INTRODUCTION

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Neural cell adhesion molecules (NCAMs) play roles in both the developing and the adult central nervous system (CNS) NCAMs have a single-pass transmembrane domain or are anchored to the cell surface via a glycosylphosphatidyl inositol linkage. Their extracellular domains contain variable numbers of immunoglobulin (Ig) and fibronectin type III (FNIII) repeats, and their intracellular domains are noncatalytic, distinguishing them from the receptor protein tyrosine kinase and phosphatase subfamilies. NCAMs participate in homophilic and/or heterophilic binding interactions and, in some cases, soluble or matrix-bound ligands have been identified. Despite the fact that NCAMs do not have catalytic activity, they are thought to participate in signaling either by extracellular interactions with catalytic subfamily members or by intracellular interactions with signal adaptors (39, 41).

NCAMs have complex, dynamic, and overlapping expression patterns during nervous system development. Many family members are also expressed widely in the adult. Thus, the expression and adhesive interaction data for a given family member can only provide clues to its function. Characterization of the phenotypes of mutants has been invaluable in determining the functions of these genes. To date, the phenotypes of individuals bearing mutations in the genes encoding four different NCAMs (NCAM, L1, DCC, and contactin) have been reported in vertebrates (1, 5, 6, 9, 10, 15, 17, 27, 37, 38, 45). Targeted ablation of these molecules substantiated other evidence that they function during development in axon guidance, fasciculation, and cell migration and linked specific developmental events to particular NCAMs. In contrast to the L1, DCC, and contactin mutants and despite widespread NCAM expression, NCAM-deficient mice have relatively mild abnormalities. This outcome allowed studies of its roles in nervous system function in the adult, which suggested that NCAM is required for one form of long-term potentiation in the hippocampus (4).

The punc gene, which encodes a new NCAM, was identified in a differential display screen of mRNA isolated from transgenic mice that overexpress the Islet-1 transcription factor. punc transcripts were found predominantly in the developing limbs and spinal cord, where its expression level was inversely correlated with that of islet-1. With four extracellular Ig domains and two FNIII repeats, Punc defined a new class within the neural Ig superfamily (31).

Here we describe studies aimed at determining the function of punc. We identified an embryonic stem (ES) cell line with a gene-trap insertion in punc. Expression of punc in the developing inner ear and localization of the mouse and human genes to syntenic regions of chromosomes 9(40) and 15q22, respectively prompted evaluation of PUNC as a candidate for the recessive deafness locus, DFNB16. Radiation hybrid mapping of PUNC relative to DFNB16 flanking markers showed that PUNC lies outside of the DFNB16 critical region and is unlikely to be responsible for this disorder. To facilitate studies of punc expression and determine its function, we generated a mouse strain with a lacZ insertion in punc. X-Gal (5-bromo-4-chloro-3-indolyl--D-galactopyranoside) staining revealed that punc expression is more complex than previously appreciated during mid-gestation. punc expression becomes progressively restricted to the brain and inner ear during late gestation and is maintained in these tissues in the adult. Comparisons of -Galactosidase (-Gal) activity and punc transcript levels in heterozygous and homozygous mutant individuals showed that Punc has a tissue-specific role in negatively regulating the level of its own transcript. punc-deficient mice were viable and fertile but had subtly impaired motor coordination that could be correlated with expression of punc in the Bergmann glia of the adult cerebellum.

	MATERIALS AND METHODS

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Gene trapping and 5'-RACE (rapid amplification of cDNA ends). The gene trap vector, pGTV1 (44), was modified by inserting an additional 51 bp of the adenovirus 2 splice acceptor upstream of the minimal acceptor sequence present in pGTV1 and by deleting pBluescript KS(+) sequences between the NotI and the proximal SspI sites to create pGTV2. The gene trap cell line, 24-B9, was isolated following electroporation of 10⁶ R1 ES cells (26) with 25 µg of pGTV2 and selection in medium containing 350 µg of G-418 (Life Technologies) per ml. Aliquots of 24-B9 cells were tested for -Gal activity before and after the application of thyroid hormone, nerve growth factor, and retinoic acid as described previously (44).

Mapping of punc. (i) Mouse. A 460-bp NcoI-to-SmaI DNA fragment (Identical to nucleotides 748 to 1203 of GenBank accession no. AF026465) isolated from the punc open reading frame was hybridized with nylon filters carrying TaqI-digested DNA samples from the Jackson Laboratory BSS Backcross panel (29). This probe detected a DNA fragment of 7.5 kbp in C57BL/6J DNA and one of 7.8 kbp in M. spretus DNA. The resulting strain distribution data were analyzed using MapMaker software (http://www.jax.org/resources/documents/cmdata).

