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. 2010 Mar 19;6(3):e1000884.
doi: 10.1371/journal.pgen.1000884.

Identification and functional analysis of the vision-specific BBS3 (ARL6) long isoform

Pamela R Pretorius  1 , Lisa M Baye, Darryl Y Nishimura, Charles C Searby, Kevin Bugge, Baoli Yang, Robert F Mullins, Edwin M Stone, Val C Sheffield, Diane C Slusarski
Affiliations

Identification and functional analysis of the vision-specific BBS3 (ARL6) long isoform

Pamela R Pretorius et al. PLoS Genet. .

Abstract

Bardet-Biedl Syndrome (BBS) is a heterogeneous syndromic form of retinal degeneration. We have identified a novel transcript of a known BBS gene, BBS3 (ARL6), which includes an additional exon. This transcript, BBS3L, is evolutionally conserved and is expressed predominantly in the eye, suggesting a specialized role in vision. Using antisense oligonucleotide knockdown in zebrafish, we previously demonstrated that bbs3 knockdown results in the cardinal features of BBS in zebrafish, including defects to the ciliated Kupffer's Vesicle and delayed retrograde melanosome transport. Unlike bbs3, knockdown of bbs3L does not result in Kupffer's Vesicle or melanosome transport defects, rather its knockdown leads to impaired visual function and mislocalization of the photopigment green cone opsin. Moreover, BBS3L RNA, but not BBS3 RNA, is sufficient to rescue both the vision defect as well as green opsin localization in the zebrafish retina. In order to demonstrate a role for Bbs3L function in the mammalian eye, we generated a Bbs3L-null mouse that presents with disruption of the normal photoreceptor architecture. Bbs3L-null mice lack key features of previously published Bbs-null mice, including obesity. These data demonstrate that the BBS3L transcript is required for proper retinal function and organization.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Identification of a second BBS3 transcript.
(A) Alignment of human BBS3 and BBS3L proteins. (B) C-terminal end alignment of human, mouse, and zebrafish BBS3L protein. Asterisks (*) indicate identical amino acids shared in all alignments, while colons (:) and periods (.) represent conserved amino acids. (C) RT–PCR tissue expression profile of zebrafish bbs3 and bbs3L transcripts in wild-type adult zebrafish tissues: fat, brain, heart, whole eye and retina. β-actin was used as a positive control. bbs3 is expressed in all adult tissues examined, while bbs3L expression is limited to the eye. (D) RT–PCR developmental expression profile of bbs3 and bbs3L at the following stages: maternal, 8–12 somites, 24, 36, 42, 48, 60, 72, 96 hpf, and 5 dpf. bbs3 is expressed throughout development, while the long form is only present by 48 hpf, correlating with photoreceptor development.
Figure 2
Figure 2. bbs3 gene structure and cardinal features of BBS knockdown in zebrafish.
(A) Schematic depicting the bbs3 gene structure and antisense oligonucleotide strategy used to target either both transcripts (bbs3 aug MO) or to target only bbs3L (bbs3 long MO) in zebrafish embryos. The bbs3 aug MO targets the start site of the gene and thus hits both transcripts, while the bbs3L MO is a splice-blocking morpholino that only targets the long form. (B) RT-PCR from staged bbs3L morphant embryos at 8–12 somites, 72 hpf, and 5 dpf. The bbs3L transcript is absent through 5 dpf injected embryos indicating successful knockdown. Note that the bbs3 transcript is unperturbed in bbs3L morphants. (C–E) Images of live zebrafish embryos at 8–10 somite stage. Scale bar 200 µm. (C) Side view of an embryo highlighting the location of the Kupffer's Vesicle (circle), the ciliated structure located in the tailbud. (D) Dorsal view of a normal sized KV from a wild-type embryo. (E) bbs3 aug MO–injected embryos with a reduced KV. (F) The percentage of embryos with KV defects (reduced or absent) in uninjected, control MO, bbs3 aug MO and bbs3 long MO injected embryos. The sample size (n) is noted on the x-axis. **Fisher's Exact test, p<0.001. (G–I) Epinephrine-induced melanosome transport of wild-type 6-day old larvae. Scale bar 100 µm. (G) Melanosome transport is observed in cells on the head of the embryos. Boxed region is magnified for (H,I). (H) Wild-type larvae prior to epinephrine treatment and (I) the endpoint at 1.4 minutes after epinephrine treatment. (J) Epinephrine-induced retrograde transport times. The sample size (n) is noted on the x-axis. **ANOVA with Tukey, p<0.01.
Figure 3
Figure 3. Vision startle response in zebrafish.
