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Human Molecular Genetics, 2003, Vol. 12, No. 10 1163-1170
DOI: 10.1093/hmg/ddg122
© 2003 Oxford University Press

Congenital hydrocephalus in hy3 mice is caused by a frameshift mutation in Hydin, a large novel gene

Brian E. Davy and Michael L. Robinson*

Division of Molecular and Human Genetics, Columbus Children's Research Institute and The Department of Pediatrics, College of Medicine and Public Health, The Ohio State University, Columbus, OH 43205, USA

Received January 20, 2003; Revised February 26, 2003; Accepted March 12, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The autosomal-recessive mutation hydrocephalus3 (hy3) results in lethal communicating hydrocephalus with perinatal onset. We recently described a hydrocephalus-inducing transgenic insertional mutation, OVE459, which represents a new allele of hy3. Direct cDNA selection performed on a wild-type mouse BAC clone spanning the OVE459 insertion locus on chromosome 8 led to the identification of two novel candidate genes, Hydin and Vac14. The transgene insertion resulted in a rearrangement of Hydin exons in OVE459 mice. Hydin consists of at least 86 exons spanning over 340 kb of genomic DNA. The full-length Hydin transcript is nearly 16 kb, encoding a putative 5099 amino acid protein. Northern analysis revealed a marked reduction of Hydin mRNA in both OVE459 and hy3 homozygotes relative to wild-type littermates. A single CG base-pair deletion in exon 15 of Hydin was discovered specifically in mice carrying the spontaneous hy3 mutant allele. This deletion creates a premature termination signal two codons downstream of the mutation, likely resulting in the loss of 89% of the full-length gene product. Within the neonatal brain, Hydin expression is confined to the ciliated ependymal cell layer lining the lateral, third and fourth ventricles. Other sites of Hydin expression include the ciliated epithelial cells lining the bronchi and oviduct, as well as in the developing spermatocytes in the testis. The Hydin gene product is not closely related to any previously identified protein, with the exception of a 314 amino acid domain with homology to caldesmon, an actin-binding protein, suggesting an interaction with the cytoskeleton.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Congenital hydrocephalus is a frequent human birth defect, occurring with an estimated incidence of 1 in 1000 live births (1,2). A significant portion of human congenital hydrocephalus is genetic in origin. Despite this, the molecular genetics of human hydrocephalus remains poorly understood. The X-linked gene, L1CAM, remains the only gene known to be involved in the development of congenital hydrocephalus in humans (3,4). While mutations in L1CAM are responsible for many, if not most, cases of X-linked hydrocephalus, there is abundant evidence that autosomal recessive forms of human hydrocephalus exist. Several cases of multiple female or mixed sex siblings having hydrocephalus have been reported where autosomal recessive hydrocephalus was suspected (5–8). The disease genes or chromosomal locations associated with these cases remain entirely unknown.

There are currently a number of genetic mouse models of hydrocephalus. Some of these are the result of induced or targeted mutations (9–13), while others are the result of spontaneous mutations (14–17). Hydrocephalus-inducing mutations in which the affected gene remains unidentified include hydrocephalus 3 (hy3), hop-sterile (hop), obstructive hydrocephalus (oh) and hydrocephalus with hop gait (hyh). The spontaneous autosomal-recessive mutation hy3, first identified more than 60 years ago by Hans Gruneberg (15), causes lethal communicating hydrocephalus with perinatal onset. Newborn hy3 homozygotes are indistinguishable from wild-type littermates but develop progressive hydrocephalus that is externally observable within the first week after birth and always lethal before 7 weeks of age.

Hydrocephalus, by definition, is a net accumulation of cerebrospinal fluid (CSF) within the ventricular system of the brain. Cerebrospinal fluid is constantly produced within the brain ventricles and drains into the subarachnoid spaces surrounding the external surface of the brain where it is reabsorbed into the venous system. The choroid plexus produces 70–80% of the total CSF volume with an additional 10–30% being produced by the bulk flow of extracellular fluid from the brain parenchyma through the ependymal cells that line the ventricular lumen (18). While the precise mechanism of hydrocephalus in hy3 homozygotes is unclear, the first histological changes have been observed in the meninges of the subarachnoid space (19). Dye injection experiments indicated that there was clear communication between the ventricles, but dye injected into hydrocephalic ventricles failed to enter the subarachnoid space (19,20). These observations led to the hypothesis that hydrocephalus in hy3 homozygotes was caused by defective CSF reabsorption.