(ii) Human. A human expressed sequence tag (EST) with 77% sequence identity to the 3' untranslated region (UTR) of mouse punc was identified (GenBank accession no. AA860555). To confirm that this EST was derived from human PUNC, 5'-RACE was performed on human fetal brain cDNA (kindly provided by Mark Keating, Howard Hughes Medical Institute) using a primer designed according to the EST sequence (SLM218, 5'-GAAGAAGATAGAATTTGTGGTG-3'). Sequence analysis of the largest 5'-RACE clone showed that the human 3' UTR was preceded by 81 bp encoding 27 amino acids. This coding region had 89% nucleotide sequence identity with mouse punc. Two primers were designed according to the human PUNC sequence (forward, 5'-GAAGAAGATAGAATTTGTGGTG-3'; reverse, 5'-AGTCAAAATGAGCAGAGTGTG-3'). A 260-bp fragment was produced by PCR amplification of human DNA as the template but was not detected with hamster DNA as the template. Chromosomal assignment was determined using the NIGMS human-rodent somatic cell hybrid mapping panel 1 (kindly provided by Mark Keating). Concordance-discordance analysis of the PCR results placed PUNC on chromosome 15. Finer mapping of PUNC was obtained by screening the Stanford G3 Radiation Hybrid Mapping Panel (Research Genetics). DNAs from 83 hybrid clones were analyzed by PCR to detect those that contained the human PUNC sequence. Two DFNB16-flanking markers, D15S1039 and D15S155 (2), were also mapped to the same panel using PCR conditions suggested by Research Genetics. The markers were localized by two-point maximum likelihood analysis (http://wwwshgc.stanford.edu/Mapping/rh/search.html).

Gene targeting. A 19-kbp DNA fragment containing exon 2 of the punc gene was isolated from a FixII library prepared from mouse strain 129/Sv genomic DNA (Stratagene). The coding sequence of punc was disrupted by insertion into the MscI site of exon 2 of a lacZ gene that carried a consensus Kozak translation initiation codon and nuclear localization signal and was followed by a PGKneobpA cassette (35). LoxP sites flanked the PGKneobpA cassette. A short stretch of synthetic oligonucleotides (5'-GGCCGCTAAGTGAGTAAGCCGCCCGCC-3') was placed in front of the lacZ sequence to ensure the closure of all three possible reading frames and to prevent production of a signal sequence--Gal fusion protein that might not have enzymatic activity (34). The final targeting vector contained 5.5 kbp of punc DNA upstream of the disruption in exon 2 and 3 kbp of punc DNA downstream of the disruption and was flanked by two thymidine kinase (TK) expression cassettes (kindly provided by Kirk Thomas, University of Utah). A total of 25 µg of the linearized vector was introduced into 10⁶ R1-45 mouse ES cells, which were grown in medium containing 380 µg of G418 per ml and 2 µM ganciclovir (23). Correctly targeted cell lines were identified using Southern blot hybridization analysis of DNA. The 5'-flanking probe was a 450-bp DNA fragment that was PCR amplified from genomic DNA using the primers SLM185 (5'-CTAGGAAACCTCTCCCTATG-3') and SLM207 (5'-GATAATCGAGCAAGATGACATG-3'). The 3'-flanking probe was a 500-bp DNA fragment that was amplified from genomic DNA using primers SLM186 (5'-GCAATGTAAGGAATTGAGCTG-3') and SLM208 (5'-TCAACCCTCACACTATGAGC-3'). The neo probe was a 630-bp PstI-to-XbaI fragment of pPGKneobpA. Thirty-three percent of the drug-resistant clones carried the targeted allele. Cells from three correctly targeted lines were diluted to 0.5 × 10⁵/ml and used to generate germ line chimeras by the one-step coculture method (19). The PGKneobpA cassette was removed from the targeted allele by mating heterozygous punc^LN mice with a strain that expresses CRE in the germ line (33). The absence of PGKneobpA sequences in the resulting offspring was confirmed by PCR analysis using internal primers that amplify a 295-bp fragment in heterozygous DNA samples (SLM10, 5'-GCCTGCTTGCCGAATATCATGG-3'; SLM74, 5'-AAACAACAGATGGCTGGCAAC-3'). In addition, carriers of the CRE transgene were identified by PCR screening with CRE-specific primers (tCreF1, 5'-GGATTTCCGTCTCTGGTGTAGC-3'; tCreR1, 5'-ACCATTGCCCCTGTTTCACTATC-3') and were not used in propagating punc^L mice.

Genotyping of mice. A PCR assay was used to genotype offspring of germ line chimeras and of intercrosses between heterozygotes. The primers (SLM24 and SLM25) that amplified a 333-bp fragment of the lacZ allele have been described previously (43). The forward primer for the wild-type punc allele was SLM136 (5'-AAATGATGATATTGCCAACCC-3'). The reverse primer for the wild-type allele was SLM137 (5'-CTACCTCCTTGCTCCGCC-3'). These punc primers amplified a fragment of 180 bp.