(A) Vision function was assayed in 5-day old embryos by testing embryos sensitivity to short blocks in light at 30 second intervals for 5 trials (adapted from Easter and Nicola 1996). Selected images from a time-lapse collection before and immediately after a one second block in light. The typical response, a distinct C-bend, is scored as a positive response as shown in time point 139 ms. ms, milliseconds. (B) Quantification of the vision startle response for each treatment. Cone-rod homeobox (crx) gene knockdown was used as a control for vision impairment. bbs3 morphants lacking either both transcripts or only the long transcript showed a statistically significant reduction in the number of responses, indicating visual impairment. Rescue experiments using wild-type human BBS3L or BBS3 RNA co-injected with the bbs3 morpholinos demonstrated that hBBS3L RNA is sufficient to rescue the vision defect associated with knockdown, while hBBS3 is not sufficient to rescue the vision defect. The sample size (n) is noted on the x-axis. **ANOVA with Tukey, p<0.01.
Figure 4
Figure 4. Green opsin mislocalization and rescue in 5-day-old bbs3 morphant zebrafish.
Immunofluorescence of green cone opsin (green) on transverse cryosections of 5-day-old embryos (A) uninjected wild-type, (B) bbs3 aug MO, (C) bbs3 aug MO and hBBS3 RNA, (D) bbs3 aug MO and hBBS3L RNA, (E) bbs3 long MO, (F) bbs3 long MO and hBBS3 RNA, and (G) bbs3 long MO and hBBS3L RNA. To-Pro3 was used to counterstain the nuclei (blue). In bbs3 aug (B) and bbs3 long morphants (E), green opsin was not restricted to the outer segment of the photoreceptors and was detected in the cell bodies of the outer nuclear layer (arrowheads and asterisks). Expression of hBBS3L RNA improved green opsin localization in both bbs3 aug (D) and bbs3L (G) morphants. Of note, hBBS3 RNA failed to rescue green opsin localization in bbs3 aug (C) and bbs3 long (F) morphant embryos (arrowheads and asterisks). OS, outer segment; ONL, outer nuclear layer. Scale Bar 10 µm. (H) The percentage of mislocalizing green opsin cells. The sample size (n) is noted on the x-axis and represents the total number of green opsin positive cells counted. **Fisher's exact test, p<0.01.
Figure 5
Figure 5. Localization of BBS3 in human and wild-type mouse retinas.
Immunohistochemistry triple labeling of cryosections taken from 8-month old wild-type mouse retinas (A–C) and human donor eyes (D–F). Localization of BBS3 (green) using an antibody generated against a central region of the mouse Bbs3 peptide, which recognizes both Bbs3 and Bbs3L (A,D). Peanut agglutinin (PNA, red) was used as a marker for cone outer segments (B,E). Nuclei were counterstained with DAPI (blue). Bbs3 was found robustly in the ganglion cell layer as well as the photoreceptor cell layer of human and mouse retinas. PR, photoreceptor; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.
Figure 6
Figure 6. Generation and initial characterization of a Bbs3L mutant mice.
(A) Schematic for the targeted alteration of the splice donor and acceptor sites of exon 8 (asterisk) found in the Bbs3L transcript. Homologous recombination leads to the inclusion of these altered sites and the loss of Bbs3L. Hematoxylin/eosin staining of cryosections from 8-month old Bbs3L+/+ (B,C) and Bbs3L−/− (E,F) mouse retinas. Disruption of the normal photoreceptor architecture was observed in Bbs3L−/− mice. Immunohistochemistry analysis of cryosections from (D) wild-type and (G) targeted mutants retinas using the Bbs3 antibody (green) and rhodopsin (red), a marker for rod photoreceptor outer segments. To-Pro3 was used as a counterstain for nuclei. PR, photoreceptor; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.
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References

    1. Green JS, Parfrey PS, Harnett JD, Farid NR, Cramer BC, et al. The cardinal manifestations of Bardet-Biedl syndrome, a form of Laurence-Moon-Biedl syndrome. N Engl J Med. 1989;321:1002–1009. - PubMed
    1. Harnett JD, Green JS, Cramer BC, Johnson G, Chafe L, et al. The spectrum of renal disease in Laurence-Moon-Biedl syndrome. N Engl J Med. 1988;319:615–618. - PubMed
    1. Bardet G. On congenital obesity syndrome with polydactyly and retinitis pigmentosa (a contribution to the study of clinical forms of hypophyseal obesity). 1920. Obes Res. 1995;3:387–399. - PubMed
    1. Biedl A. A pair of siblings with adiposo-genital dystrophy. 1922. Obes Res. 1995;3:404. - PubMed
    1. Elbedour K, Zucker N, Zalzstein E, Barki Y, Carmi R. Cardiac abnormalities in the Bardet-Biedl syndrome: echocardiographic studies of 22 patients. Am J Med Genet. 1994;52:164–169. - PubMed

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