We recently described the transgenic insertional mutation Tg(Bdnf)459Ove (OVE459), representing a new mutation allelic to hy3 (21). To clone genomic DNA flanking the transgene-insertion site, a lambda phage library constructed from OVE459 homozygous genomic DNA was screened by hybridization using a transgene-specific probe. Two phage clones, BAA and CAA, representing opposite sides of the insertion site were identified and sequenced. Unique genomic sequences from both phage clones were found on a 120 kb wild-type mouse BAC clone, CITB-CJ7-218P4 (BAC 218P4). In addition, a PCR polymorphism between C57BL/6 DNA and Mus spretus, D8Mlr1, present on the CAA phage clone, did not recombine with D8Mit151 using the Jackson Laboratory Backcross DNA Panel Mapping Resource. This places the transgene insertion site at 54 cM on the mouse genome informatics consensus linkage map of chromosome 8. The corresponding chromosomal region in the human genome, 16q22, has been associated with at least one case of human hydrocephalus (22). Here, we report a candidate gene approach to identify the relevant gene in the hy3 mouse model of congenital hydrocephalus.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The genomic sequences flanking the OVE459 transgene insertion site are present on the wild-type BAC clone 218P4 (21). We chose two independent approaches to identify expressed sequences present on BAC 218P4. First, we used the entire BAC clone as a probe to screen an arrayed I.M.A.G.E. Consortium mouse cDNA filter set consisting of over 180 000 non-redundant clones from several different libraries. One clone (I.M.A.G.E. ID no. 312752) from a Life Technology cDNA library, thought to be from mouse brain, hybridized to BAC 218P4 under stringent conditions (unpublished data). This cDNA library was later discovered to be of rat origin. Nonetheless, we completely sequenced this EST (GenBank accession no. AY220476) and designated this gene candidate 1. The second approach utilized cDNA derived from neonatal head mRNA as the input for direct cDNA selection using BAC 218P4 as the driver. cDNA clones representing partial transcripts of two different genes were isolated. Seven exons matched the sequenced EST representing candidate 1, and 30 exons were identified for a gene designated candidate 2.

Neither candidate gene was present in its entirety on BAC 218P4. A screen to identify larger BAC clones encompassing the transgene insertion site led to the identification of BAC RP23-21B7 (BAC 21B7), which was approximately twice the size and fully encompassing the genomic sequence of BAC 218P4. BAC 21B7 was sequenced by the Trans-NIH Mouse Sequencing Consortium to facilitate the identification of additional exons from these candidate genes, but this BAC also failed to contain the entire genomic sequence of either candidate gene. The sequence of BAC 21B7 made it possible to identify the homologous region of the assembled human genome on chromosome 16. The map of the hy3 candidate region was constructed after comparing the sequence of this segment of chromosome 16 with the subsequent release of the assembled mouse genome (Fig. 1). In addition to candidates 1 and 2, the genome assemblies allowed us to determine the positions and relative orientations of two other genes, Calretinin (Calb2) and a gene encoding a novel putative protein (BC025546 and FLJ11171 in the mouse and human assemblies, respectively).



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  		Figure 1. The hy3/OVE459 candidate region on mouse chromosome 8 (A) and the corresponding region on human chromosome 16 (B). (A) Four genes located at 8D3 are situated near the OVE459 transgene insertion site. Genes, arranged on the chromosome from centromere (cen) to telomere (tel), are represented by boxes with transcriptional direction indicated by arrows. BAC clones described in the text are represented as solid horizontal bars beneath the chromosome, and solid bars above the chromosome represent the locations of the genomic DNA present on phage clones CAA and BAA that flank the transgene insertion site in OVE459 mice. Upward pointing arrows indicate the positions of the polymorphic genetic markers D8Mlr1 and D8Mit151. Calretinin (CalB2) and a gene defined by its putative protein (accession BC025546) lie upstream of Hydin (Candidate 2) and Vac14 (Candidate 1) lies downstream of Hydin. (B) The hy3/OVE459 candidate region corresponds to human chromosome 16q22. The order and orientation of candidate genes through this region are conserved in the human genome. BAC clones are represented as solid horizontal bars beneath the chromosome.

