Background Dynein axonemal assembly factors (DNAAF) are essential for the assembly and transport of dynein motor complexes, which are crucial for the movement of cilia. Mutations in genes encoding these proteins often lead to motile ciliopathies called primary ciliary dyskinesia (PCD). In humans, loss-of-function mutations of Cilia and flagella-associated protein 300 (CFAP300, also known as DNAAF17) have been reported to cause PCD. The function of CFAP300 during embryogenesis, however, has not been reported. We carried out functional studies using zebrafish to understand its function during vertebrate development. Methods The expression of cfap300 mRNA during zebrafish embryogenesis was identified using whole-mount in situ hybridization (WISH). A knockout zebrafish line was created using TALEN and the function of cfap300 during embryogenesis was studied. cfap300 mRNA is highly expressed in the zebrafish pronephros. Hence, the effect of cfap300 knock-out on zebrafish pronephros segmentation, multiciliated cell (MCC) and Corpuscle of Stannius (CS) development was studied by analysing the expression of marker genes in these segments and cells using WISH. The gene expression during CS gland development was analysed using RT-qPCR and digital PCR. Results In zebrafish embryos, cfap300 is expressed in multiple ciliated organs, beginning at 10 hpf in Kupffer’s vesicle and later in the notochord, pronephros, otic placode, floor plate, brain, and tail bud. cfap300 mutants develop normally and show no nephron segmentation defects, but exhibit impaired CS development. The CS gland is an endocrine gland in teleost fish, including zebrafish, that secretes Stanniocalcin 1 (Stc1) hormone and regulates calcium homeostasis. The CS gland originates by transdifferentiation of pronephric tubule epithelial cells at the distal early (DE) and distal late (DL) boundary. Mechanistically, hnf1ba expression is increased in the cfap300 mutants, which in turn drives transcription of cdh17 and leads to impaired CS precursor transdifferentiation. In cfap300 mutants, knockdown of hnf1ba or knockout of cdh17 restores CS gland formation. Conclusions These results uncover a Cfap300–Hnf1ba–Cdh17 pathway that regulates epithelial-to-endocrine transdifferentiation, revealing a novel, lineage-specific role for a DNAAF protein in endocrine organogenesis.

Cfap300 regulates the transdifferentiation of Corpuscle of Stannius cells in zebrafish

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2026年5月1日

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Usharani Nayak,
Kalyani Sahoo,
Praveen Barrodia,
Rajeeb K. Swain

摘要

Background

Dynein axonemal assembly factors (DNAAF) are essential for the assembly and transport of dynein motor complexes, which are crucial for the movement of cilia. Mutations in genes encoding these proteins often lead to motile ciliopathies called primary ciliary dyskinesia (PCD). In humans, loss-of-function mutations of Cilia and flagella-associated protein 300 (CFAP300, also known as DNAAF17) have been reported to cause PCD. The function of CFAP300 during embryogenesis, however, has not been reported. We carried out functional studies using zebrafish to understand its function during vertebrate development.

Methods

The expression of cfap300 mRNA during zebrafish embryogenesis was identified using whole-mount in situ hybridization (WISH). A knockout zebrafish line was created using TALEN and the function of cfap300 during embryogenesis was studied. cfap300 mRNA is highly expressed in the zebrafish pronephros. Hence, the effect of cfap300 knock-out on zebrafish pronephros segmentation, multiciliated cell (MCC) and Corpuscle of Stannius (CS) development was studied by analysing the expression of marker genes in these segments and cells using WISH. The gene expression during CS gland development was analysed using RT-qPCR and digital PCR.

Results

In zebrafish embryos, cfap300 is expressed in multiple ciliated organs, beginning at 10 hpf in Kupffer’s vesicle and later in the notochord, pronephros, otic placode, floor plate, brain, and tail bud. cfap300 mutants develop normally and show no nephron segmentation defects, but exhibit impaired CS development. The CS gland is an endocrine gland in teleost fish, including zebrafish, that secretes Stanniocalcin 1 (Stc1) hormone and regulates calcium homeostasis. The CS gland originates by transdifferentiation of pronephric tubule epithelial cells at the distal early (DE) and distal late (DL) boundary. Mechanistically, hnf1ba expression is increased in the cfap300 mutants, which in turn drives transcription of cdh17 and leads to impaired CS precursor transdifferentiation. In cfap300 mutants, knockdown of hnf1ba or knockout of cdh17 restores CS gland formation.

Conclusions

These results uncover a Cfap300–Hnf1ba–Cdh17 pathway that regulates epithelial-to-endocrine transdifferentiation, revealing a novel, lineage-specific role for a DNAAF protein in endocrine organogenesis.