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Identification of FOXJ1 effectors during ciliogenesis in the fetal respiratory epithelium and embryonic left-right organizer of the mouse - Lung subscreen B

ABSTRACT: Formation of motile cilia in vertebrate embryos is essential for proper development and tissue function. Key regulators of motile ciliogenesis are the transcription factors FOXJ1 and NOTO, which are conserved throughout vertebrates. Downstream target genes of FOXJ1 have been identified in a variety of species, organs and cultured cell lines; in murine embryonic and foetal tissues, however, FOXJ1 and NOTO effectors have not been comprehensively analysed and our knowledge of the downstream genetic programme driving motile ciliogenesis in the mammalian lung and ventral node is fragmentary. We compared genome-wide expression profiles of undifferentiated E14.5 vs. abundantly ciliated E18.5 micro-dissected airway epithelia as well as Foxj1+ vs. Foxj1-deficient foetal (E16.5) lungs of the mouse using microarray hybridisation. 326 genes deregulated in both screens are candidates for FOXJ1-dependent, ciliogenesis-associated factors at the endogenous onset of motile ciliogenesis in the lung, including 123 genes that have not been linked to ciliogenesis before; 46% of these novel factors lack known homologues outside mammals. Microarray screening of Noto+ vs. Noto null early headfold embryos (E7.75) identified 59 of the lung candidates as NOTO/FOXJ1-dependent factors in the embryonic left-right organiser that carries a different subtype of motile cilia. For several uncharacterised factors from this small overlap – including 1700012B09Rik, 1700026L06Rik and Fam183b – we provide extended experimental evidence for a ciliary function.

ORGANISM(S): Mus musculus

PROVIDER: GSE89582 | GEO | 2017/09/18

SECONDARY ACCESSION(S): PRJNA352627

REPOSITORIES: GEO

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Identification of FOXJ1 effectors during ciliogenesis in the fetal respiratory epithelium and embryonic left-right organizer of the mouse - Lung subscreen A

Project description:Formation of motile cilia in vertebrate embryos is essential for proper development and tissue function. Key regulators of motile ciliogenesis are the transcription factors FOXJ1 and NOTO, which are conserved throughout vertebrates. Downstream target genes of FOXJ1 have been identified in a variety of species, organs and cultured cell lines; in murine embryonic and foetal tissues, however, FOXJ1 and NOTO effectors have not been comprehensively analysed and our knowledge of the downstream genetic programme driving motile ciliogenesis in the mammalian lung and ventral node is fragmentary. We compared genome-wide expression profiles of undifferentiated E14.5 vs. abundantly ciliated E18.5 micro-dissected airway epithelia as well as Foxj1+ vs. Foxj1-deficient foetal (E16.5) lungs of the mouse using microarray hybridisation. 326 genes deregulated in both screens are candidates for FOXJ1-dependent, ciliogenesis-associated factors at the endogenous onset of motile ciliogenesis in the lung, including 123 genes that have not been linked to ciliogenesis before; 46% of these novel factors lack known homologues outside mammals. Microarray screening of Noto+ vs. Noto null early headfold embryos (E7.75) identified 59 of the lung candidates as NOTO/FOXJ1-dependent factors in the embryonic left-right organiser that carries a different subtype of motile cilia. For several uncharacterised factors from this small overlap – including 1700012B09Rik, 1700026L06Rik and Fam183b – we provide extended experimental evidence for a ciliary function.

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Identification of FOXJ1 effectors during ciliogenesis in the fetal respiratory epithelium and embryonic left-right organizer of the mouse - Noto screen

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Transcription profiling of Xenopus epithelial explants injected with the intracellular domain of Notch (ICD) to block the formation of multi-ciliate cells, either alone or along with FoxJ1

Project description:Analysis of epithelial explants injected with the intracellular domain of Notch (ICD) to block the formation of multi-ciliate cells, either alone or along with FoxJ1. FoxJ1 misexpression leads to the induction fo ectopic cilia in Xenopus laevis epithelia. Results show which genes are affected by FoxJ1 during the induction of ectopic cilia. Ciliated cells that produce a leftward fluid flow have been proposed to mediate left-right patterning in many vertebrate embryos. These cilia combine features of primary sensory and motile cilia, but how such cilia are specified is unknown. We address this issue by analyzing the Xenopus and Zebrafish homologs of FoxJ1, a forkhead transcription factor necessary for ciliogenesis in multi-ciliate cells of the mouse. We show that the cilia that underlie left-right patterning on the Xenopus gastrocoel roof plate (GRP) and Zebrafish Kupfferâs vesicle (KV) are severely shortened or fail to form in FoxJ1 morphants. We also show that misexpressing XFoxJ1 is sufficient to induce ectopic GRP-like cilia formation in frog embryos. Microarray analysis indicates that XFoxJ1 induces the formation of cilia by upregulating the expression of motile cilia genes. These results indicate that FoxJ1 is a critical determinant in specifying cilia used in left-right patterning. Experiment Overall Design: 2-cell stage Xenopus embryos were injected with synthetic mRNA encoding ICD alone or along with FoxJ1. At stage 9/10 the presumptive ectoderm was cut off the embryo and cultured on a fibronectin coated coverslip. At stage 22 RNA was harvested from explants and used as the starting material for arrays.

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Project description:Analysis of epithelial explants injected with the intracellular domain of Notch (ICD) to block the formation of multi-ciliate cells, either alone or along with FoxJ1. FoxJ1 misexpression leads to the induction fo ectopic cilia in Xenopus laevis epithelia. Results show which genes are affected by FoxJ1 during the induction of ectopic cilia. Ciliated cells that produce a leftward fluid flow have been proposed to mediate left-right patterning in many vertebrate embryos. These cilia combine features of primary sensory and motile cilia, but how such cilia are specified is unknown. We address this issue by analyzing the Xenopus and Zebrafish homologs of FoxJ1, a forkhead transcription factor necessary for ciliogenesis in multi-ciliate cells of the mouse. We show that the cilia that underlie left-right patterning on the Xenopus gastrocoel roof plate (GRP) and Zebrafish Kupffer’s vesicle (KV) are severely shortened or fail to form in FoxJ1 morphants. We also show that misexpressing XFoxJ1 is sufficient to induce ectopic GRP-like cilia formation in frog embryos. Microarray analysis indicates that XFoxJ1 induces the formation of cilia by upregulating the expression of motile cilia genes. These results indicate that FoxJ1 is a critical determinant in specifying cilia used in left-right patterning. Keywords: Cilia Induction

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Project description:Adenosine N6-methylation (m6A) is one of the most pervasive mRNA modifications, and yet the physiological significance of m6A removal (demethylation) remains elusive. Here we report the m6A demethylase FTO functions as a conserved regulator of motile ciliogenesis. Mechanistically, FTO demethylates and thereby stabilizes the mRNA encoding the master ciliary transcription factor FOXJ1. Depletion of Fto in Xenopus laevis embryos caused widespread motile cilia defects, and Foxj1 was identified as one of the major phenocritical targets. In primary human airway epithelium, FTO depletion also led to FOXJ1 mRNA destabilization and a severe loss of ciliated cells with an increase of neighboring goblet cells. Consistently, Fto knockout mice showed strong asthma-like phenotypes upon allergen challenge owing to defective ciliated cells in the airway epithelium. Altogether, our study reveals a conserved role of the FTO-FOXJ1 axis in embryonic and homeostatic motile ciliogenesis.

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