 
Candidate 1 is a novel 19 exon gene beginning 12.7 kb distal to the transgene insertion site. Portions of the human orthologue of candidate 1 were initially described as HTLV-I Tax1-binding protein (TRX), but our results indicate that the reported cDNA sequence was partial and contained reading frame errors (23). Candidate 1 covers a genomic region of 102 kb in the mouse. A ubiquitously expressed 3.1 kb candidate 1 transcript encodes a protein that is highly conserved among all eukaryotic organisms sequenced from yeast to human. Candidate 1 is the mouse orthologue of the Saccharomyces cerevisiae gene Vac14 and, based on this homology, candidate 1 was renamed Vac14 and VAC14 in both the mouse and human genomes, respectively. Yeast Vac14p regulates the lipid kinase Fab1p and is required for the osmotic stress-induced increase of phosphatidylinositol 3,5-bisphosphate (24).

The transgene in line OVE459 inserted within candidate gene 2. For this and a number of other reasons detailed below, we have named this gene Hydin (for hydrocephalus-inducing). Hydin spans over 340 kb of genomic DNA and consists of at least 86 exons. The 56 exons not isolated by cDNA selection were predicted by mouse–human homology and gene prediction software using sequence information compiled from the public and Celera genomic databases. Predicted exons were then confirmed by RT–PCR. We have included an additional alternatively spliced exon (exon 2) which we have been unable to experimentally confirm. This exon consists of 182 bp and was included because it is surrounded by appropriate splice consensus sites and is present in a neonatal mouse lung EST (GenBank accession no. BB664150). The full-length 87 exon (including exon 2) Hydin transcript (GenBank accession no. AY173049) is ~15.9 kb and encodes a putative protein of 5099 amino acids. Despite the presence of several upstream AUG codons, the length of the putative protein is based on choosing an AUG codon in exon 3 that is both consistent with translation initiation (25) and conserved in the human Hydin orthologue on chromosome 16.

Reconstruction of the transgenic locus using the phage clones BAA and CAA indicated that the order of exons within Hydin is disrupted in line OVE459 (Fig. 2). PCR amplification of all 87 Hydin exons from OVE459 homozygous DNA (unpublished data) demonstrated that the transgene-induced genomic rearrangement at this locus is more complex than a simple deletion. While the precise nature of this rearrangement remains unclear, the disruption of Hydin by the transgene prompted an in-depth analysis of Hydin expression in OVE459 and hy3 mutant mice.



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  		Figure 2. Genomic structure of wild-type Hydin (top) and the OVE459 transgene insertion locus (bottom). The 87 exons of Hydin are indicated by vertical lines. In some cases, exons are too close together to be individually resolved at the scale represented. The location of the putative translation initiation codon in exon three is indicated. The phage clones BAA and CAA, which flank the multi-copy transgene (TG) insertion site, are separated by 51 kb in the wild-type genome. The transgene array in line OVE459 is situated between exons 85 (proximal) and 46 (distal), indicating a genomic rearrangement at the transgene insertion site.

 
While the Hydin transcript is readily detected in neonatal brain by RT–PCR, Hydin messages are much more abundant in testis and a signal by northern blotting has only been achieved from testis RNA. Northern analysis was performed to address potential differences in abundance or size of Hydin transcripts between wild-type and homozygous mutant mice (Fig. 3A). A single putative full-length transcript running well above the 9 kb RNA marker is detected in wild-type RNA using a probe derived from exons 21–24. In contrast, this transcript is undetectable in both hy3 and OVE459 homozygotes. The close proximity of Vac14 to the transgene insertion site made it a reasonable possibility that Vac14 expression is altered in homozygous OVE459 mice. No differences in Vac14 transcript size or abundance were detected between wild-type or either homozygous mutant strain (Fig. 3B). Amplification of Hydin mRNA by RT–PCR from both hy3 and OVE459 homozygous mutants (unpublished data) indicated that Hydin mRNA is not completely absent in these animals, but the northern data suggests that these transcripts are either unstable or not full-length.



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  		Figure 3. Northern analysis of Hydin and Vac14 in hy3 and OVE459 homozygous mutant and wild-type littermates. (A) Total testis RNA was blotted and hybridized with a radiolabeled Hydin-specific probe spanning exons 21–24. A single transcript much larger than the 9 kb RNA marker (M) is detected in both hy3 age 21 days (P21) and OVE459 (P20) wild-type animals, but is undetectable in hy3 and OVE459 homozygous mutants. The ß-actin RNA loading control is shown below the Hydin blot. (B) PolyA+ brain RNA was blotted and hybridized to a Vac14-specific probe. No difference in Vac14 mRNA size or abundance was observed between FVB/N, and homozygous mutant OVE459 or hy3 animals.

