We have screened for retinoic acid–inducible gene trapping in mouse embryonic stem (ES) cells using the retroviral vector ROSAβgeo*, which transduces a promoterless βgeo*
(a fusion gene of β-galactosidase
and neomycin phosphotransferase II
) as a reporter (Komada et al., 2000
fhcrc.org/labs/soriano/trap.html). Northern blot analysis confirmed that one of the lines, ROSA62, exhibited a 17-fold induction of the reporter gene expression after 6-h treatment with 1 μM all-trans retinoic acid (unpublished data). This strong induction led us to characterize the gene trap mutation in detail. As the expression of the ROSA62
gene is mostly restricted to neurons in mice (see below), this induction might reflect the ability of retinoic acid to induce neuronal differentiation of ES cells.
Neuromuscular defects in ROSA62 mutant mice
Germline chimeric mice were generated by injecting ROSA62 mutant ES cells into blastocysts, and were crossed to 129/S4 and C57BL/6J mice to derive mutants. Heterozygotes exhibited no overt phenotype. Viable homozygous mutant mice were recovered from intercrossing of heterozygotes according to Mendelian expectations and were mostly of normal size. However, by 3 wk of age, homozygous mutants were distinguishable from wild-type and heterozygous mutant littermates by fine tremors. Until 2–3 mo of age, the homozygotes were grossly normal except for the tremor. They also exhibited clasping of the hindlimbs when held by the tail, a hallmark of ataxia. As they grew older, the phenotype became more severe. By 6–10 mo of age, they exhibited continuous contraction of hindlimb skeletal muscle and were not able to walk. The same phenotype was observed on congenic 129/S4, mixed 129/S4 x C57BL/6J, and congenic C57BL/6J (>10-generation backcross) genetic background. The following analyses were performed on mixed 129/S4x C57BL/6J genetic background.
Expression pattern of the ROSA62 gene in mice
Expression of β-galactosidase activity in gene trap mutant mice is driven by the promoter of the trapped gene (Friedrich and Soriano, 1991
). Therefore, ROSA62 heterozygous mutant embryos and adult tissues were isolated and stained with X-gal to determine the expression pattern of the trapped gene.
In embryonic day 10.5 embryos, expression was restricted to cranial and dorsal root ganglia ( A). In adult mice, expression was detected in many regions of the brain, with the highest level in the hippocampus and cerebellum ( B). Expression was also detected in the spinal cord ( C). In contrast, no β-galactosidase activity was detected in skeletal muscle (unpublished data), suggesting that the phenotype of ROSA62 mutant mice is primarily due to a defect in neurons but not skeletal muscle.
Figure 1. Expression pattern of the ROSA62 gene in mice. Expression pattern of the ROSA62 gene was assessed by X-gal staining of heterozygous mutant mice. (A) Embryonic day 10.5 embryo. Expression is detected in the dorsal root ganglia (DRG) and trigeminal ganglia (more ...)
Other than the neuronal tissues, ROSA62 expression was detected in the pancreas ( D) and testis ( E), but not in the heart, lung, liver, kidney, spleen, stomach, small intestine, colon, ovary, and uterus (unpublished data). In the pancreas, expression was restricted to the islet of Langerhans ( D), whereas in the testis, it was detected in round and elongated spermatids ( E). However, no histological abnormalities were observed in any of these tissues of the homozygotes (unpublished data). The homozygous mutant males reproduced very inefficiently when mated with wild-type females. However, this is likely to reflect a mating problem as litter sizes were normal.
Mutation in βIV-spectrin in ROSA62 mutant mice
To identify the gene trapped in this mutant, the ROSA62–
fusion cDNA was cloned from ROSA62 mutant ES cells by 5′-RACE, and full-length cDNAs were subsequently cloned by screening cDNA libraries. Sequencing of the cDNAs revealed that the trapped gene encodes βIV-spectrin, for which the human homologue has been recently identified (Berghs et al., 2000
; Tse et al., 2001
). Two βIV-spectrin splice isoforms, Σ1 and Σ6, were cloned ( A). βIVΣ1-spectrin was 2561 amino acids long, consisted of an NH2
-terminal actin-binding domain, 17 spectrin repeats, a variable region, and a COOH-terminal plecstrin homology (PH) domain, and had 95% overall sequence identity to its human homologue. The actin-binding domain, the entire spectrin repeats, and the PH domain of βIVΣ1-spectrin were ~70, 40, and 55% identical in amino acid sequences, respectively, to those of βI-, βII-, and βIII-spectrins, with the highest sequence identity to βII-spectrin.
