the telencephalon; (c) median view of the brain. WNT8B expression domains are represented by solid black shading as follows: expression in the dorsomedial wall of the telencephalon (a and b); expression in the mammillary and retromammillary regions of the posterior hypothalamus (c, middle); expression in the dorsal thalamus (c, lower). The figures were adapted from O'Rahilly and Muller (3) with the permission of Wiley-Liss. Indicated regions are as follows: AH, adenohypophysis; Archi, archicortex (forebrain region including the hippocampus); Amyg. nuclei, amigdaloid nuclei; Ep, epiphysis; Hipp, hippocampus; l-v f, lateral ventricle foramen; M, midbrain; MA, mammillary area; Med. em., median eminence; Med. wall of left hemisphere, median wall of left hemisphere; NH, neurohypophysis; RM, retromammillary area; Thal.d., thalamus, dorsal. Neuroepithelium of the dorsal thalamus. During Carnegie stage 15 the developing diencephalon is seen to be separated by different sulci into epithalamus, dorsal thalamus, ventral thalamus, subthalamus and hypothalamus (dorsal to ventral; see ref. 3). In the embryos we studied at this stage the dorsal thalamus is incompletely separated from the ventral thalamus, but WNT8B transcripts could be seen to be confined to a patch of cells of the dorsal thalamus (Fig. 4a and d; see also Fig. 5c for a schematic representation). In subsequent embryonic stages (CS16-CS21) extension of the thalamus as well as some stratification occurs. However, WNT8B expression was restricted to the germinative neuroepithelium of the dorsal thalamus. In the early stages (CS15 and CS16) expression of WNT8B is also detected in the diencephalon-telencephalon junction (Fig. 4e) and along the roof of the midbrain (Fig. 4b). This expression domain could not be detected after Carnegie stage 16 (37 post-ovulatory days). We also detected faint signals in a patch of cells in the caudal midbrain just abutting the midbrain-hindbrain border throughout the stages we studied, but we did not detect any WNT8B transcripts in the hindbrain or spinal cord.
DISCUSSION
In the present study we have reported extensive structural characterization and embryonic expression patterns of a novel mammalian Wnt gene which may have an important role in development of certain forebrain structures, notably the hippocampus. The WNT8B gene is a member of a large family of genes encoding intercellular signalling molecules, which are believed in some cases to act as morphogens (20). Individual vertebrate Wnt genes are, typically, expressed in a variety of organ systems and tissues of the developing embryo. In contrast, embryonic expression of the mammalian Wnt8b gene is very highly restricted, being confined to the brain, predominantly to highly delimited regions of the forebrain. The expression domains observed in human and mouse embryos appear to be strictly conserved, unlike those of human and mouse orthologues for some other Wnt genes. For example, midbrain expression of the human WNT7A gene is confined to the dorsolateral regions, with no expression detected on the ventral floor (our unpublished observations), whereas midbrain expression of mouse Wnt7a is restricted to the ventral floor and part of the lateral walls and is absent from the dorsal regions (4; our unpublished observations and unpublished data from S. Lee and A. McMahon). Furthermore, although mammalian Wnt8b expression shows some similarities to expression of Wnt8b in other vertebrates [chicken, zebrafish and Xenopus Wnt8b are expressed mainly in the developing forebrain and forebrain-midbrain boundary (14-16)] there are also some important species differences. For example, unlike the human and mouse orthologues zebrafish wnt8b is expressed strongly in the hindbrain (rhombomeres 3 and 5) and chicken wnt8b is also expressed in the hindbrain (14,16). However, the information concerning Wnt8b expression in other species is not as detailed as the information available for the mammalian Wnt8b gene, thereby preventing meaningful interspecies comparisons of expression patterns.
The highly restricted Wnt8b expression patterns in the developing mammalian forebrain constitute hitherto unrecognized subdivisions of anatomical structures (Fig. 5). In recent years there have been considerable advances in our understanding of the inductive processes and molecular signals involved in patterning of the vertebrate brain, notably the hindbrain and to a lesser extent the midbrain. In the former case restricted patterns of expression of a variety of genes, such as Hox genes, have been associated with transverse segmentation along the anterio-posterior axis into rhombomeres, whereas in midbrain restricted expression patterns of certain genes have been tightly linked to the activity of a signalling region in the isthmus (1). Transverse segmentation of the developing vertebrate forebrain into neuromeres (prosomeres), analogous to the rhombomeres of hindbrain, plus longitudinal segmentation of prosomeres have also been proposed (2,21,22 and references therein). Some evidence supporting the prosomere models has come from morphological observations, such as the presence of horizontal bulges and sulci along the medial walls of the third ventricle of the diencephalon, but the major support comes from region-restricted patterns of expression for a variety of developmental control genes. Many of the latter encode presumptive transcription factors (21-23) or molecules involved in intercellular signalling or cell adhesion (22,24), including some members of the Wnt gene family, such as Wnt3, Wnt3a and Wnt7b, which show sharp boundaries of expression in the developing mouse forebrain (4,25). The patterns of forebrain WNT8B expression that we observe in human/mouse embryos are also consistent with the prosomere model (2): expression is confined to the P3 and P4 prosomeres (a ventral region including the mammillary and retromamillary regions, plus a dorsal region of P4 which represents the hippocampal primordia). However, the relationship of the proposed prosomeres to later structures is unclear and there is increasing evidence that environmental cues play a role in neuronal migration and differentiation (26 and references therein). The picture should become clearer with further examination of gene expression patterns and studies to identify the developmental relationships between early and later forebrain structures.
