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FoxK mediates TGF-beta signalling during midgut differentiation in flies.

Casas-Tinto S, Gomez-Velazquez M, Granadino B, Fernandez-Funez P - J. Cell Biol. (2008)

Bottom Line: Genet.This regulatory activity does not require direct labial activation by the TGF-beta effector Mad.Thus, we propose that the combined activity of the TGF-beta target genes FoxK and Dfos is critical for the direct activation of lab in the endoderm.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, University of Texas Medical Branch, Galveston, TX 77555, USA. scasas@cnio.es

ABSTRACT
Inductive signals across germ layers are important for the development of the endoderm in vertebrates and invertebrates (Tam, P.P., M. Kanai-Azuma, and Y. Kanai. 2003. Curr. Opin. Genet. Dev. 13:393-400; Nakagoshi, H. 2005. Dev. Growth Differ. 47:383-392). In flies, the visceral mesoderm secretes signaling molecules that diffuse into the underlying midgut endoderm, where conserved signaling cascades activate the Hox gene labial, which is important for the differentiation of copper cells (Bienz, M. 1997. Curr. Opin. Genet. Dev. 7:683-688). We present here a Drosophila melanogaster gene of the Fox family of transcription factors, FoxK, that mediates transforming growth factor beta (TGF-beta) signaling in the embryonic midgut endoderm. FoxK mutant embryos fail to generate midgut constrictions and lack Labial in the endoderm. Our observations suggest that TGF-beta signaling directly regulates FoxK through functional Smad/Mad-binding sites, whereas FoxK, in turn, regulates labial expression. We also describe a new cooperative activity of the transcription factors FoxK and Dfos/AP-1 that regulates labial expression in the midgut endoderm. This regulatory activity does not require direct labial activation by the TGF-beta effector Mad. Thus, we propose that the combined activity of the TGF-beta target genes FoxK and Dfos is critical for the direct activation of lab in the endoderm.

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Molecular characterization of FoxK mutant alleles. (A) The P element EP(3)3428 is inserted in 676 bp 5′ of the ATG (0) of FoxK. Red arrowheads indicate the primers used for sequencing exons 2–5. (B) Both FoxK44 and FoxK16 carry a deletion of 2 bp at the insertion site of EP(3)3428 (−676ΔTA). FoxK44 flies also contain a reinsertion of a fragment of the P element in exon 3 (green) that generates a premature Stop codon. In FoxK16, a deletion in exon 2 generates a new ORF (purple) containing a Stop codon. (C) Southern blot hybridized with a probe covering the entire FoxK coding region shows an extra band of 2.6 Kb in FoxK44 and FoxK16 (arrow). (D and E) Stage 15 FoxK16 homozygous embryos do not stain with anti-FoxK (D, arrow), but the ventral nerve cord stains with anti-Elav and shows normal morphology (E, arrow).
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fig4: Molecular characterization of FoxK mutant alleles. (A) The P element EP(3)3428 is inserted in 676 bp 5′ of the ATG (0) of FoxK. Red arrowheads indicate the primers used for sequencing exons 2–5. (B) Both FoxK44 and FoxK16 carry a deletion of 2 bp at the insertion site of EP(3)3428 (−676ΔTA). FoxK44 flies also contain a reinsertion of a fragment of the P element in exon 3 (green) that generates a premature Stop codon. In FoxK16, a deletion in exon 2 generates a new ORF (purple) containing a Stop codon. (C) Southern blot hybridized with a probe covering the entire FoxK coding region shows an extra band of 2.6 Kb in FoxK44 and FoxK16 (arrow). (D and E) Stage 15 FoxK16 homozygous embryos do not stain with anti-FoxK (D, arrow), but the ventral nerve cord stains with anti-Elav and shows normal morphology (E, arrow).

Mentions: To elucidate the function of FoxK in Drosophila, we generated FoxK loss-of-function alleles by imprecise excision of a P element inserted 676 bp upstream of the ATG for FoxK (Fig. 4 A). We recovered two FoxK mutant alleles that resulted in recessive lethal chromosomes. To ensure that the lethality of the FoxK alleles was contained in the FoxK region, we confirmed that a chromosomal duplication of FoxK recovered the viability of FoxK16 and FoxK44 homozygous flies. To molecularly characterize these new FoxK alleles, we analyzed genomic DNA from FoxK16 and FoxK44 flies by Southern blot with a probe covering the entire FoxK coding region. DNA samples from FoxK16 and FoxK44 heterozygous flies showed an unexpected band suggestive of a chromosomal aberration within FoxK (Fig. 4 C, arrow). To delimitate the affected region, we sequenced the central region of FoxK using specific primers for exons 3–5 (Fig. 4 A, red arrowheads). We confirmed that FoxK44 contains a partial reinsertion of the P element in exon 3, creating a Stop codon 28 nucleotides after the insertion (Fig. 4 B). The truncated protein produced by FoxK44 retained the FHA domain, but lacked the FH domain. Next, to identify the molecular changes associated with FoxK16, we sequenced exons 2–5 and identified a deficiency of 962 bp affecting exons 2 and 3 (Fig. 4 B). Four extra nucleotides (TCTG) in the 3′ sequence adjacent to the deficiency changed the ORF. Consequently, FoxK16 encoded for a chimeric polypeptide that shared the first 26 amino acids with FoxK, but the predicted new frame eliminated both the FH and FHA domains and introduced 66 new amino acids (Fig. 4 B).