 
The reduction of Hydin mRNA in OVE459 homozygotes probably resulted from transgene-induced structural changes at this locus, but it was not clear why the spontaneous hy3 mutants should also exhibit such a reduction. All 87 exons of Hydin were sequenced in homozygous hy3 mice. A deletion of a single CG base pair in exon 15 was detected, resulting in a premature termination signal two codons downstream from the mutation (Fig. 4A). The consequence of this deletion is a predicted loss of more than 89% of the full-length Hydin gene product in hy3 homozygotes. The presence of a termination codon at this location likely destabilizes the message via nonsense-mediated decay (26).



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  		Figure 4. The hy3 allele of Hydin contains a single base pair deletion in exon 15. (A) Sequence comparison of Hydin exon 15 between wild-type (+/+) and hy3 homozygous mutant (-/-) littermates. An RT–PCR product spanning exon 15 was generated from wild-type and hy3 homozygous mutant brain RNA and sequenced. The hy3 allele (bottom) is missing a cytosine between positions 2163 and 2166 of the Hydin transcript, resulting in a premature termination signal two codons downstream of the mutation. (B) The wild-type and hy3 alleles of Hydin can be distinguished by allele-specific PCR. Using a wild-type-specific sense primer, amplification products are generated from wild-type (+/+) and hy3 heterozygous (+/-) genomic DNA, but not from homozygous mutant hy3 (-/-) genomic DNA. Using an hy3-specific sense primer, amplification products are generated from hy3 +/- and -/- genomic DNA, but not hy3 +/+ genomic DNA.

 
The deletion made it possible to distinguish wild-type, heterozygous and homozygous hy3 animals by allele-specific PCR (Fig. 4B). Two sense primers specific to the wild-type or hy3 allele were independently used with a common antisense primer specific to intron 15. Amplification products were generated only from wild-type and heterozygous hy3 genomic DNA using the primer set containing the wild-type sense primer. Similarly, when using the primer pair containing the hy3-specific sense primer, amplification products were generated using hy3 heterozygous or homozygous genomic DNA, but not wild-type DNA. This established the first molecular test for the hy3 allele.

Hydin is expressed within the embryonic and neonatal brain in structures known to be important for CSF homeostasis. In situ hybridization in the embryonic day 15 (E15) brain revealed Hydin transcripts in the developing choroid plexus (unpublished data). Hydin expression in newborn brain is observed exclusively in the ependymal cells, a specialized ciliated epithelium lining the lateral, third and fourth ventricles (Fig. 5A, B and C, respectively). Interestingly, Hydin expression is not uniform throughout the entire ependymal surface. For example, in the neonate, Hydin expression in the lateral ventricles is restricted to the medial ventricular surface (Fig. 5A). This differential expression pattern may represent different developmental stages of ependymal cell maturation. Hydin mRNA is also detected in several other tissues including the bronchi of the lung, spermatocytes within the semeniferous tubules of the testis and the lining of the oviduct, extending out to the fimbriae (Fig. 6A, B and C, respectively). Like the ependymal cells in the brain, these other sites of Hydin expression are either ciliated epithelia (bronchi and oviduct) or have components common to cilia (spermatocyte flagella).



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  		Figure 5. Analysis of Hydin expression pattern in the newborn FVB/N brain by in situ hybridization. Hemotoxylin-stained sections (left) are shown with the corresponding dark-field images (right). In the newborn brain, Hydin is specifically expressed in the ependymal cells lining the lateral (A), third (B) and fourth (C) ventricles. Asymmetric distribution of Hydin mRNA is demonstrated in the lateral ventricles (LV), where the transcript is only present in the ependymal cells of the medial ventricular surface. The scale bar in (A) and (B) are 500 and 50  µm, respectively. The scale bar in (B) also applies to (C).

