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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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FoxK and Dfos regulate Lab independently of Mad. FoxK (A, arrow) and Dfos (B, arrow) colocalize in the midgut endoderm of wild-type embryos (C, arrow). Homozygous FoxK16 embryos lack FoxK (D) but accumulate Dfos (E, arrow). (F) Dfos1/Sro mutant embryos maintain FoxK expression in the endoderm (arrow). Wild-type expression of FoxK (G, arrow), Lab (H, arrowhead), and pSmad (I, arrow) in the midgut endoderm in stage 15 embryos. A Z-axis projection of all the endoderm is shown. The arrowhead in I indicates pSmad staining in PS3. (J–L) FoxK and Dfos coexpression in the endoderm (48Y-Gal4) induces anterior expansion of Lab (K, arrow). The arrowhead indicates the normal position of Lab. Coexpression of FoxK and tkvDN (M, green) in the endoderm results in loss of Lab (N, arrowhead) and pSmad in PS7 (O, arrow). Coexpression of FoxK, Dfos, and tkvDN in the endoderm restores midgut constrictions (P, arrowhead) and Lab accumulation (Q, arrowhead), whereas pSmad expression is still missing in PS7 (R, arrow). Note that the expression of pSmad in PS3 is still present (R, arrowhead).
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fig8: FoxK and Dfos regulate Lab independently of Mad. FoxK (A, arrow) and Dfos (B, arrow) colocalize in the midgut endoderm of wild-type embryos (C, arrow). Homozygous FoxK16 embryos lack FoxK (D) but accumulate Dfos (E, arrow). (F) Dfos1/Sro mutant embryos maintain FoxK expression in the endoderm (arrow). Wild-type expression of FoxK (G, arrow), Lab (H, arrowhead), and pSmad (I, arrow) in the midgut endoderm in stage 15 embryos. A Z-axis projection of all the endoderm is shown. The arrowhead in I indicates pSmad staining in PS3. (J–L) FoxK and Dfos coexpression in the endoderm (48Y-Gal4) induces anterior expansion of Lab (K, arrow). The arrowhead indicates the normal position of Lab. Coexpression of FoxK and tkvDN (M, green) in the endoderm results in loss of Lab (N, arrowhead) and pSmad in PS7 (O, arrow). Coexpression of FoxK, Dfos, and tkvDN in the endoderm restores midgut constrictions (P, arrowhead) and Lab accumulation (Q, arrowhead), whereas pSmad expression is still missing in PS7 (R, arrow). Note that the expression of pSmad in PS3 is still present (R, arrowhead).

Mentions: FoxK and Dfos are two transcription factors that (a) are regulated by Dpp, (b) colocalize in the midgut endoderm (Fig. 7, A–C), (c) are required for lab expression and endoderm differentiation (Fig. 5 L; Riese et al., 1997), and (d) contain functional binding sites in the lab regulatory region (Szuts and Bienz, 2000; this study). Still, neither FoxK nor Dfos induce ectopic accumulation of Lab when overexpressed in the endoderm (Fig. 6 N; Riese et al., 1997). To better understand how FoxK and Dfos work in the endoderm, we first studied the possible cross-regulation between these two transcription factors. We found no changes in Dfos expression in flies mutant for FoxK or in flies overexpressing FoxK in the endoderm (Fig. 8, D and E, only FoxK loss-of-function is shown). Similarly, we found no changes in FoxK expression in embryos mutant for Dfos or in flies overexpressing Dfos in the endoderm (Fig. 8 F, only Dfos loss-of-function is shown). In all, these experiments ruled out mutual regulation between FoxK and Dfos. We next investigated the potential functional interaction of FoxK and Dfos by coexpressing both transcription factors in the endoderm. Remarkably, FoxK/Dfos coexpression induced the anterior expansion of the Lab domain (Fig. 8, compare J–L with G and H). Because FoxK and Dfos can drive ectopic lab expression when coexpressed, but not separately, these transcription factors may function cooperatively to regulate lab in the 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)

