Knockdown of Bardet-Biedl Syndrome Gene BBS9/PTHB1 Leads to Cilia Defects

Bardet-Biedl Syndrome (BBS, MIM#209900) is a genetically heterogeneous disorder with pleiotropic phenotypes that include retinopathy, mental retardation, obesity and renal abnormalities. Of the 15 genes identified so far, seven encode core proteins that form a stable complex called BBSome, which is implicated in trafficking of proteins to cilia. Though BBS9 (also known as PTHB1) is reportedly a component of BBSome, its direct function has not yet been elucidated. Using zebrafish as a model, we show that knockdown of bbs9 with specific antisense morpholinos leads to developmental abnormalities in retina and brain including hydrocephaly that are consistent with the core phenotypes observed in syndromic ciliopathies. Knockdown of bbs9 also causes reduced number and length of cilia in Kupffer's vesicle. We also demonstrate that an orthologous human BBS9 mRNA, but not one carrying a missense mutation identified in BBS patients, can rescue the bbs9 morphant phenotype. Consistent with these findings, knockdown of Bbs9 in mouse IMCD3 cells results in the absence of cilia. Our studies suggest a key conserved role of BBS9 in biogenesis and/or function of cilia in zebrafish and mammals

Long wavelength-sensing cones of zebrafish retina exhibit multiple layers of transcriptional heterogeneity

IntroductionUnderstanding how photoreceptor genes are regulated is important for investigating retinal development and disease. While much is known about gene regulation in cones, the mechanism by which tandemly-replicated opsins, such as human long wavelength-sensitive and middle wavelength-sensitive opsins, are differentially regulated remains elusive. In this study, we aimed to further our understanding of transcriptional heterogeneity in cones that express tandemly-replicated opsins and the regulation of such differential expression using zebrafish, which express the tandemly-replicated opsins lws1 and lws2.MethodsWe performed bulk and single cell RNA-Seq of LWS1 and LWS2 cones, evaluated expression patterns of selected genes of interest using multiplex fluorescence in situ hybridization, and used exogenous thyroid hormone (TH) treatments to test selected genes for potential control by thyroid hormone: a potent, endogenous regulator of lws1 and lws2 expression.ResultsOur studies indicate that additional transcriptional differences beyond opsin expression exist between LWS1 and LWS2 cones. Bulk RNA-Seq results showed 95 transcripts enriched in LWS1 cones and 186 transcripts enriched in LWS2 cones (FC > 2, FDR < 0.05). In situ hybridization results also reveal underlying heterogeneity within the lws1- and lws2-expressing populations. This heterogeneity is evident in cones of mature zebrafish, and further heterogeneity is revealed in transcriptional responses to TH treatments.DiscussionWe found some evidence of coordinate regulation of lws opsins and other genes by exogenous TH in LWS1 vs. LWS2 cones, as well as evidence of gene regulation not mediated by TH. The transcriptional differences between LWS1 and LWS2 cones are likely controlled by multiple signals, including TH

Why do banks promise to pay par on demand?

We survey the theories of why banks promise to pay par on demand and examine evidence about the conditions under which banks have promised to pay the par value of deposits and banknotes on demand when holding only fractional reserves. The theoretical literature can be broadly divided into four strands: liquidity provision, asymmetric information, legal restrictions, and a medium of exchange. We assume that it is not zero cost to make a promise to redeem a liability at par value on demand. If so, then the conditions in the theories that result in par redemption are possible explanations of why banks promise to pay par on demand. If the explanation based on customers’ demand for liquidity is correct, payment of deposits at par will be promised when banks hold assets that are illiquid in the short run. If the asymmetric-information explanation based on the difficulty of valuing assets is correct, the marketability of banks’ assets determines whether banks promise to pay par. If the legal restrictions explanation of par redemption is correct, banks will not promise to pay par if they are not required to do so. If the transaction explanation is correct, banks will promise to pay par value only if the deposits are used in transactions. After the survey of the theoretical literature, we examine the history of banking in several countries in different eras: fourth-century Athens, medieval Italy, Japan, and free banking and money market mutual funds in the United States. We find that all of the theories can explain some of the observed banking arrangements, and none explain all of them

Human mRNA rescues zebrafish <i>bbs9</i>-spMO phenotype.

<p>h<i>W</i> and h<i>M</i> represent wild type and mutant human mRNA, respectively. The arrows indicate eye phenotype. (<b>A</b>) The uninjected control (top) and <i>bbs9</i>-spMO alone injected (bottom) zebrafish at 72 hpf. (<b>B</b>) Rescue of <i>bbs9</i>-spMO eye phenotype by hW 100 pg (top), but not by lower dose of 50 pg (bottom). (<b>C</b>) The <i>bbs9</i>-spMO phenotype is not rescued by h<i>M</i> as the eye defect remains in the morphants. (<b>D</b>) The quantification of embryos' eye size at 72 hpf in rescue experiment using human mRNAs co-injected with <i>bbs9</i>-spMO. X-axis shows category of embryos scored. Y-axis shows the eye size in pixels. Data are presented as mean ± SEM. Statistically significant and non-significant observations are indicated with p value and n.s., respectively.</p

Exon 5-targeted <i>bbs9</i> splice morpholino affects eye development independent of p53 pathway.