 


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  		Figure 6. Analysis of Hydin expression in adult FVB/N mice. Hydin is expressed in the bronchial (Br) lining of the lung (A), spermatocytes within the semeniferous tubules of the testis (B) and the lining of the oviduct (C). In (C), oviduct (Ov), fimbriae (F) and uterus (U) are indicated. The scale bar in (A) equals 50 µm, and also applies to (B) and (C).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Using a combination of methods, we have identified the location and genomic structure of two novel genes, Hydin and Vac14, on mouse chromosome 8. We have also determined that the transgene insertion event in line OVE459 induced a genomic rearrangement, including a breakpoint within Hydin. Additionally, a frameshift mutation was discovered in exon 15 of the spontaneous hy3 mutant allele of Hydin. While Vac14 transcripts appeared unaltered in both OVE459 and hy3 homozygotes, the full-length Hydin transcript was undetectable by northern analysis in these mutant mice. Finally, within the brain, Hydin is expressed specifically in the cells lining the ventricles. These observations provide strong evidence that Hydin is the relevant gene for the development of hydrocephalus in both hy3 and OVE459 mice.

The order and orientation of genes surrounding Hydin on mouse chromosome 8 is conserved on human chromosome 16q22. Interestingly, a human fibroblast cell line isolated from a hydrocephalic patient was shown to carry a (4;16)(q35;q22.1) translocation (22). In an attempt to define the translocation breakpoint, Sakuragawa and Yokoyama (27) detected a rearranged 1.2 Mb NotI fragment using a calretinin (CALB2)-specific probe. Based on this information, these authors suggested that the human homologue of the gene disrupted by the hy3 mutation would lie within 1.2 Mb of calretinin. In agreement with this prediction, the current public mouse and human genome assemblies place Hydin 99 and 160 kb from calretinin in the mouse and human genomes, respectively (Fig. 1). While a relationship between HYDIN and human hydrocephalus has not been established, it will be interesting to determine if the hydrocephalus-associated (4;16)(q35;q22.1) translocation breakpoint is within the HYDIN gene.

Hydin is likely to be an ancient gene as there is evidence that Hydin expression is conserved in both vertebrate and invertebrate animals. While we have yet to experimentally confirm all human HYDIN exons, our predicted HYDIN protein exhibits an overall 76% identity and 86% similarity to the predicted 5099 amino acid mouse Hydin protein. We have also identified ESTs corresponding to portions of mouse Hydin from Rattus norvegicus, Sus scrofa, Bos taurus, Silurana tropicalis and Ciona intestinalis that, when translated, exhibit 95, 85, 77, 58 and 47% amino acid identity to mouse Hydin, respectively.

The function of the Hydin gene product is unknown, but the expression pattern of the Hydin transcript is suggestive of a role in the formation, function or maintenance of cilia, cilia-like structures or ciliated epithelium. Despite its large size, few conserved domains have been identified in the putative Hydin protein. A predicted transmembrane domain resides between amino acids 675–697. Amino acids 2258–2572 share significant similarity with caldesmon [e value of 4e-12 (28) compared with human caldesmon (GenBank accession no. Q05682) amino acids 258–566], a widely expressed actin-binding protein thought to be important in cytoskeletal assembly and stabilization as well as regulation of smooth muscle contraction (29). Taken together, the pattern of Hydin expression coupled with the caldesmon homology domain support a possible role for Hydin in cellular movement.

In mice, cilia dysfunction and hydrocephalus are features of null mutations in Spag6 (11), Hfh4 (12), Polaris (30), Mdnah5 (13) and Pol {lambda} (31). Additionally, ciliary dyskinesia is associated with hydrocephalus in rats (32), dogs (33) and humans (34–39). Hydrocephalus is thought to result from the overproduction of CSF, blockage of CSF flow, or defects in the reabsorption of CSF in the subarachnoid space surrounding the brain. Ependymal cells clearly play a role in CSF production, and may aid in CSF circulation, but the precise role of ependymal cilia in CSF homeostasis is unclear. A phenotypic examination of mice harbouring the hyh mutation describes a denudation of the ependymal layer that precedes the onset of hydrocephalus, suggesting that ependymal defects can serve as a primary cause of hydrocephalus (40).

Several groups have investigated the cause of hydrocephalus in hy3 homozygous mice. Previous investigations suggest that the hy3 mutation leads to communicating hydrocephalus as a result of a physiological defect involving CSF absorption in the subarachnoid space (19,20). Other studies reported a significant increase in the extracellular space (presumably the result of oedema) of the white matter in the cytoplasmic sheath surrounding many axons, and severe oedema in the subependymal layer (20,41–43). Investigations of the ependymal surface of hydrocephalic hy3 homozygotes reported a progressive loss of ependymal cilia and eventually the ependymal cells as examination moved from the basal portions, to the lateral walls, to the roof of the lateral ventricles (44,45). These authors concluded that cilia and ependymal loss were the result, not the cause, of hydrocephalus (45).