Figure 2. Characterization of βIV-spectrin and its gene products. (A) Schematic diagram of the βIVΣ1- and Σ6-spectrin isoforms. The gene trap vector was inserted between exons encoding the spectrin repeat 12 of the βIV-spectrin (more ...)
The other isoform has not been identified in human and was termed βIVΣ6-spectrin in this study (Σ2–5 isoforms have been reported in humans) (Berghs et al., 2000
; Tse et al., 2001
). This novel isoform lacked the NH2
-terminal actin-binding domain, the spectrin repeats 1–9, and part of spectrin repeat 10 ( A). The βIV
cDNA has a distinct 81-bp sequence upstream of nucleotide 4079 of βIV
, which is derived from an alternative exon ( B). An in-frame translation stop codon is located 72 bp upstream of the ATG encoding methionine 1321 of the Σ1 isoform, suggesting that this ATG is used as an translation initiation codon for βIV
( B). Two more distinct βIV
cDNAs with a different 5′ noncoding region diverging upstream of nucleotide 4079 of βIV
were also cloned (unpublished data), indicating that the second exon for βIV
can be alternatively spliced from three distinct first exons. The original 5′-RACE product cloned from ROSA62 mutant ES cells encoded the Σ6 isoform, and its sequence indicated that the gene trap vector was inserted between exons that encode the spectrin repeat 12 of βIVΣ1-spectrin ( A). We did not isolate cDNAs encoding the Σ2, Σ3, Σ4, and Σ5 isoforms identified in human.
Expression of βIV-spectrin in mice and its absence in ROSA62 mutant
To determine if the gene trap mutation leads to a null allele, Northern and Western blot analyses were performed. By Northern blot analysis with a βIV-spectrin cDNA fragment encoding the 3′ region (3′-probe in A), four βIV-spectrin transcripts (~9, 7, 6.5, and 5 kb in size) were detected in the brain of wild-type and heterozygous mutant mice ( C). Using a 5′-probe specific to βIVΣ1-spectrin ( A), only the 9-kb transcript was detected ( C). As the Σ1 and Σ6 cDNAs we cloned are 8.7 and 4.7 kb long, respectively, mRNAs encoding these isoforms likely correspond to the 9- and 5-kb transcripts, respectively. In the homozygous mutant, all of the transcripts detected with the 3′-probe were absent ( C). Using the 5′-probe, a transcript which is ~9 kb in size was detected (unpublished data). As this transcript did not hybridize with the 3′ probe, it likely represents the fusion transcript between the 5′ end of βIV-spectrin and the reporter. Western blot analysis was performed using a polyclonal antibody raised against the variable region of βIV-spectrin which has no homology to other proteins. In the wild-type brain, the antibody detected a minor ~300-kD band and a major ~160-kD band ( D). The faint band of ~140 kD may be another splice isoform or a degradation product. From the calculated molecular mass (289 kD for Σ1 and 141 kD for Σ6), the ~300- and ~160-kD bands most likely correspond to βIVΣ1- and Σ6-spectrins, respectively. Interestingly, the truncated Σ6 isoform was much highly expressed than the full-length Σ1 isoform both at the level of mRNA as well as protein. The same bands were detected in the heterozygotes, but were less abundant, and they were absent from the homozygous mutant extract ( D). A truncated βIV-spectrin containing the NH2-terminal portion might conceivably be produced in the homozygotes. However, the absence of any mRNAs and proteins detected by the 3′ probe and the antibody, respectively, as well as the gene trap insertion site indicates that it lacks the spectrin repeats 12–17, the variable region, and the PH domain. Therefore, the gene trap insertion has most likely created a functionally null allele of the βIV-spectrin gene.