Northern blotting. Total RNA was isolated from tissues or staged embryos as described above. mRNA was purified using an Oligotex mRNA isolation kit (Qiagen). Five micrograms of each mRNA sample was separated in a formaldehyde-agarose gel, transferred to nylon membranes, and hybridized with ³²P-labeled probes as described earlier (43). The 3' punc probe was the 1.2-kb cDNA fragment described above, and the 5' punc probe was a 164-bp cDNA fragment obtained by PCR using forward primer SLM179 (5'-GGTCTGGGCCATTCTGCTG-3') and reverse primer SLM180 (5'-GTGGTGTGGGTGCCCTCTG-3'). The simian virus 40 (SV40) probe was prepared from viral DNA. Hybridization with a chicken -actin cDNA fragment served as a loading control. punc transcript levels were quantified by densitometry (Personal Densitometer SI; Amersham Pharmacia Biotech) of the hybridization signals on the X-ray films and normalized to -actin hybridization signals.

In situ hybridization. A 540-bp fragment of punc cDNA (nucleotides 603 to 1203 of GenBank accession no. AF026465) was cloned into pBluescript vectors KS() and SK(). Sense and antisense digoxigenin-labeled riboprobes were synthesized using T7 RNA polymerase from the respective vectors. Whole E9.5 and E10.5 embryos were processed for in situ hybridization as described elsewhere (14) and photographed. Stained embryos were then embedded in gelatin and cryostat sectioned at 10 µm as described earlier (36).

X-Gal staining and immunohistochemistry. To localize -Gal activity in embryos, heterozygotes were intercrossed, and all of the littermates from each pregnant female were fixed and stained with X-Gal under identical conditions and photographed as described previously (43). All embryos at E14.5 and older were manually hemisected halfway through the fixation step, and all staining was terminated before any background signal was detected in wild-type embryos. For the punc^LN strain, we examined two to four homozygous mutants and four to eight heterozygotes at each day of development between E8.5 and P0, except E16.5, which was omitted from the study. For the punc^L heterozygous intercrosses, we examined two to four homozygotes and four to eight heterozygous embryos at E11.5, E13.3, and E15.5 only.

Behavioral phenotyping. (i) General. Wild-type, heterozygous, and homozygous mutant mice used for behavioral tests were obtained by intercrossing heterozygotes. The parental punc^LN heterozygotes were F₁ offspring of the original germ line chimeric mice. The parental punc^L heterozygotes were offspring of the F₁ animals crossed to the CRE-deleter strain (33). Animals were tested at approximately 6 to 8 weeks of age in a procedure room separate from their home cage room.

(ii) Motor coordination. Mice were placed on a Rotarod (diameter, 3.2 cm; Accelerating Rota-Rod for Mice 7650; Biological Research Apparatus), while it was turning at a fixed speed. Female mice were tested at 27 rpm, while males were tested at 31 rpm (maximum speed). These speeds were chosen based on preliminary testing of wild-type mice. Use of the accelerating mode (0 to 31 rpm, maximum acceleration setting) for the Rotarod was unsatisfactory because wild-type mice could stay on the rod for the full test period at the first trial. Thus, we sought to determine a fixed speed at which a few mice could remain on the rod for only a few seconds at the first trial. This condition allowed discrimination of differing abilities over time. Retention time on the rod was recorded in seconds, up to a maximum of 300 during two trials per day for five consecutive days (20). The test population was composed of 15 female and 19 male wild types, 17 female and 19 male heterozygotes, and 17 female and 19 male homozygous mutants.

Other tests. Auditory brainstem response thresholds for click and 8-, 16-, and 32-kHz tone pip stimuli were measured by using hardware and software from Intelligent Hearing Systems according to the methods described by Zheng et al. (46). Swimming behavior was tested according to the method of Marshall and Berrios (24). Sensitivity to temperature was tested according to the method of Dahme et al. (7). Olfaction was tested according to the method of Cremer et al. (6).

	RESULTS

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Isolation of a gene trap cell line with a lacZ insertion in punc. A mouse ES cell line, designated 24-B9, was isolated from an ongoing gene trap screen that has been described previously (44). -Gal activity was detected in undifferentiated 24-B9 cells and was insensitive to the application of a variety of different growth factors and hormones. Chimeric embryos prepared using 24-B9 cells and analyzed at E11.5 and E13.5 exhibited -Gal activity in a variety of locations, including the otic epithelium, facial mesenchyme, ventral neural tube, and limb mesenchyme (data not shown). Southern blot hybridization analysis showed that a single copy of the gene trap vector was integrated into the genome of 24-B9 cells (data not shown), demonstrating that the pattern of -Gal activity observed in chimeric embryos reflected the activity of a single gene. 5'-RACE was used to isolate 68 bp of endogenous trapped gene sequences located at the 5' end of lacZ mRNA isolated from 24-B9 cells (Fig. 1A). The trapped sequences were not in frame with respect to the lacZ gene, suggesting that translation of the hybrid lacZ mRNA was initiated from the Kozak consensus ATG that is present on the gene trap vector (44).