A limitation of these reports was that examinations were only conducted on hy3 mice well after hydrocephalus had begun. Hydrocephalus is known to develop in hy3 homozygotes around the time of birth, but none of these studies examined hy3 mice prior to 10 days of age. This makes it difficult to determine primary versus secondary aspects of hydrocephalus. Reabsorption of CSF in the subarachnoid space is likely compromised due to increases in intracranial pressure resulting from hydrocephalus. Our ability to distinguish wild-type from hy3/hy3 littermates will facilitate the phenotypic analysis of hy3 animals prior to the onset of hydrocephalus. The ependymal cell-specific expression pattern of Hydin suggests a need to revise the current thinking on the pathogenic mechanism of hydrocephalus in these mice.

Clinically, most human hydrocephalus is thought to result from a blockage of CSF flow and/or insufficient CSF absorption in the subarachnoid space (46). CSF overproduction, as in the case of choroid plexus papillomas, is thought to be a rare cause of human hydrocephalus (46,47). Overproduction of CSF has, however, been suggested as the primary cause of hydrocephalus in mice homozygous for a targeted mutation in the transcription factor E2F5 (48). Interestingly, the expression pattern of E2F5 in the choroid plexus and ependymal cell layer is similar to that of Hydin (48). Even if the origin of hydrocephalus lies in the ependyma, secondary CSF blockage or insufficient subarachnoid reabsorption may occur. Homozygous hyh mice experience ependymal cell loss prior to obstruction of the cerebral aqueduct (40). Homozygous hy3 mice develop a defect in CSF reabsorption early (19) and a blockage in the cerebral aqueduct late (43) in hydrocephalus pathogenesis. Considering the accumulating evidence from mouse mutations, including hy3, where ependymal cells are the likely targets, it is reasonable to expect that ependymal cell dysfunction may be a frequent initiating event in human autosomal-recessive hydrocephalus.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Genomic DNA clones
The mouse BAC CITB-CJ7-218P4, from strain 129SV, was obtained from Research Genetics (Huntsville, AL, USA) and BAC RPCI23-21B7, from strain C57BL/6, was obtained from Pieter de Jong (Children's Hospital Oakland Research Institute).

Mice
OVE459 mice were originally produced in the laboratory of Paul A. Overbeek (Baylor College of Medicine) and have been maintained on an FVB/N inbred background. B6CBACa-Aw-J/A-hy3/+ mice were originally purchased from the Jackson Laboratory and have been back-crossed to FVB/N mice to N12 and are maintained as a FVB/N congenic hy3/+ stock. All animal procedures were performed in accordance with the National Research Council Guide for the Care and Use of Laboratory Animals under the IACUC approved protocol 0997AR.

IMAGE Consortium cDNA array screening
Thirty nanograms of agarose-purified CITB-CJ7-218P4 genomic insert were radiolabelled and hybridized to High Density IMAGE Consortium mouse cDNA filters (Genome Systems, FCDNA-1611) as described (49). The hybridizing clone (IMAGE ID 312752) was purchased from Genome Systems.

Direct cDNA selection
cDNA selection was carried out as described (50). A cDNA population derived from pooled E17 and P0 head polyA+ RNA was hybridized to biotinylated BAC 218P4 restriction fragments. Following a second round of enrichment, selected cDNA fragments were cloned into pSTBlue-1 (Novagen) and sequenced.

Northern blot analysis
To detect the Hydin transcript, total testis RNA was isolated from P21 wild-type and homozygous mutant hy3 littermates or P20 wild-type and homozygous mutant OVE459 littermates using Trizol reagent (Gibco-BRL). Fifteen micrograms of total RNA per lane were run under standard formamide denaturing conditions. The Hydin-specific probe consisted of a 444 bp RT–PCR product amplified from newborn FVB/N total brain RNA using the primer pair C3-2a (5'-ACCCCGTCAGGATGGAGTTGTA-3') and C3-2b (5'-CCCAGTTGTTCAGGTCGTTTCT-3'). To detect the Vac14 transcript, 5 µg of polyA+ newborn brain RNA per lane was run under standard formamide denaturing conditions. The Vac14-specific probe consisted of a 426 bp RT–PCR product amplified from newborn FVB/N total brain RNA using the primer pair C1-2S (5'-GGCCTATGATGACCGCAAGAAAAG-3') and C1-2A (5'-TGAAGTAAGATGGGGAAGAGGCT-3'). Probes were labelled with 32P-dCTP using a random prime labelling system (Gibco/BRL).