Subcellular localization of βIV-spectrin
Although spectrin was originally identified as the major component of the plasma membrane skeleton, it is also associated with various intracellular organelles and is implicated in maintaining organelle structures and membrane trafficking (for review see De Matteis and Morrow, 2000
). Therefore, subcellular localization of endogenous βIV-spectrin was examined by immunofluorescence staining first in ES cells (the only cell line among those tested expressing βIV-spectrin) using the anti–βIV-spectrin antibody. When plated on gelatinized cover glass, ES cells often formed an epithelia-like monolayer of cells that adhered to each other. In these cells, the anti–βIV-spectrin antibody stained subdomains of the adherens junctions which were also stained for F-actin and β-catenin ( , A–A′′; unpublished data). βIV-spectrin completely colocalized with another adherens junction protein, vinculin (, B-B′′). These results suggest that βIV-spectrin functions as a component of the plasma membrane skeleton.
Figure 3. Subcellular localization of βIV-spectrin. Cultured ES cells (A–A′′ and B–B′′), dentate gyrus (C–C′′) and cerebellum (D–D′′) of the brain, and sciatic (more ...)
To understand the function of βIV-spectrin in physiological cell types, its subcellular localization was next examined in neurons in sections of mouse brain, spinal cord, and sciatic nerve. In agreement with the X-gal staining pattern of the heterozygous mutant brain ( B), strong anti–βIV-spectrin staining was observed in the dentate gyrus ( C) and cerebellum ( D). This staining pattern coincided with that of 480/270 kD ankyrin-G (′ and D′), a marker of AIS and NR (Kordeli et al., 1995
). Colocalization of βIV-spectrin with ankyrin-G was also detected in neurons in other regions of the brain and spinal cord (unpublished data). In sciatic nerves, βIV-spectrin colocalized with Nav
1.6, a member of the VGSC α-subunit family that is concentrated at NR (Caldwell et al., 2000
; , E-E′′). These results indicate that βIV-spectrin localizes to AIS and NR of neurons in both central and peripheral nervous systems.
Binding of βIV-spectrin to ankyrin-G
βI- and βII-spectrins have been shown to bind ankyrin-R and ankyrin-B (Davis and Bennett, 1990
; Kennedy et al., 1991
). This fact, together with colocalization of βIV-spectrin with ankyrin-G at AIS and NR, suggested that βIV-spectrin might bind ankyrin-G. To test this possibility, Myc-tagged βIVΣ6-spectrin (Myc-βIVΣ6) was cotransfected with green fluorescent protein (GFP), GFP-tagged 270-kD ankyrin-G (AnkG-GFP), or GFP-tagged 220-kD ankyrin-B (AnkB-GFP) into COS-7 cells. Transfected cells were lysed and immunoprecipitated, and then immunoblotted with anti-Myc or anti-GFP antibody. Whereas Myc-βIVΣ6 was expressed at a similar level in the three transfectants ( C), GFP and AnkB-GFP were much more highly expressed than AnkG-GFP ( A). Consistent with the presence in βIVΣ6-spectrin of the spectrin repeat 15, which has been mapped as an ankyrin-binding domain in βI- and βII-spectrins (Kennedy et al., 1991
), the anti-Myc antibody coprecipitated both AnkG-GFP and AnkB-GFP but not GFP alone ( B). However, significantly more AnkG-GFP was brought down than AnkB-GFP ( B). Similarly, AnkG-GFP coprecipitated Myc-βIVΣ6 more efficiently than AnkB-GFP ( D). These results indicate that βIV-spectrin binds to ankyrin-G with high affinity, and to ankyrin-B to a lesser extent.
Figure 4. Binding of βIV-spectrin to ankyrin. COS-7 cells were transfected with Myc-tagged βIVΣ6-spectrin (Myc-βIVΣ6) together with GFP (lanes 1), GFP-tagged 270 kD ankyrin-G (AnkG-GFP; lanes 2), or GFP-tagged 220 (more ...)
Clustering of ankyrin-G and VGSC at AIS and NR in βIV-spectrin–null neurons
The subcellular localization of βIV-spectrin as well as binding to ankyrin-G raised the possibility that it is involved in regulating the localization of ankyrin-G and thus ankyrin-G-binding membrane proteins to AIS and NR. To test this possibility, we examined ankyrin-G and VGSC localization in βIV-spectrin–null neurons.