FoxK and Dfos regulate Lab independently of Mad. FoxK (A, arrow) and Dfos (B, arrow) colocalize in the midgut endoderm of wild-type embryos (C, arrow). Homozygous FoxK16 embryos lack FoxK (D) but accumulate Dfos (E, arrow). (F) Dfos1/Sro mutant embryos maintain FoxK expression in the endoderm (arrow). Wild-type expression of FoxK (G, arrow), Lab (H, arrowhead), and pSmad (I, arrow) in the midgut endoderm in stage 15 embryos. A Z-axis projection of all the endoderm is shown. The arrowhead in I indicates pSmad staining in PS3. (J–L) FoxK and Dfos coexpression in the endoderm (48Y-Gal4) induces anterior expansion of Lab (K, arrow). The arrowhead indicates the normal position of Lab. Coexpression of FoxK and tkvDN (M, green) in the endoderm results in loss of Lab (N, arrowhead) and pSmad in PS7 (O, arrow). Coexpression of FoxK, Dfos, and tkvDN in the endoderm restores midgut constrictions (P, arrowhead) and Lab accumulation (Q, arrowhead), whereas pSmad expression is still missing in PS7 (R, arrow). Note that the expression of pSmad in PS3 is still present (R, arrowhead).
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fig8: FoxK and Dfos regulate Lab independently of Mad. FoxK (A, arrow) and Dfos (B, arrow) colocalize in the midgut endoderm of wild-type embryos (C, arrow). Homozygous FoxK16 embryos lack FoxK (D) but accumulate Dfos (E, arrow). (F) Dfos1/Sro mutant embryos maintain FoxK expression in the endoderm (arrow). Wild-type expression of FoxK (G, arrow), Lab (H, arrowhead), and pSmad (I, arrow) in the midgut endoderm in stage 15 embryos. A Z-axis projection of all the endoderm is shown. The arrowhead in I indicates pSmad staining in PS3. (J–L) FoxK and Dfos coexpression in the endoderm (48Y-Gal4) induces anterior expansion of Lab (K, arrow). The arrowhead indicates the normal position of Lab. Coexpression of FoxK and tkvDN (M, green) in the endoderm results in loss of Lab (N, arrowhead) and pSmad in PS7 (O, arrow). Coexpression of FoxK, Dfos, and tkvDN in the endoderm restores midgut constrictions (P, arrowhead) and Lab accumulation (Q, arrowhead), whereas pSmad expression is still missing in PS7 (R, arrow). Note that the expression of pSmad in PS3 is still present (R, arrowhead).
Mentions: FoxK and Dfos are two transcription factors that (a) are regulated by Dpp, (b) colocalize in the midgut endoderm (Fig. 7, A–C), (c) are required for lab expression and endoderm differentiation (Fig. 5 L; Riese et al., 1997), and (d) contain functional binding sites in the lab regulatory region (Szuts and Bienz, 2000; this study). Still, neither FoxK nor Dfos induce ectopic accumulation of Lab when overexpressed in the endoderm (Fig. 6 N; Riese et al., 1997). To better understand how FoxK and Dfos work in the endoderm, we first studied the possible cross-regulation between these two transcription factors. We found no changes in Dfos expression in flies mutant for FoxK or in flies overexpressing FoxK in the endoderm (Fig. 8, D and E, only FoxK loss-of-function is shown). Similarly, we found no changes in FoxK expression in embryos mutant for Dfos or in flies overexpressing Dfos in the endoderm (Fig. 8 F, only Dfos loss-of-function is shown). In all, these experiments ruled out mutual regulation between FoxK and Dfos. We next investigated the potential functional interaction of FoxK and Dfos by coexpressing both transcription factors in the endoderm. Remarkably, FoxK/Dfos coexpression induced the anterior expansion of the Lab domain (Fig. 8, compare J–L with G and H). Because FoxK and Dfos can drive ectopic lab expression when coexpressed, but not separately, these transcription factors may function cooperatively to regulate lab in the 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