<p>(<b>A</b>) At 24 hpf, the <i>p53</i>-atgMO (1.5 ng) alone injection did not elicit a phenotype. The <i>bbs9</i>-spMO (1 ng) injection alone caused developmental defects in the eye, brain and tail of morphants. However, co-injection of <i>p53</i>-atgMO reduced the defects seen by the <i>bbs9</i>-spMO injection alone, though mild eye defect remained the tail becomes normal (bottom panel). (<b>B</b>) Higher magnification of morphants' head region. Top, middle and bottom rows are 24-, 48- and 72-hpf, respectively. Left and right column of panels are <i>p53</i>-atgMO without and with <i>bbs9</i>-spMO, respectively. At 48 hpf the effect of <i>bbs9</i>-spMO injection on eye size visible (compare the arrows). The <i>bbs9</i>-spMO injection also resulted in hydrocephalous (compare the arrow heads). The defects seen at 48 hpf are weaker at 72 hpf. (<b>C</b>) The gel photograph of RT-PCR showing exon-skipping by <i>bbs9</i>-spMO. mRNA isolated from individual embryos was used for RT-PCR. U, C (4 and 6 ng) and B (1, 4, 6 ng) represent un-injected, control, and <i>bbs9</i>-spMO, respectively. Splice blocking gave an additional smaller (marked e5skip) band along with the original WT band. The bottom panel shows β-actin control for respective samples. (<b>D</b>) Quantification of the effect of morpholino(s) injection on eye size. X-axis shows the morpholinos used and time (hpf) of scoring. Y-axis shows eye size in pixels (mean ± SEM).</p

Zebrafish <i>bbs9</i> gene: structural comparison and expression pattern.

<p>(<b>A</b>) A comparison of <i>BBS9</i> exon:intron structure between human (<i>H. sapiens</i>, top blue), mouse (<i>M. musculus</i>, middle black) and zebrafish (<i>D. rerio</i>, bottom gray/black). The filled and open boxes indicate coding exons and UTRs, respectively. The blue and black boxes represent validated exons. The gray boxes represent exons present in provisional sequence XM_002664792.1. Exons 2 to 8 are highly conserved across species (boxed area within hatched square). The yellow arrow points to yellow mark on exon 5, which represents the missense mutation G→A (p.G141R) in human BBS9 protein. Under the zebrafish <i>bbs9</i> transcript, the red line represents <i>bbs9</i>-spMO targeting site at intron4:exon5 boundary. (<b>B</b>) The protein sequence alignment (clustalW) between human (NP_940820.1), mouse (NP_848502.1) and predicted zebrafish BBS9 (904 amino acids). Exon 5 is highlighted (yellow), and the position of missense mutation (p.G141R) in human is highlighted by a black rectangle. The bar coding on top of the sequences represents degree of conservation (red and blue represent maximum and minimum conservation, respectively). (<b>C, D</b>) <i>In situ</i> hybridization analysis at 11 hpf and 15 hpf. Left and right panels represent the sense and anti-sense probes generated from <i>bbs9</i> cDNA. (<b>E</b>) <i>In situ</i> hybridization analysis at 48 hpf. Expression of <i>bbs9</i> in the eye, brain and somites gives a strong signal with the anti-sense probe compared to the background signal from the sense probe. Compare the strong signal in the head regions (arrows). Left and right panels represent lateral and dorsal views, respectively.</p

In Vitro Modeling Using Ciliopathy-Patient-Derived Cells Reveals Distinct Cilia Dysfunctions Caused by CEP290 Mutations

Mutations in CEP290, a transition zone protein in primary cilia, cause diverse ciliopathies, including Leber congenital amaurosis (LCA) and Joubert-syndrome and related disorders (JSRD). We examined cilia biogenesis and function in cells derived from CEP290-LCA and CEP290-JSRD patients. CEP290 protein was reduced in LCA fibroblasts with no detectable impact on cilia; however, optic cups derived from induced pluripotent stem cells (iPSCs) of CEP290-LCA patients displayed less developed photoreceptor cilia. Lack of CEP290 in JSRD fibroblasts resulted in abnormal cilia and decreased ciliogenesis. We observed selectively reduced localization of ADCY3 and ARL13B. Notably, Hedgehog signaling was augmented in CEP290-JSRD because of enhanced ciliary transport of Smoothened and GPR161. These results demonstrate a direct correlation between the extent of ciliogenesis defects in fibroblasts and photoreceptors with phenotypic severity in JSRD and LCA, respectively, and strengthen the role of CEP290 as a selective ciliary gatekeeper for transport of signaling molecules in and out of the cilium