As expected from the Northern and Western blot analyses (), no anti–βIV-spectrin staining was detected above background level in the βIV-spectrin mutant cerebellar Purkinje and hippocampal pyramidal neurons () . Compared with wild-type neurons where ankyrin-G colocalized with βIV-spectrin at AIS (, A′, C, C′, and E), staining for ankyrin-G was undetectable or very faint at these sites in most βIV-spectrin–null neurons (′, D′, and F). In mutant Purkinje cells with faint anti–ankyrin-G staining, staining was not restricted to the AIS but was spread over the rest of the axon (unpublished data). Although anti–ankyrin-G staining was still restricted to the AIS in some neurons of the mutant, it was much weaker than in wild-type neurons ().
Figure 5. Clustering of ankyrin-G at AIS of βIV-spectrin–null cerebellar and hippocampal neurons. Cerebellum (A, A′, B, and B′) and hippocampus (C, C′, D, and D′) of wild-type (A, A′, C, and C') and βIV-spectrin (more ...)
The localization of VGSC was examined using an anti–pan-sodium channel antibody that recognizes all the known vertebrate sodium channel isoforms (Rasband et al., 1999
). In wild-type mice, VGSC colocalized with βIV-spectrin at AIS of cerebral cortex ( and A′) and hippocampal pyramidal (′) neurons. However, in βIV-spectrin
mutant mice, VGSC staining was very faint in the cerebral cortex (′) and mostly undetectable in the hippocampus (′). Similar mislocalization was also observed for a specific sodium channel isoform Nav
1.6 (′), which normally localizes to AIS in Purkinje cells (′; S. Jenkins and V. Bennett, personal communication).
Figure 6. Clustering of VGSC at AIS of βIV-spectrin–null cerebral, hippocampal, and cerebellar neurons. Cerebral cortex (A, A′, B, and B′), hippocampus (C, C′, D, and D′), and cerebellum (E, E′, F, and F′) (more ...)
Next, we examined the localization of Nav1.6 at NR of the peripheral nervous system. In the βIV-spectrin–null sciatic nerves, Nav1.6-positive but βIV-spectrin–negative NR were detected ( , B-B′′). However, the number of Nav1.6-positive NR was less than in the wild-type littermate in which the NR were also positive for βIV-spectrin (, A–A′′). The number of Nav1.6-positive NR was quantified in sciatic nerve sections of wild-type and the mutant mice. Counting of nine randomly chosen 0.1-mm2 fields from three mice of each genotype revealed that the numbers were 98 ± 23 and 44 ± 15 (mean ± SD) per field in the wild-type and the mutant, respectively (55% reduction in the mutant; C). In addition, anti-Nav1.6 staining of the NR was often fainter in the mutant than in the wild-type control (′ and B′, insets). Phase contrast microscopy showed that the number as well as the morphology of NR was unaffected in the mutant (unpublished data).
Figure 7. Clustering of Nav1.6 at NR of βIV-spectrin–null sciatic nerves. Double staining of wild-type (A, A′, and A′′) and βIV-spectrin–null (B, B′, and B′′) sciatic nerves with anti–βIV-spectrin (more ...)
Taken together, these results indicate that normal localization of ankyrin-G and VGSC to AIS and NR requires βIV-spectrin in its membrane skeleton.
Localization of βIV-spectrin at AIS of ankyrin-G–null neurons
Next, we examined whether localization of βIV-spectrin to AIS requires ankyrin-G. To test this, we examined the localization of βIV-spectrin in cerebellar neurons of mutant mice generated by Zhou et al. (1998)
in which the cerebellum-specific form of ankyrin-G is knocked out. Compared with wild-type cerebellum where AIS of both Purkinje and granular neurons were positive for ankyrin-G and βIV-spectrin ( , A′, and D), βIV-spectrin staining as well as ankyrin-G staining was mostly lost in the knockout mice (′, and E). βIV-spectrin localization was normal in the mutant in other regions than the cerebellum, such as the hippocampus, where ankyrin-G was expressed (′; unpublished data), indicating that ankyrin-G is a prerequisite for the correct βIV-spectrin localization in AIS.
Figure 8. Localization of βIV-spectrin at AIS of ankyrin-G–null neurons. Cerebellum (A, A′, B, and B′) and hippocampus (C and C′) of wild-type (A and A′) and cerebellum-specific ankyrin-G– null (B, B′, (more ...)