View larger version (31K): [in this window] [in a new window]   FIG. 1.   Characterization and mapping of punc gene, which encodes a member of the neural cell adhesion molecule family. (A) Diagram of the gene trap vector insertion in 24-B9 ES cells. The gene trap vector, composed of a splice acceptor (sa), lacZ gene (lacZpA), and PGKneobpA cassette (neo) inserted between exons 2 and 3 (hatched boxes) of punc. The nucleotide (upper line) and the corresponding protein sequence (lower line) near the splice junction joining punc exon 2 to the vector sequences is indicated below the diagram. Capitalized letters in the nucleotide sequence indicate punc sequence, whereas lowercase letters indicate the vector sequence. The arrow indicates the position of the splice junction. Only 43 of the 68 bp of punc sequence identified by 5'-RACE are shown. (B) Predicted domain structure of Punc protein. Ig and FNIII domains are indicated by loops and boxes, respectively. The sites at which Punc was disrupted by the gene trap vector (GT) or the targeting vector (TV) are marked by arrows. SP, signal peptide; TM, transmembrane domain. (C) Nucleotide sequence (upper line) and corresponding amino acid sequence (lower line) of the alternative 5' punc exon (GenBank accession no. AF236125). An in-frame stop codon (star) preceding the open reading frame is underlined. An arrow indicates the splice junction between the alternative exon and exon 2. (D) At the top of the panel is a map figure from the Jackson BSS backcross showing part of mouse chromosome 9. The map is depicted with the centromere toward the top. A 3-cM scale bar is shown to the right of the figure. Loci mapping to the same position are listed in alphabetical order. Missing typings were inferred from surrounding data where the assignment was unambiguous. Raw data were from the Jackson Laboratory (http://www.jax.org/resources/documents/cmdata). At the bottom of the panel is a haplotype figure from the Jackson Laboratory BSS backcross showing part of chromosome 9 with loci linked to punc. Loci are listed in order with the most proximal at the top. The black boxes represent the C57BL6/JEi allele, and the white boxes represent the SPRET/Ei allele. The number of animals with each haplotype is given at the bottom of each column of boxes. The percent recombination (R) between adjacent loci is given to the right of the figure, along with the standard error (SE) for each value. Missing typings were inferred from surrounding data where the assignment was unambiguous. (E) Map figure of human chromosome 15 showing the localization of PUNC. Vertical bars on the left and right indicate the regions where TPM1 and DFNB16, respectively, are located. Arrows indicate the location of loci linked to the two DFNB16 flanking markers. The horizontal bar indicates the region where PUNC and its linked loci were mapped.

Chromosomal localization of punc and elimination of PUNC as a candidate for DFNB16. Mouse punc was mapped by hybridizing a punc cDNA fragment to the TaqI-digested Jackson Laboratories BSS interspecific backcross mapping panel (29). Analysis of the strain distribution pattern of a TaqI polymorphism showed that punc is located in the middle of chromosome 9, approximately 40 centimorgans (cM) from the centromere, in the vicinity of Tpm1 (Fig 1D). This result is consistent with the mapping of punc to the D-E1 region of chromosome 9 by fluorescence in situ hybridization (30) but provides higher resolution.

Expression of punc mRNA. To determine the relative levels of punc expression during development, a stage-specific Northern blot was prepared from wild-type E9.5 to newborn embryo mRNA and hybridized with the 1.2-kbp punc cDNA fragment (Fig. 2). Strong expression of a 5-kb transcript was detected in E9.5 and E10.5 embryos. Levels of this transcript decreased gradually from E11.5 and were almost extinguished by E15.5, suggesting that punc could function during mid-gestation (Fig. 2A). The quantity of punc transcripts present in several adult mouse tissues was also determined. Northern blot hybridization analysis of mRNA derived from brain, heart, kidney, liver, muscle, spinal cord, spleen, and thymus showed similarly low levels of the 5-kb punc transcript (Fig. 2B).

View larger version (59K): [in this window] [in a new window]   FIG. 2.   Northern blot analysis of punc expression at embryonic stages and in adult tissues. (A) Stage-specific Northern blot of whole-embryo mRNA. (B) Northern blot of adult tissue mRNA. Each lane contains 5 µg of the indicated mRNA. A 1.2-kb punc cDNA was used as a probe. The lower panels show the results of subsequent hybridization with a -actin probe. The positions of size markers are indicated on the left.