Hydin sequencing
Eighty six of the predicted 87 Hydin exons were confirmed by generating overlapping RT–PCR products of the predicted size from FVB/N P0 brain RNA (primer sequences available upon request). Each of the 87 exons, including flanking intronic sequences, were then PCR-amplified from hy3 homozygous genomic DNA using Pfu Turbo polymerase (Stratagene) and gel-purified using Ultrafree-DA columns (Millipore). Purified PCR products were sequenced using Big Dye Sequencing chemistry (Applied Biosystems) with the same primers used for the initial amplification (primer sequences available upon request). The two sequences for each exon were assembled using the Seqman program (DNA Star) and compared to ENSEMBL's mouse chromosome eight genomic contig, 8.110000001–8.111000000 version 8.3c.1 using the pairwise BLAST program (28). The frameshift in exon 15 was detected by amplification of genomic DNA using the primer set 243454-F (5'-TGTTGATCAAGGGAGGCTACTGG-3') and 243454-R (5'-ACGGCATTCCCTATCACTGTCCTT-3'). For further verification, RT–PCR products spanning exon 15 were generated using the primer pair 5 prime-F6 (5'-CTTGGGGGCCTGCTTTGTCTTC-3') and 5 prime-R6: (5'-TTTGGGTTTTGTGGTAGGCATTTC-3') from FVB/N and homozygous hy3 P0 brain RNA and sequenced using Big Dye Sequencing chemistry (Applied Biosystems). The frameshift mutation in exon 15 was also detected in archival hy3 DNA obtained from the Jackson Laboratory DNA Resource.

Hydin allele-specific PCR
Tail DNA from wildtype, heterozygous and homozygous hy3 mice was prepared as described (51). Two µL of each sample was used as PCR template using the primer pairs Cand2 wt sense (5'-CTTCCAGGATTTCCCCAT-3') and Cand2 genotype anti (5'-TGTGATCTCAGAGGCTTAGT-3') and independently using primers Cand2 hy3 sense (5'-CTTCCAGGATTTCCC-ATA-3') and Cand2 genotype anti. The final concentration of each primer was 0.8 µM. To increase the specificity of the reaction, an oligonucleotide antisense to the specific sense primer was added to each reaction at a final concentration of 0.8 µM (52). PCR conditions were 94°C for 40 s, 55.5°C for 40 s, 72° for 40 s for 35 cycles. The amplification product was 410 and 409 bp in length for the wild-type and hy3 alleles, respectively.

In situ hybridization
In situ hybridization was carried out as described (53). Five-micrometer sections of 4% paraformaldehyde-fixed, paraffin-embedded tissue were subjected to hybridization using a cocktail of two 35S-labelled antisense or sense RNA probes derived from RT–PCR products using primer pair C2-12a (5'-GAGAACAAGGTCCTATTTTG-3') and C2-12b (5'-AGTCCAGAACTCTTCCGTG-3'), and primer pair 3 prime-1a (5'-TCTACGAGGTGGAGTTGAAT-3') and 3 prime-1b (5'-GTGTGGGGTGTTGGTGTA-3').


   ACKNOWLEDGEMENTS
 
The authors would like to acknowledge Christian M. Rizo and Shrinivas Hebsur for technical assistance. The authors wish to thank Dr Thomas Sferra, Dr Carlton Bates, Dr David Cunningham and Dr Michael Weinstein for critical review of the manuscript and Dr Lois Maltais of the Jackson Laboratory for assistance with officially naming Hydin. DNA sequencing was accomplished with the assistance of Huachun Zhong with the advice and assistance of Dr Robert Munson. The DNA sequencing Core Facility at Columbus Children's Research Institute is supported in part by the NIH grant HD34615. Genomic sequence databases were provided by both Celera Genomics and the Mouse Genome Sequencing Consortium. This work was supported by a grant (6-FY00-280) from the March of Dimes Birth Defects Foundation.


   FOOTNOTES
 
* To whom correspondence should be addressed at: Division of Molecular and Human Genetics, Columbus Children's Research Institute, 700 Children's Drive, Columbus, OH 43205, USA. Tel: +1 6147222764; Fax: +1 6147222716; Email: robinsom{at}pediatrics.ohio-state.edu Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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