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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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Dpp directly regulates FoxK in the endoderm. (A) Transheterozygous combinations of dpp+/− and FoxK+/− mutant alleles result in lethality. (B and C) A control embryo (stage 15) shows normal Lab (arrowhead) and FoxK (green; arrows) accumulation in the endoderm. (D–F) dpp overexpression in the visceral mesoderm (24B-Gal4) induces ectopic Lab (arrowhead) and FoxK (arrow) in the endoderm. Merged panel is shown in F. (G–I) Expression of tkvDN in the endoderm (48Y-Gal4) eliminates both Lab (arrowhead) and FoxK (arrow) in the endoderm. Merged panel is shown in I. (J–L) Homozygous FoxK16 embryos that also overexpress dpp in the visceral mesoderm lack Lab expression in the endoderm (arrowhead). K shows negative FoxK staining and the merged image is in L. (M) EMSA performed with a genomic-derived probe (Oligo-Mad) containing multiple Mad-binding sites (right) and protein extracts from S2 cells transfected with combinations of Mad, Med, and tkvact constructs. The cellular extracts from nontransfected cells (S2) or cells transfected with Mad and Med constructs resulted in a small shift of the Oligo-Mad (arrow). Extracts expressing tkvact produced a stronger binding, but extracts expressing all three constructs resulted in the strongest binding to the Oligo-Mad probe. The lanes with no cell extract (−) and the use of an unspecific probe (GAS) produced no shift. The free oligonucleotides and oligonucleotide complexes are indicated by the arrowhead. (N) The 24-B-Gal4 strain induces GFP expression in the mesoderm (arrow).
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fig7: Dpp directly regulates FoxK in the endoderm. (A) Transheterozygous combinations of dpp+/− and FoxK+/− mutant alleles result in lethality. (B and C) A control embryo (stage 15) shows normal Lab (arrowhead) and FoxK (green; arrows) accumulation in the endoderm. (D–F) dpp overexpression in the visceral mesoderm (24B-Gal4) induces ectopic Lab (arrowhead) and FoxK (arrow) in the endoderm. Merged panel is shown in F. (G–I) Expression of tkvDN in the endoderm (48Y-Gal4) eliminates both Lab (arrowhead) and FoxK (arrow) in the endoderm. Merged panel is shown in I. (J–L) Homozygous FoxK16 embryos that also overexpress dpp in the visceral mesoderm lack Lab expression in the endoderm (arrowhead). K shows negative FoxK staining and the merged image is in L. (M) EMSA performed with a genomic-derived probe (Oligo-Mad) containing multiple Mad-binding sites (right) and protein extracts from S2 cells transfected with combinations of Mad, Med, and tkvact constructs. The cellular extracts from nontransfected cells (S2) or cells transfected with Mad and Med constructs resulted in a small shift of the Oligo-Mad (arrow). Extracts expressing tkvact produced a stronger binding, but extracts expressing all three constructs resulted in the strongest binding to the Oligo-Mad probe. The lanes with no cell extract (−) and the use of an unspecific probe (GAS) produced no shift. The free oligonucleotides and oligonucleotide complexes are indicated by the arrowhead. (N) The 24-B-Gal4 strain induces GFP expression in the mesoderm (arrow).

Mentions: Because both FoxK and Dpp regulate lab in the midgut and their loss-of-function leads to midgut developmental arrest, we investigated the functional interaction between dpp and FoxK. First, we generated double heterozygous combinations dpp+/−; FoxK+/− and found that the combinations with strong dpp alleles resulted in synthetic lethality, supporting the functional interaction between dpp and FoxK (Fig. 7 A). Next, we asked whether FoxK functioned under the control of the Dpp signaling cascade in midgut endoderm. As shown previously (Staehling-Hampton and Hoffmann, 1994), ectopic expression of dpp in the visceral mesoderm leads to ectopic Lab accumulation in the endoderm (Fig. 7, B and D) and also resulted in increased levels of FoxK in the endoderm (Fig. 7, C and E). Conversely, embryos overexpressing a dominant-negative form of the Dpp type I receptor thickveins (tkvDN) in the endoderm showed low levels of both Lab and FoxK in the endoderm (Fig. 7, H and I). Collectively, these observations suggested that Dpp activity in the visceral mesoderm regulates FoxK expression in the adjacent midgut endoderm.