Gene targeting of punc. To determine the role of punc, a mouse strain carrying a mutation in the gene was required. Since fluorescence in situ hybridization analysis revealed that a high proportion of 24-B9 ES cells had an abnormal chromosome number accompanied by loss of the Y chromosome, that cell line was not a suitable candidate for germ line transmission of the gene trap punc allele (data not shown). Instead, the punc gene was disrupted by homologous recombination (Fig. 3). A targeting vector was designed to disrupt punc expression by insertion of a reporter gene (nls-lacZ) and a loxP-flanked PGKneobpA expression cassette into the Msc1 site of exon 2 (Fig. 3A). This site is located within sequences that encode the first Ig domain (Fig. 1B). To ensure activity of the -Gal enzyme, production of a protein with both a signal sequence and a nuclear localization signal was prevented by placing a short stretch of synthetic oligonucleotides containing stop codons in all three reading frames immediately upstream of nls-lacZ. Thus, -Gal activity from this construct was only expected to reflect punc transcription and was expected to be similar to the gene trap insertion except that -Gal would be located in the nucleus rather than the cytoplasm. Two thymidine kinase expression cassettes were also included in the final construct for negative selection. The vector was introduced into R1-45 ES cells, which were cultured under standard selective conditions. Clones of targeted cells were identified by Southern blot hybridization analysis. As predicted, a 5'-flanking probe detected a 16-kb EcoRV fragment in DNA isolated from R1-45 cells, whereas DNA isolated from targeted clones contained both the 16-kb EcoRV fragment and an 11-kb EcoRV fragment (Fig. 3B). This result was confirmed by using a 3'-flanking probe (data not shown). In addition, hybridization with a neo probe confirmed that the new sequences were present at a single site in the genome (data not shown).

View larger version (42K): [in this window] [in a new window]   FIG. 3.   Gene targeting of punc. (A) The targeting vector is depicted on the first line. Exon 2 of punc was disrupted by insertion of a nuclear localized lacZ (nls-lacZpA), followed by a PGKneobpA expression cassette flanked by two loxP sites (lx). Thymidine kinase expression cassettes (TK1 and TK2) were placed at either end of the punc DNA (horizontal line). Hatched boxes indicate punc exons. The wild-type allele is shown on the second line. Shaded boxes indicate the probes used in Southern blot analysis. The locations of primers used to detect the wild-type allele (positions 136 and 137) are indicated with arrows. The third line shows the lacZ/neo targeted allele (LN). The locations of primers used to detect the mutant allele (24, 25) are indicated by arrows. The fourth line shows the lacZ targeted allele (L) after Cre-mediated removal of the PGKneobpA cassette. Key restriction enzyme recognition sites are indicated (RI and RV, EcoRI and EcoRV). (B) Southern blot hybridization analysis of DNA extracted from ES cell clones or from mouse tails. DNA was digested with EcoRV and probed with the 5' probe. Genotypes (+/+, wild type; +/, heterozygous; /, homozygous mutant) are indicated above each lane. The sizes of the bands are indicated. (C) PCR analysis of DNA extracted from yolk sacs. Each DNA sample was amplified with the wild-type and mutant primer pairs, which gave rise to bands of 180 and 330 bp, respectively. (D) Northern blot analysis of mRNA extracted from E11.5 mouse embryos of each punc genotype and hybridized sequentially with a 3' punc probe and a -actin probe.

Mice homozygous for punc^LN or punc^L are viable and fertile. Heterozygous punc^LN or punc^L animals did not exhibit any overt abnormalities when compared with their wild-type littermates. To assess whether punc has an essential embryonic function, heterozygous punc^LN mice were intercrossed, and the offspring were genotyped. Of 131 E8.5-to-E17.5 embryos, 32 (24%) were wild type, 71 (54%) were heterozygous, and 28 (21%) were homozygous mutants, consistent with a normal Mendelian distribution. Of 109 adult offspring of the intercross, 23 (21%) were wild type, 55 (50%) were heterozygous, and 31 (28%) were homozygous mutants, again suggesting a normal Mendelian distribution. The homozygous mutant offspring on a mixed 129/Sv and C57BL/6 genetic backgound were overtly normal; they gained weight normally, and both sexes were fertile. Similar results were obtained with the punc^L strain (data not shown). Therefore, punc does not have an essential embryonic or postnatal function.

Localization of punc in embryos and adults. To determine the tissues that express punc, which might provide clues to its function, we examined the patterns of -Gal activity in heterozygous and homozygous punc^LN embryos using X-Gal staining (Fig. 4). No -Gal activity was detected in embryos younger than E9.5. Since Salbaum reported punc expression in the neuroectoderm at E7.5 and E8.5 (31), and E8.5 punc^LN heterozygotes and homozygotes did not exhibit any -Gal activity, RT-PCR analysis of E8.5 heterozygous RNA was performed. Both punc and lacZ transcripts were readily detected and whole-mount in situ hybridization using a punc probe supported the RT-PCR results (data not shown). Thus, it is possible that the absence of X-Gal staining at E8.5 was caused by a delay in the translation of lacZ mRNA and/or in the assembly of active -Gal tetramers.