FoxK mediates TGF-beta signalling during midgut differentiation in flies.

Casas-Tinto S, Gomez-Velazquez M, Granadino B, Fernandez-Funez P - J. Cell Biol. (2008)

Molecular characterization of FoxK mutant alleles. (A) The P element EP(3)3428 is inserted in 676 bp 5′ of the ATG (0) of FoxK. Red arrowheads indicate the primers used for sequencing exons 2–5. (B) Both FoxK44 and FoxK16 carry a deletion of 2 bp at the insertion site of EP(3)3428 (−676ΔTA). FoxK44 flies also contain a reinsertion of a fragment of the P element in exon 3 (green) that generates a premature Stop codon. In FoxK16, a deletion in exon 2 generates a new ORF (purple) containing a Stop codon. (C) Southern blot hybridized with a probe covering the entire FoxK coding region shows an extra band of 2.6 Kb in FoxK44 and FoxK16 (arrow). (D and E) Stage 15 FoxK16 homozygous embryos do not stain with anti-FoxK (D, arrow), but the ventral nerve cord stains with anti-Elav and shows normal morphology (E, arrow).
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Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2600746&req=5

fig4: Molecular characterization of FoxK mutant alleles. (A) The P element EP(3)3428 is inserted in 676 bp 5′ of the ATG (0) of FoxK. Red arrowheads indicate the primers used for sequencing exons 2–5. (B) Both FoxK44 and FoxK16 carry a deletion of 2 bp at the insertion site of EP(3)3428 (−676ΔTA). FoxK44 flies also contain a reinsertion of a fragment of the P element in exon 3 (green) that generates a premature Stop codon. In FoxK16, a deletion in exon 2 generates a new ORF (purple) containing a Stop codon. (C) Southern blot hybridized with a probe covering the entire FoxK coding region shows an extra band of 2.6 Kb in FoxK44 and FoxK16 (arrow). (D and E) Stage 15 FoxK16 homozygous embryos do not stain with anti-FoxK (D, arrow), but the ventral nerve cord stains with anti-Elav and shows normal morphology (E, arrow).
Mentions: To elucidate the function of FoxK in Drosophila, we generated FoxK loss-of-function alleles by imprecise excision of a P element inserted 676 bp upstream of the ATG for FoxK (Fig. 4 A). We recovered two FoxK mutant alleles that resulted in recessive lethal chromosomes. To ensure that the lethality of the FoxK alleles was contained in the FoxK region, we confirmed that a chromosomal duplication of FoxK recovered the viability of FoxK16 and FoxK44 homozygous flies. To molecularly characterize these new FoxK alleles, we analyzed genomic DNA from FoxK16 and FoxK44 flies by Southern blot with a probe covering the entire FoxK coding region. DNA samples from FoxK16 and FoxK44 heterozygous flies showed an unexpected band suggestive of a chromosomal aberration within FoxK (Fig. 4 C, arrow). To delimitate the affected region, we sequenced the central region of FoxK using specific primers for exons 3–5 (Fig. 4 A, red arrowheads). We confirmed that FoxK44 contains a partial reinsertion of the P element in exon 3, creating a Stop codon 28 nucleotides after the insertion (Fig. 4 B). The truncated protein produced by FoxK44 retained the FHA domain, but lacked the FH domain. Next, to identify the molecular changes associated with FoxK16, we sequenced exons 2–5 and identified a deficiency of 962 bp affecting exons 2 and 3 (Fig. 4 B). Four extra nucleotides (TCTG) in the 3′ sequence adjacent to the deficiency changed the ORF. Consequently, FoxK16 encoded for a chimeric polypeptide that shared the first 26 amino acids with FoxK, but the predicted new frame eliminated both the FH and FHA domains and introduced 66 new amino acids (Fig. 4 B).

Bottom Line: Genet.This regulatory activity does not require direct labial activation by the TGF-beta effector Mad.Thus, we propose that the combined activity of the TGF-beta target genes FoxK and Dfos is critical for the direct activation of lab in the endoderm.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, University of Texas Medical Branch, Galveston, TX 77555, USA. scasas@cnio.es

ABSTRACT
Inductive signals across germ layers are important for the development of the endoderm in vertebrates and invertebrates (Tam, P.P., M. Kanai-Azuma, and Y. Kanai. 2003. Curr. Opin. Genet. Dev. 13:393-400; Nakagoshi, H. 2005. Dev. Growth Differ. 47:383-392). In flies, the visceral mesoderm secretes signaling molecules that diffuse into the underlying midgut endoderm, where conserved signaling cascades activate the Hox gene labial, which is important for the differentiation of copper cells (Bienz, M. 1997. Curr. Opin. Genet. Dev. 7:683-688). We present here a Drosophila melanogaster gene of the Fox family of transcription factors, FoxK, that mediates transforming growth factor beta (TGF-beta) signaling in the embryonic midgut endoderm. FoxK mutant embryos fail to generate midgut constrictions and lack Labial in the endoderm. Our observations suggest that TGF-beta signaling directly regulates FoxK through functional Smad/Mad-binding sites, whereas FoxK, in turn, regulates labial expression. We also describe a new cooperative activity of the transcription factors FoxK and Dfos/AP-1 that regulates labial expression in the midgut endoderm. This regulatory activity does not require direct labial activation by the TGF-beta effector Mad. Thus, we propose that the combined activity of the TGF-beta target genes FoxK and Dfos is critical for the direct activation of lab in the endoderm.

Show MeSH
Related in: MedlinePlus