Genetic interaction between Bardet-Biedl syndrome genes and implications for limb patterning - PubMed (original) (raw)

. 2008 Jul 1;17(13):1956-67.

doi: 10.1093/hmg/ddn093. Epub 2008 Apr 1.

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Genetic interaction between Bardet-Biedl syndrome genes and implications for limb patterning

Marwan K Tayeh et al. Hum Mol Genet. 2008.

Abstract

Bardet-Biedl syndrome (BBS) is a pleiotropic, genetically heterogeneous disorder characterized by obesity, retinopathy, polydactyly, cognitive impairment, renal and cardiac anomalies, as well as hypertension and diabetes. Multiple genes are known to independently cause BBS. These genes do not appear to code for the same functional category of proteins; yet, mutation of each results in a similar phenotype. Gene knockdown of different BBS genes in zebrafish shows strikingly overlapping phenotypes including defective melanosome transport and disruption of the ciliated Kupffer's vesicle. Here, we demonstrate that individual knockdown of bbs1 and bbs3 results in the same prototypical phenotypes as reported previously for other BBS genes. We utilize the zebrafish system to comprehensively determine whether simultaneous pair-wise knockdown of BBS genes reveals genetic interactions between BBS genes. Using this approach, we demonstrate eight genetic interactions between a subset of BBS genes. The synergistic relationships between distinct combinations are not due to functional redundancy but indicate specific interactions within a multi-subunit BBS complex. In addition, we utilize the zebrafish model system to investigate limb development. Human polydactyly is a cardinal feature of BBS not reproduced in BBS-mouse models. We evaluated zebrafish fin bud patterning and observed altered Sonic hedgehog (shh) expression and subsequent changes to fin skeletal elements. The SHH fin bud phenotype was also used to confirm specific genetic interactions between BBS genes. This study reveals an in vivo requirement for BBS function in limb bud patterning. Our results provide important new insights into the mechanism and biological significance of BBS.

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Figures

Figure 1.

Figure 1.

bbs3 knockdown phenotypes in zebrafish. (A–C) Photographs of live zebrafish embryos at the 10–13 somite stage. (A) KV (dashed box) is a ciliated vesicle located in the tail bud region in a wild-type embryo. (B) Higher magnification of control KV (arrowhead). (C) bbs3 MO-injected embryo with reduced KV (arrowhead). Magnification: (A) 5×; (B and C) 10×. (D) The percentage of zebrafish KV defects (reduced or absent) generated in MO and control sets. (E) Percentage of heart laterality defects observed in bbs3 morphants and controls. (F–H) Epinephrine-induced melanosome retrograde transport, dorsal anterior view of 5-day-old larvae. (F) Wild-type larvae prior to epinephrine treatment and (G) at the endpoint of 1.5 min after epinephrine treatment. (H) bbs3 morphant larvae showing a delayed response after 3.0 min of epinephrine treatment. (I) Graphical representation of epinephrine-induced melanosome retrograde transport times demonstrating a dose-dependent delay in bbs3 knockdown when compared with wild-type and control-injected embryos. Treatment and sample size noted on the _x_-axis and percentage noted at the top of the bar. *P < 0.001.

Figure 2.

Figure 2.

Genetic interaction between eight BBS genes (bbs1_–_bbs8). (A) Twenty-eight subphenotypic pair-wise combinations of BBS MO demonstrated only eight synergistic interactions, noted as ‘+’ sign. Genetic interactions result in (B) KV disruption and (C) epinephrine-induced melanosome retrograde transport delay when compared with control and low-dose BBS MO-injected embryos. Specific RNA can rescue KV disruption and melanosome retrograde transport delay. Control MOs were added when necessary to maintain uniform final concentration of MO in the injection mix. *P < 0.001; Cont, control.

Figure 3.

Figure 3.

Medium-dose knockdown of genetically interacting BBS genes results in an increased severity and penetrance of BBS knockdown phenotypes, including (A) KV disruption and (B) epinephrine-induced melanosome retrograde transport when compared with wild-type, control injected and embryos injected with MO combinations of bbs genes that do not genetically interact. Treatment and numbers of embryos noted on the x axis and values on the top of the bars. *P < 0.001; Cont, control.

Figure 4.

Figure 4.

Zone of polarizing activity (ZPA) of the pectoral fin bud and Sonic hedgehog (SHH). (A) Schematic representation of zebrafish fin bud prior to 48 h.p.f. ZPA is localized posteriorly and can be labeled with shh expression. Whole-mount in situ hybridization using shh as a probe in the pectoral fin bud of 42 h.p.f. (B) wild-type, (C) bbs7 morphant and (D) low-dose bbs1 MO with bbs7 MO combination. Arrowhead denotes shh expression in the limb bud on one side, and the area of expression is outlined on the other side. (E) Graphical representation of the measured area (µm2) of shh expression domain in the pectoral fin bud of BBS morphants and wild-type. *P < 0.001.

Figure 5.

Figure 5.

Zebrafish pectoral fin cartilage staining. (A) Schematic representation of the fin structures: Cl as a dorsoventral line (arrow); Sc distal to Cl and proximal to fin. (B–D) Alcian Blue staining of the pectoral fin shows prototypical cartilage structure in 6-day-old (B) wild-type larvae, whereas (C) bbs7 morphant larvae and (D) low-dose combination of bbs1 MO with bbs7 MO show enlarged Sc structure with two to three cell lines at the proximal posterior region of Sc (arrowhead).

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