The basis for the stringent regulation of WNT8B expression remains unknown. Genetic studies in Drosophila have indicated regulation of wingless expression by several homeodomain-encoding genes [including distal-less, even-skipped, gooseberry and fushi-tarazu (8,27)] and so regulation of vertebrate Wnt gene expression could be expected to be carried out by related genes. In support of this some vertebrate homeobox genes have the spatiotemporal expression patterns expected of Wnt gene regulators, e.g. expression of the Pax2 homeobox gene precedes and encompasses the Wnt1 expression domain in the developing mouse neural plate (28). Other evidence comes from identifying homeodomain binding sites in murine Wnt genes. A well-defined homeodomain binding site has been characterized in the 3[prime] enhancer sequence of the mouse Wnt1 gene (29). In addition, an intron in the mouse Wnt5a gene appears to have a series of repeated sequence motifs which are specifically bound in vitro by the homeodomain of the product of the Msx1 gene, a gene whose expression overlaps that of Wnt5a in mouse embryos (30). In the case of Wnt8b candidate regulatory genes could include the Otx and Emx homeobox genes, which are expressed in the same forebrain structures as Wnt8b (31). The Emx2 gene, like Wnt8b, is expressed in the hippocampal primordia of the archicortex and specifies a homeodomain which, like the Dlx2 homeodomain, can bind to the same core motif as that recognized by the Msx1 homeodomain (TAAT/ATTA) and also to an additional AT-rich motif, TTTAT/ATAAA (32). The Emx2 and Dlx2 proteins also interact specifically with the homeobox binding site in the Wnt1 enhancer, whereas classical Hox homeodomains do not (29). In the case of the WNT8B gene there are many examples of the same sequence motifs recognized by these homeodomain proteins. For example, the short intron 2 has 10 examples of the TAAT/ATTA motif and four of the TTTAT/ATAAA motif (data not shown; see EMBL accession no. Y11108 for the genomic sequence).
The precise functional significance of WNT8B expression remains to be elucidated. Within the restricted domains of forebrain expression WNT8B transcripts are detected only in the germinative neuroepithelium throughout neurogenesis, suggesting that this gene might play an important role in cell proliferation, as previously reported for the mouse Wnt1 gene (33). Zebrafish and Xenopus wnt8b gene expression is also prominent in the developing forebrain (14,15) and in these species the hippocampus is a comparatively more prominent forebrain component than in mammals. It is tempting to speculate, therefore, that the Wnt8b gene is important for hippocampus development and differentiation. Its potential role in the developing dorsal thalamus and posterior hypothalamus is uncertain. The restriction of WNT8B expression to only small components of these structures may reflect some functional subdivision and further studies in later stages of development are required to clarify the importance of WNT8B expression in formation of diencephalic nuclei.
Possible pathogenic consequences of mutation in the WNT8B gene remain to be identified. We previously mapped this gene to 10q24 (13), a region which is known to be the locus for a variety of inherited disorders, including infantile onset spinocerebellar ataxia, autosomal dominant progressive external ophthalmoplegia, glioblastoma multiforme, split hand/foot malformation type 3 and partial epilepsy (34-39). Of these, the partial epilepsy condition is one for which the WNT8B gene is a plausible candidate gene. Although this disorder shows an autosomal dominant mode of inheritance while phenotypes resulting from mutations in mouse Wnt genes are normally seen only in homozygotes, the example of holoprosencephaly due to mutations in the Sonic hedgehog gene should serve as a cautionary precedent: the human phenotype is expressed in heterozygotes, but in mouse the reported phenotypes are recessive (40,41). It should be noted that individuals with medial temporal lobe epilepsy have hippocampal atrophy, hippocampal and dentate gyrus neuronal cell loss, lower neuronal cell density and associated neocortical malformations (42-45). While hippocampal cell loss could happen as a result of prolonged febrile convulsions (46), we cannot exclude that cell loss could also happen during embryogenesis as a result of malfunction of genes that control cell proliferation and migration in the hippocampus. The WNT8B gene is expressed amongst others in the primordial hippocampus in the period when future neurons are generated and migrate to their target destinations. If WNT8B plays an important role in cell proliferation, as has been shown in the case of Wnt1 (33,47), mutations causing abnormal WNT8B expression could lead to hippocampal abnormalities.
