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The forkhead protein Foxj1 specifies node-like cilia in Xenopus and zebrafish embryos.

Stubbs JL, Oishi I, Izpisúa Belmonte JC, Kintner C - Nat. Genet. (2008)

Bottom Line: We also show that misexpressing Foxj1 is sufficient to induce ectopic GRP-like cilia formation in frog embryos.Microarray analysis indicates that Xenopus Foxj1 induces the formation of cilia by upregulating the expression of motile cilia genes.These results indicate that Foxj1 is a critical determinant in the specification of cilia used in left-right patterning.

View Article: PubMed Central - PubMed

Affiliation: The Salk Institute for Biological Studies, San Diego, CA 92186, USA.

ABSTRACT
It has been proposed that ciliated cells that produce a leftward fluid flow mediate left-right patterning in many vertebrate embryos. The cilia on these cells combine features of primary sensory and motile cilia, but how this cilia subtype is specified is unknown. We address this issue by analyzing the Xenopus and zebrafish homologs of Foxj1, a forkhead transcription factor necessary for ciliogenesis in multiciliated 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 are severely shortened or fail to form in Foxj1 morphants. We also show that misexpressing Foxj1 is sufficient to induce ectopic GRP-like cilia formation in frog embryos. Microarray analysis indicates that Xenopus Foxj1 induces the formation of cilia by upregulating the expression of motile cilia genes. These results indicate that Foxj1 is a critical determinant in the specification of cilia used in left-right patterning.

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Validation of gene expression regulated by FoxJ1.(A-C) Shown is the roof of the gastrocoel in stage 17 embryos after staining for the expression of Tektin-t (A,A′), PF16 (B,B′) and L-R dynein (C,C′) RNA using whole-mount, in situ hybridization. Expression (Red-Blue stain) in the posterior GRP is marked with an arrow. Top panels show staining in uninjected embryos while lower panels shows that in embryos injected twice in one blastomere at the two-cell stage with XFoxJ1 RNA. Injected side is oriented to the right. (D) Embryos were injected at the two-cell stage with the indicated RNAs or with XFoxJ1 or control morpholinos. At stage 10, the ectoderm was isolated, cultured on fibronectin-coated glass to stage 22, and then extracted for total RNA. The levels of Tektin-t, PF16 (Spag6), or L-R dynein RNA was measured in each sample using quantitative PCR, and normalized relative to a ubiquitously expressed control RNA, ODC. Values for each experimental condition is an average of three measurements, and are expressed on a logarithmic plot as a ratio to the average value obtained with a control. Uninjected controls were used for the RNA injected samples and a control morpholino sample was used as a control for FoxJ1 morpholino injection.
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Figure 5: Validation of gene expression regulated by FoxJ1.(A-C) Shown is the roof of the gastrocoel in stage 17 embryos after staining for the expression of Tektin-t (A,A′), PF16 (B,B′) and L-R dynein (C,C′) RNA using whole-mount, in situ hybridization. Expression (Red-Blue stain) in the posterior GRP is marked with an arrow. Top panels show staining in uninjected embryos while lower panels shows that in embryos injected twice in one blastomere at the two-cell stage with XFoxJ1 RNA. Injected side is oriented to the right. (D) Embryos were injected at the two-cell stage with the indicated RNAs or with XFoxJ1 or control morpholinos. At stage 10, the ectoderm was isolated, cultured on fibronectin-coated glass to stage 22, and then extracted for total RNA. The levels of Tektin-t, PF16 (Spag6), or L-R dynein RNA was measured in each sample using quantitative PCR, and normalized relative to a ubiquitously expressed control RNA, ODC. Values for each experimental condition is an average of three measurements, and are expressed on a logarithmic plot as a ratio to the average value obtained with a control. Uninjected controls were used for the RNA injected samples and a control morpholino sample was used as a control for FoxJ1 morpholino injection.

Mentions: To validate the results from the microarray analysis, we examined the expression of several genes on the FoxJ1 upregulated list, focusing on three likely to be critical for cilia motility based on mouse mutants, namely PF16, Tektin-t and LR-dynein30-32. When examined by whole mount, in situ hybridization, all three genes are expressed in the multi-ciliate cells in the skin, are lost when multi-ciliate cells are eliminated by expressing ICD, or when ciliogenesis is blocked in the multi-ciliate cells by injecting the XFoxJ1MO (Data not shown, Supplementary Fig. 8). Thus, all three genes are expressed in multi-ciliate cells in a FoxJ1-dependent manner. All three genes are expressed in the GRP (Fig. 5A-C), and are markedly upregulated ectopically in embryos misexpressing FoxJ1 (Fig. 5A-C, Supplementary Figs. 7E-G, and 8). To confirm these observations quantitatively real-time PCR was used to measure RNA levels of PF16, Tektin-t and LR-dynein when the embryonic epithelium is explanted on fibronectin-coated glass (Fig. 5D). In explants injected with ICD RNA alone, the expression levels of these three cilia genes dropped approximately 8-fold relative to control, inline with the idea that these genes are expressed in multi-ciliate cells. Moreover, in explants that express both FoxJ1 and ICD, the levels of RNA encoding these three cilia proteins increased approximately 5-10 fold over the levels found in control larval skin and 50 fold or more relative to ICD injected samples. Thus, these results indicate that when XFoxJ1 induces node-like cilia in ectopic locations, it upregulates the expression of genes involved in cilia motility.


