A CRISPR-based screen for Hedgehog signaling provides insights into ciliary function and ciliopathies

Abstract

The primary cilium organizes Hedgehog signaling and shapes embryonic development, and its dysregulation is the unifying cause of ciliopathies. We conducted a functional genomic screen for Hedgehog signaling by engineering antibiotic-based selection of Hedgehog-responsive cells and applying genome-wide CRISPR-mediated gene disruption. The screen robustly identifies factors required for ciliary signaling with few false positives or false negatives. Characterization of hit genes uncovers novel components of several ciliary structures, including a protein complex containing δ- and ε-tubulin that is required for centriole maintenance. The screen also provides an unbiased tool for classifying ciliopathies and reveals that many congenital heart disorders are caused by loss of ciliary signaling. Collectively, our study enables a systematic analysis of ciliary function and of ciliopathies and also defines a versatile platform for dissecting signaling pathways through CRISPR-based screening.

Centriolar satellites are acentriolar assemblies of centrosomal proteins

“…Note that this and all subsequent analyses were performed on human orthologues of the chicken proteins from CS‐WT. Venn diagrams showing the number of published CS components (illustrated in the table in C) detected in each of the indicated datasets. Table depicts previously reported CS components and their detection in the indicated datasets. Venn diagram showing the number of centrosomal proteins based on the human centrosome proteome dataset (Jakobsen et al , ) detected in each of the indicated datasets. Note that the majority of the proteins detected in both CS‐WT and PCM1‐BioID are centrosomal proteins (33/43). Venn diagram showing the overlap between proteins identified in CS‐WT and in a functional screen for positive regulators of Hedgehog signalling (Breslow et al , ).…”

“…In addition to the BBSome, CS‐WT contained several disease‐linked regulators of ciliogenesis including IFT74, KLHL7, CEP41 and CENPF, which had no previous links to centriolar satellites, or with the exception of CEP41, to centrosomes. Functional cilia are crucial for Hedgehog signalling; indeed, a recent genome‐wide screen for Hedgehog regulators identified many ciliogenesis‐related factors (Breslow et al , ). Remarkably, 30 of these overlapped with the CS‐WT proteome, including TEDC1, a new regulator of centriole assembly and ciliogenesis (Fig EV2E).…”

Meckel syndrome: Clinical and mutation profile in six fetuses

“…In addition to the classical features of MKS, the fetus (F1‐II:4) had holoprosencephaly, single ventricle of heart and bilateral bowing of tibia and fibula (Table S3). The pathogenic variants in TXNDC15 cause aberrant localization of TMEM67 to the transition zone, reduced number of ciliated cells and abnormal ciliary morphology . TMEM67 encodes for a protein which is involved in formation of primary cilium by centriole migration to the apical membrane and pathogenic variants in TMEM67 are known to cause MKS 3 …”

FAM92A Underlies Nonsyndromic Postaxial Polydactyly in Humans and an Abnormal Limb and Digit Skeletal Phenotype in Mice

“…When FAM92A is knocked down in cells, an impaired ciliogenesis is observed. (37,39) FAM92A localization was detected at the transition zone of the primary cilium in IMCD3 cells. (39) In addition, FAM92A interacts with Cby1 (Chibby1) at the ciliary base to promote ciliogenesis via regulation of membrane-remodeling processes.…”

“…(37,39) FAM92A localization was detected at the transition zone of the primary cilium in IMCD3 cells. (39) In addition, FAM92A interacts with Cby1 (Chibby1) at the ciliary base to promote ciliogenesis via regulation of membrane-remodeling processes. (37) Cby1 knockout mice show several hallmarks of ciliary defects, including chronic upper airway infection, subfertility, and polycystic kidneys as well as polydactyly and hydrocephalus at low frequencies.…”