View larger version (58K): [in this window] [in a new window]   FIG. 4.   Sites of -Gal activity as revealed by X-Gal staining of embryos heterozygous or homozygous mutant for targeted punc alleles. (A, B, and D to O) X-Gal-stained whole embryos. Embryos in panels I to O were hemisected prior to staining. The age, genotype, and punc strain for each embryo is shown at the bottom of the panel. Heterozygotes are indicated by +/, and homozygous mutants are indicated by /. The lacZ/neo insertion is indicated by LN, and the lacZ insertion is indicated by L. (C) In situ hybridization of a punc probe with an E10.5 wild-type embryo. (B') Transverse section (10 µm) through the brain of the embryo shown in panel B. (C') Transverse section (10 µm) through the brain of the embryo shown in panel C. Structures mentioned in the text are indicated with arrows and are abbreviated as follows: forebrain (fb), midbrain (mb), hindbrain (hb), spinal cord (sc), forelimb (fl), hindlimb (hl), DRG (drg), branchial arch (ba), telencephalon (t), diencephalon (d), metenecphalon (m), trigeminal ganglion (Gv), cerebellum (cb) and cerebellar cortex (Cbc). Objective magnifications used for photography were as follows: A, ×4; B, ×2.5; C, ×2.5; and B' and C', ×6.6 (scale bar indicates 0.2 mm) and D to F, ×1.6; G and H, ×1.2; I to L, ×1; M, ×1.2; N, ×1; and O, ×3.2.

View larger version (65K): [in this window] [in a new window]   FIG. 5.   X-Gal staining of adult tissues showing that punc is expressed in the adult brain and inner ear. The genotype and punc strain for each sample is indicated at the bottom of the panels (heterozygotes, +/; homozygous mutants, /). The lacZ/neo insertion is indicated by LN, and the lacZ insertion is indicated by L. (A to D) X-Gal-stained sagittal brain slices (1 mm) showing punc expression in the cerebellar cortex (Cbc), the CA2 region of the hippocampus (CA2), and the olfactory bulb (OB). A, C, and D, objective magnification of ×2.0; B, objective magnification of ×0.8. (E) Section (10 µm) of slice in panel D showing small -Gal-expressing cells in the position of Bergmann glia (BG) between the Purkinje cell bodies (PC). The Purkinje cell layer (PCL) is indicated. Scale bar, 5 µm. (F) Cryosection (10 µm) of X-Gal-stained puncL/L cerebellum immunostained with antibody against GFAP (brown) showing colocalization of -Gal activity with this marker of Bergmann glia (BG). Scale bar, 5 µm. (G and H) Medial view of whole X-Gal-stained inner ears showing -Gal activity in the spiral ganglion (SG) and support cells of the Organ of Corti (OC). Objective magnification of ×3 .2. (I) Section (10 µm) of inner ear in panel H showing -Gal-expressing neurons in the spiral ganglion. Objective magnification of ×40. (J) Section (10 µm) of inner ear in panel H showing -Gal activity in the pillar cells, which are indicated with arrows. Objective magnification of ×40.

punc expression is negatively autoregulated. Visual comparisons of the quantity of X-Gal precipitate produced by punc^LN heterozygous and homozygous mutant littermate embryos processed and stained under identical conditions suggested that in some tissues of homozygotes, -Gal activity was more than twofold higher than the levels found in heterozygotes. For example, -Gal activity in the DRG anterior to the hindlimb was relatively strong in both E11.5 and E15.5 in punc^LN/LN embryos, yet its expression level in punc^LN/+ embryos varied from very weak to undetectable (compare Fig. 4E and K with Fig. 4D and J). This difference in DRG -Gal activity between genotypes was also seen in E12.5 to E14.5 embryos (data not shown). Moreover, striking differences in the quantity of X-Gal staining intensity were also observed between punc^LN/LN and punc^LN/+ brain and inner ear samples (compare Fig. 5A with C and Fig. 5G with H). Similar observations were made when the X-Gal precipitate produced by punc^L heterozygotes was compared with that of punc^L homozygous littermates (data not shown). In contrast, many of the X-Gal-stained regions, particularly those in young embryos, had the expected twofold difference in staining intensity between heterozygous and homozygous samples (compare the limbs in Figs. 4D and E). These observations suggested that punc transcription or message stability might normally be negatively autoregulated in some tissues.

View larger version (45K): [in this window] [in a new window]   FIG. 6.   punc transcription is negatively autoregulated. Northern blot analysis of 5 µg of mRNA isolated from E11.5 embryos of the indicated genotypes (+/+, wild type; +/, heterozygous; /, homozygous mutant). (A) Hybridization of the blot with the 5' punc probe. (B) Hybridization of the blot with the SV40 probe. The lower panels show the results of a subsequent hybridization with -actin. The positions and the relative sizes of the probes (thick lines) are indicated below the Northern blots in schematic diagrams of the wild-type and punc/lacZ transcripts. The hatched boxes represent punc sequences, and the open boxes represent lacZ sequences.

Mice homozygous for punc^LN performed poorly in a test of motor coordination. Further characterization of the punc mutant strains focused on adult behaviors potentially affected by punc/lacZ-expressing cells. To assess inner ear function, animals of all three genotypes were tested for threshold shifts in the auditory brainstem response, for the righting reflex, and for swimming posture. To assess olfaction, behavior in the presence of attractive versus repellant bedding was observed. No differences in performance between animals of different genotypes were found in the results of any of these tests (data not shown). Furthermore, comparisons of the gross morphology and innervation of the inner ear in heterozygous and homozygous mutants at P0 revealed no differences (data not shown).