MATERIALS AND METHODS
Genomic clone isolation and characterization
Two WNT8B genomic clones were isolated by conventional screening of the ICI YAC library (13). DNA from one clone, 21C-F9,was partially digested with Sau3AI, then ligated to [lambda] Fix II replacement vector arms (Stratagene) which had been partially filled to allow ligation of overhanging ends to take place. In vitro packaging was carried out using Gigapack III Gold Packaging extract from Stratagene. Approximately 105 plaques were plated and hybridized with partial cDNA probes which covered the 900 bp cDNA product (13; see also Fig. 1). The probes were labelled with [[alpha]-32P]dCTP following the random priming method (49). Hybridization washes were conducted at 0.1× SSC and 0.1% SDS. Four strongly positive clones were selected and purified as described (49). DNA was digested with a variety of enzymes and after Southern blotting and rehybridization with the same cDNA probes positive restriction fragments were identified. These fragments were subcloned into the pZERO cloning vector (Invitrogen, Leeks, The Netherlands). Insert DNA was sequenced using oligonucleotide primers derived from known cDNA sequence (see below). Exon-intron boundaries of the WNT8B gene were largely determined by sequencing two adjacent XbaI genomic subclones, a 4.1 kb fragment linking XbaI sites in introns 1 and 4 and a 1.97 kb XbaI fragment spanning an interval from the middle of intron 4 to the 3[prime]-end.
cDNA structure
We supplemented the partial WNT8B cDNA sequence (13) by retrieving the 5[prime]-end of the transcript using RACE (rapid amplification of cDNA ends) on human fetal brain cDNA (Marathon ready cDNA kit; Clontech Laboratories, Palo Alto, CA). The first amplification was carried out using the gene-specific primer GSP1 (5[prime]-GCTGCCCGTGCATCCTGTCC-3[prime]) and the AP1 primer provided with the RACE kit. Aliquots of 1 µl PCR product were diluted 250 times and 5 µl of this dilution were used as template in a nested secondary reaction with a second gene-specific primer (GSP2, 5[prime]-GGGAGTCATCACAGCCACAG-3[prime]) and AP2. In both PCRs the cycling conditions were as follows: 94°C for 1 min and 30 cycles of 94°C for 30 s, 60°C for 30 s and 68°C for 4 min. The amplification product was cloned into the TA cloning vector (Invitrogen). Sequencing of inserts was performed using an ABI automated sequencer and standard dideoxynucleotide sequencing methods. The 3[prime]-end sequence, including the 3[prime]-end of the coding sequence plus the untranslated region, was defined by sequencing the 1.97 kb XbaI genomic subclone (see above).
DNA sequence analysis
DNA sequence analysis was largely carried out using the GCG (Wisconsin Computer Package) programs. PILEUP and PRIMEGEN were used for multiple alignment and primer design; BESTFIT and FASTA were used for sequence comparison (49-51). The PROTEAN program (part of the DNASTAR package) was used for protein sequence analysis (52).
In situ hybridization on human and mouse embryo sections
Collection and use of human embryos was carried out after ethical permission had been obtained from the Joint Ethics Committee of the Newcastle Health Authority and with the appropriate signed consents. Embryos were collected following either surgically (53) or medically (RU486) (54) induced termination of pregnancy, staged by microscopic examination according to the Carnegie classification system (55), fixed in 4% paraformaldehyde in phosphate-buffered saline and embedded in paraffin (56). Sense and antisense probes were prepared by linearizing plasmid recombinants containing human WNT8B cDNA (a 900 bp long sequence containing most of the coding sequence; see Fig. 1) or mouse Wnt8b cDNA (a 700 bp sequence containing 600 bp of the coding sequence and 100 bp of the 3[prime]-untranslated sequence; a gift of Dr John Mason, University of Edinburgh) and transcribing with the appropriate RNA polymerases: T7 or Sp6 in the case of the human WNT8B and T7 or T3 in the case of the mouse Wnt8b cDNA. Transcripts were labelled in vitro with [[alpha]-35S]ATP and hybridized to tissue sections in conventional hybridization buffer (5 × 104 c.p.m./µl) (55). After high stringency washes slides were coated in Ilford photographic emulsion and exposed for 10 days before being developed and photographed.
ACKNOWLEDGEMENTS
We are grateful to Professor Andy McMahon (Harvard University) and Dr John Mason (University of Edinburgh) for communicating unpublished data. We would like to thank the Wellcome Trust and the Special Trustees of the Royal Victoria Infirmary, Newcastle, for supporting our work on Wnt gene studies and the UK Medical Research Council for additional support.
REFERENCES
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