The forkhead protein Foxj1 specifies node-like cilia in Xenopus and zebrafish embryos.

Stubbs JL, Oishi I, Izpisúa Belmonte JC, Kintner C - Nat. Genet. (2008)

Validation of gene expression regulated by FoxJ1.(A-C) Shown is the roof of the gastrocoel in stage 17 embryos after staining for the expression of Tektin-t (A,A′), PF16 (B,B′) and L-R dynein (C,C′) RNA using whole-mount, in situ hybridization. Expression (Red-Blue stain) in the posterior GRP is marked with an arrow. Top panels show staining in uninjected embryos while lower panels shows that in embryos injected twice in one blastomere at the two-cell stage with XFoxJ1 RNA. Injected side is oriented to the right. (D) Embryos were injected at the two-cell stage with the indicated RNAs or with XFoxJ1 or control morpholinos. At stage 10, the ectoderm was isolated, cultured on fibronectin-coated glass to stage 22, and then extracted for total RNA. The levels of Tektin-t, PF16 (Spag6), or L-R dynein RNA was measured in each sample using quantitative PCR, and normalized relative to a ubiquitously expressed control RNA, ODC. Values for each experimental condition is an average of three measurements, and are expressed on a logarithmic plot as a ratio to the average value obtained with a control. Uninjected controls were used for the RNA injected samples and a control morpholino sample was used as a control for FoxJ1 morpholino injection.
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Related In: Results  -  Collection

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Figure 5: Validation of gene expression regulated by FoxJ1.(A-C) Shown is the roof of the gastrocoel in stage 17 embryos after staining for the expression of Tektin-t (A,A′), PF16 (B,B′) and L-R dynein (C,C′) RNA using whole-mount, in situ hybridization. Expression (Red-Blue stain) in the posterior GRP is marked with an arrow. Top panels show staining in uninjected embryos while lower panels shows that in embryos injected twice in one blastomere at the two-cell stage with XFoxJ1 RNA. Injected side is oriented to the right. (D) Embryos were injected at the two-cell stage with the indicated RNAs or with XFoxJ1 or control morpholinos. At stage 10, the ectoderm was isolated, cultured on fibronectin-coated glass to stage 22, and then extracted for total RNA. The levels of Tektin-t, PF16 (Spag6), or L-R dynein RNA was measured in each sample using quantitative PCR, and normalized relative to a ubiquitously expressed control RNA, ODC. Values for each experimental condition is an average of three measurements, and are expressed on a logarithmic plot as a ratio to the average value obtained with a control. Uninjected controls were used for the RNA injected samples and a control morpholino sample was used as a control for FoxJ1 morpholino injection.
Mentions: To validate the results from the microarray analysis, we examined the expression of several genes on the FoxJ1 upregulated list, focusing on three likely to be critical for cilia motility based on mouse mutants, namely PF16, Tektin-t and LR-dynein30-32. When examined by whole mount, in situ hybridization, all three genes are expressed in the multi-ciliate cells in the skin, are lost when multi-ciliate cells are eliminated by expressing ICD, or when ciliogenesis is blocked in the multi-ciliate cells by injecting the XFoxJ1MO (Data not shown, Supplementary Fig. 8). Thus, all three genes are expressed in multi-ciliate cells in a FoxJ1-dependent manner. All three genes are expressed in the GRP (Fig. 5A-C), and are markedly upregulated ectopically in embryos misexpressing FoxJ1 (Fig. 5A-C, Supplementary Figs. 7E-G, and 8). To confirm these observations quantitatively real-time PCR was used to measure RNA levels of PF16, Tektin-t and LR-dynein when the embryonic epithelium is explanted on fibronectin-coated glass (Fig. 5D). In explants injected with ICD RNA alone, the expression levels of these three cilia genes dropped approximately 8-fold relative to control, inline with the idea that these genes are expressed in multi-ciliate cells. Moreover, in explants that express both FoxJ1 and ICD, the levels of RNA encoding these three cilia proteins increased approximately 5-10 fold over the levels found in control larval skin and 50 fold or more relative to ICD injected samples. Thus, these results indicate that when XFoxJ1 induces node-like cilia in ectopic locations, it upregulates the expression of genes involved in cilia motility.

Bottom Line: We also show that misexpressing Foxj1 is sufficient to induce ectopic GRP-like cilia formation in frog embryos.Microarray analysis indicates that Xenopus Foxj1 induces the formation of cilia by upregulating the expression of motile cilia genes.These results indicate that Foxj1 is a critical determinant in the specification of cilia used in left-right patterning.

View Article: PubMed Central - PubMed

Affiliation: The Salk Institute for Biological Studies, San Diego, CA 92186, USA.

ABSTRACT
It has been proposed that ciliated cells that produce a leftward fluid flow mediate left-right patterning in many vertebrate embryos. The cilia on these cells combine features of primary sensory and motile cilia, but how this cilia subtype is specified is unknown. We address this issue by analyzing the Xenopus and zebrafish homologs of Foxj1, a forkhead transcription factor necessary for ciliogenesis in multiciliated 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 are severely shortened or fail to form in Foxj1 morphants. We also show that misexpressing Foxj1 is sufficient to induce ectopic GRP-like cilia formation in frog embryos. Microarray analysis indicates that Xenopus Foxj1 induces the formation of cilia by upregulating the expression of motile cilia genes. These results indicate that Foxj1 is a critical determinant in the specification of cilia used in left-right patterning.

Show MeSH
Related in: MedlinePlus