View larger version (46K): [in this window] [in a new window]   FIG. 7.   punc mutant mice have impaired motor coordination. (A) The average retention times on the Rotarod for males and females are shown for each trial and genotype. Wild-type (female, n = 15; male, n = 19), heterozygous (female, n = 17; male, n = 19), and homozygous mutant (female, n = 17; male, n = 19) groups are indicated by open, gray, and black boxes, respectively. Bars above each box indicate one standard error of the mean. (B) Statistical analysis of the Rotarod data separated by sex. The graphs show the probability of retention on the rod with respect to time for animals of each genotype: wild type (+/+, dotted line), heterozygotes (+/, upper solid line), and homozygous mutants (/, lower solid line). P values shown are for the comparison of wild-type to homozygous mutant animals. The difference between the wild-type and heterozygous curves was not statistically significant for either sex.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Characterization of the gene disrupted by the 24-B9 gene trap insertion led to the identification of mouse punc, which encodes a member of the Ig superfamily. Punc protein, with an extracellular domain composed of four Ig repeats and two FNIII repeats and a single transmembrane domain is predicted to have structural properties similar to NCAMs. To investigate punc function, we generated mouse strains with either a lacZ/neo or a lacZ insertion exon 2 of the punc gene. Experimental evidence and theoretical considerations argue that the punc alleles are functionally null. First, the inserted DNA abolished expression of any normally sized punc mRNA in homozygous mutant embryos from both stains. The aberrantly short punc-containing mRNA found in mutant embryos was expressed at very low levels, was likely to have initiated from within the lacZ sequences, and did not contain a consensus in-frame translation initiation codon. Thus, even if this 5'-truncated punc transcript could be translated starting with a nonconsensus AUG, the resulting protein would lack a signal sequence and the entire first Ig domain, which are encoded upstream of the inserted DNA. Therefore, it is unlikely that the mutant punc alleles produce functional Punc protein.

Northern blot analysis showed that punc mRNA is expressed strongly between E9.5 and E11.5. After this time, the proportion of punc transcripts in whole-embryo mRNA decreases precipitously and punc transcripts are detected at very low levels in older embryos and in a variety of adult tissues. Construction of the transcriptional fusion between punc and lacZ permitted an extensive survey of the pattern of punc expression, as revealed by -Gal activity, during embryogenesis and in the adult. Between E9.5 and E11.5, -Gal activity in whole embryos coincided with the punc RNA in situ analysis of tissue sections published by Salbaum (31). We found very strong -Gal activity in the CNS, limbs, and branchial arches. Sections taken through the heads of X-Gal-stained embryos at E11.5 revealed the otic epithelium to be an additional site of -Gal activity (data not shown). Although punc mRNA was difficult to detect in samples isolated from whole embryos older than E11.5, X-Gal staining of punc^LN embryos revealed that punc continues to be expressed throughout development. Its expression domain is progressively restricted, becoming confined to the brain and inner ear by E16.5.

Despite the strong and dynamically changing expression of punc in early embryos, no morphologic abnormalities were found in homozygous mutant embryos. This suggests that its embryonic function is subtle and/or redundant. Analysis of appropriate double-mutant combinations could reveal an embryonic role for Punc. Other noncatalytic Ig superfamily members, such as DCC (18), are particularly good candidates. Since netrin-1, a ligand for DCC, is required for normal inner ear development (32), it is possible that DCC plays a role in ear development. Thus, the double mutant combination of dcc with punc might reveal a role for punc in inner ear development or in other tissues that express both genes.

Notably, we found that punc gene expression is negatively autoregulated. In some regions of homozygous mutant embryos, particularly the spinal cord and dorsal root ganglia of embryos at E11.5 and older, the -Gal activity increased more than twofold compared with the same region in heterozygous littermates stained under identical conditions. This increased -Gal activity was also noted in the spiral ganglion and the cerebellum of adult homozygous mutants. The results of Northern hybridization of E11.5 mRNA with a variety of probes support this observation. The 5' punc probe, which detects all transcripts initiated from the punc promoter, revealed that, relative to wild-type embryos, punc heterozygotes have fourfold-increased levels of the wild-type punc transcript and that punc homozygotes have eightfold-increased levels of the hybrid punc/lacZ transcript. It is also notable that the 3' punc probe, which can only detect wild-type transcripts, revealed that wild-type and heterozygous punc embryos have similar amounts of punc mRNA. In the absence of autoregulation, heterozygotes would be expected to have half as much punc as do wild-type animals. Taken together, these results suggest that in some tissues Punc may play a dose-dependent role in negatively regulating its own transcription or mRNA stability. The difference between the ratio of wild-type transcript detected in heterozygous versus wild-type mRNA using the 5' and 3' probes (fourfold versus even) may reflect differences in the stability of wild-type punc mRNA in tissues that are or are not subject to autoregulation.