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)

Dpp directly regulates FoxK in the endoderm. (A) Transheterozygous combinations of dpp+/− and FoxK+/− mutant alleles result in lethality. (B and C) A control embryo (stage 15) shows normal Lab (arrowhead) and FoxK (green; arrows) accumulation in the endoderm. (D–F) dpp overexpression in the visceral mesoderm (24B-Gal4) induces ectopic Lab (arrowhead) and FoxK (arrow) in the endoderm. Merged panel is shown in F. (G–I) Expression of tkvDN in the endoderm (48Y-Gal4) eliminates both Lab (arrowhead) and FoxK (arrow) in the endoderm. Merged panel is shown in I. (J–L) Homozygous FoxK16 embryos that also overexpress dpp in the visceral mesoderm lack Lab expression in the endoderm (arrowhead). K shows negative FoxK staining and the merged image is in L. (M) EMSA performed with a genomic-derived probe (Oligo-Mad) containing multiple Mad-binding sites (right) and protein extracts from S2 cells transfected with combinations of Mad, Med, and tkvact constructs. The cellular extracts from nontransfected cells (S2) or cells transfected with Mad and Med constructs resulted in a small shift of the Oligo-Mad (arrow). Extracts expressing tkvact produced a stronger binding, but extracts expressing all three constructs resulted in the strongest binding to the Oligo-Mad probe. The lanes with no cell extract (−) and the use of an unspecific probe (GAS) produced no shift. The free oligonucleotides and oligonucleotide complexes are indicated by the arrowhead. (N) The 24-B-Gal4 strain induces GFP expression in the mesoderm (arrow).
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Related In: Results  -  Collection

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fig7: Dpp directly regulates FoxK in the endoderm. (A) Transheterozygous combinations of dpp+/− and FoxK+/− mutant alleles result in lethality. (B and C) A control embryo (stage 15) shows normal Lab (arrowhead) and FoxK (green; arrows) accumulation in the endoderm. (D–F) dpp overexpression in the visceral mesoderm (24B-Gal4) induces ectopic Lab (arrowhead) and FoxK (arrow) in the endoderm. Merged panel is shown in F. (G–I) Expression of tkvDN in the endoderm (48Y-Gal4) eliminates both Lab (arrowhead) and FoxK (arrow) in the endoderm. Merged panel is shown in I. (J–L) Homozygous FoxK16 embryos that also overexpress dpp in the visceral mesoderm lack Lab expression in the endoderm (arrowhead). K shows negative FoxK staining and the merged image is in L. (M) EMSA performed with a genomic-derived probe (Oligo-Mad) containing multiple Mad-binding sites (right) and protein extracts from S2 cells transfected with combinations of Mad, Med, and tkvact constructs. The cellular extracts from nontransfected cells (S2) or cells transfected with Mad and Med constructs resulted in a small shift of the Oligo-Mad (arrow). Extracts expressing tkvact produced a stronger binding, but extracts expressing all three constructs resulted in the strongest binding to the Oligo-Mad probe. The lanes with no cell extract (−) and the use of an unspecific probe (GAS) produced no shift. The free oligonucleotides and oligonucleotide complexes are indicated by the arrowhead. (N) The 24-B-Gal4 strain induces GFP expression in the mesoderm (arrow).
Mentions: Because both FoxK and Dpp regulate lab in the midgut and their loss-of-function leads to midgut developmental arrest, we investigated the functional interaction between dpp and FoxK. First, we generated double heterozygous combinations dpp+/−; FoxK+/− and found that the combinations with strong dpp alleles resulted in synthetic lethality, supporting the functional interaction between dpp and FoxK (Fig. 7 A). Next, we asked whether FoxK functioned under the control of the Dpp signaling cascade in midgut endoderm. As shown previously (Staehling-Hampton and Hoffmann, 1994), ectopic expression of dpp in the visceral mesoderm leads to ectopic Lab accumulation in the endoderm (Fig. 7, B and D) and also resulted in increased levels of FoxK in the endoderm (Fig. 7, C and E). Conversely, embryos overexpressing a dominant-negative form of the Dpp type I receptor thickveins (tkvDN) in the endoderm showed low levels of both Lab and FoxK in the endoderm (Fig. 7, H and I). Collectively, these observations suggested that Dpp activity in the visceral mesoderm regulates FoxK expression in the adjacent midgut endoderm.

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