Since the major form of Punc is predicted to be found at the cell surface, it is likely that the proposed autoregulation is indirect, as illustrated in the model (Fig. 8). Although the intracellular domain of Punc does not include any recognizable motifs that would suggest a link to a known signaling pathway, it is possible that Punc could interact directly with one of those pathways. Alternatively, Punc may interact with another transmembrane protein that has signaling activity as, for example, some Ig superfamily members are thought to initiate signaling to the nucleus by interacting with fibroblast growth factor receptors (25, 41). As the increased -Gal activity in homozygous mutants is restricted to certain locations, it is possible that this autoregulatory phenomenon is dependent on the reception of an as-yet-unidentified signal and/or ligand (Fig. 8B). In this scenario, the proposed Punc ligand is not expected to be expressed at all sites of Punc expression and, at those sites, punc expression is not autoregulated (Fig. 8A). The physiological significance of punc autoregulation is unclear. Mice with a lacZ insertion in ncam were not reported to have this property (15), but it will be interesting to learn whether any other members of neural Ig superfamily display similar regulation.

View larger version (42K): [in this window] [in a new window]   FIG. 8.   Model for negative autoregulation of punc gene expression. Punc protein is symbolized by a line with four Ig domains (half circles) and two FNIII domains (boxes) located outside the cell body (pink). For each cell, the intesity of color shading the nucleus (shades of blue or plain white) represents the relative level of -Gal activity. (A) In most tissues and in all sites of punc expression prior to E11.5, the punc RNA level, as detected by the nuclear -Gal activity, is proportional to the number of copies of the targeted punc/lacZ allele. (B) In Punc-expressing cells that are exposed to a Punc ligand (red X), a signal (red line with a bar at the end) is sent to the cell to reduce the level of punc RNA. In this diagram, the effect of the Punc-mediated signal is depicted as acting in the nucleus at the level of transcription initiation, but posttranscriptional mechanisms are not excluded.

punc expression was observed in only two adult tissues: the inner ear and the brain. Mice that were homozygous for either of the punc mutant alleles had normal inner ear structure as well as normal balance, posture, and auditory brainstem response thresholds, suggesting that if punc plays a role in the adult inner ear, its function has not been discovered or is redundant. Expression of punc in the cerebellum prompted testing of motor coordination in the homozygous mutants. We found that their performance on the Rotarod was significantly impaired relative to their wild-type or heterozygous littermates. Since the mutants increased their retention times on the Rotarod with experience, it seems that they were capable of learning the task, but that they did not do so as effectively as the controls. This suggests a role for Punc in the control of motor coordination, although a role in learning or memory cannot be rigorously excluded.

How might punc play a role in motor coordination? The punc mutant is quite different from the classic mouse mutants that have impaired motor coordination (12) in that the punc mutant cerebellum is grossly normal with respect to size, cytoarchitecture, and foliation. Double-labeling of X-Gal-stained nuclei in the Purkinje cell layer with antibodies directed against GFAP showed that punc is expressed by Bergmann glia. During cerebellar development, Bergmann glia play a role in neuronal migration. In the adult, Bergmann glia are intimately associated with Purkinje cells, which provide the major cerebellar output. The cell bodies of Bergmann glia are located among the Purkinje cell bodies and the Bergmann glia fibers extend between the Purkinje cell dendrites (8, 28). Therefore, Bergmann glia are in a position to interact directly with and potentially modulate the function of Purkinje cells in the adult. In addition, Bergmann glia fibers surround the excitatory synapses made by granule cell climbing and parallel fibers with Purkinje cell dendrites (28) and electrical stimulation of parallel fibers affects the intracellular calcium concentration in microdomains of Bergmann glia fibers (13). Thus, the loss of Bergmann glia-expressed Punc from the cell surface might disrupt interactions between Bergmann glia and Purkinje cell bodies and/or dendrites or granule cell axons and account for the observed defects in motor coordination in punc^LN homozygotes. The relevance of adhesive contacts between neurons and glia to neuronal function is underscored by the reduction in hippocampal long-term potentiation seen in transgenic mice that ectopically express L1 in astrocytes (22).

Two other genes expressed in Bergmann glia, namely, glast, which encodes a glutamate transporter, and vimentin, which encodes an intermediate filament protein, also cause mild motor discoordination when mutated. The motor discoordination of glast mutant homozygotes could not be correlated with any structural abnormalities of the cerebellum but was associated with persistent multiple climbing fiber innervation of Purkinje cells (42). Ultrastructural studies of the cerebellum of vimentin-negative mice revealed subtle abnormalities of the Bergmann glia and Purkinje cells (3, 11). Although we did not detect any morphologic abnormalities of the cerebellum in the punc mutants by light microscopy, it is possible that an ultrastructural study focused on the relationships between Purkinje and granule neurons and Bergmann glia would be revealing. Finally, the intriguing similarity between the punc and vimentin mutant phenotypes could be manifest simply because these genes act in the same cell type. However, given that the many cell adhesion molecules of the integrin and cadherin classes function through interactions with the cytoskeleton and that some CAMS may function similarly (16), it would be interesting to determine whether Punc provides a link to the Bergmann glia intermediate filament cytoskeleton via vimentin.