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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 regulates Lab expression in midgut endoderm. (A and B) Lab (red; arrowhead) and FoxK (green; arrow) partially colocalize in midgut endoderm in a wild-type embryo. (C) Lab does not accumulate in the endoderm in FoxK16 homozygous embryos (arrowhead). (D) GFP accumulates in the endoderm under the control of 48Y-Gal4 (arrow). (E and F) Silencing of FoxK transcripts in the endoderm with an RNA interference construct (48Y-Gal4/UAS-FoxKi) also eliminates Lab expression (E, arrowhead). (G–I) Overexpression of FoxK in the endoderm (arrow) does not induce ectopic Lab accumulation (red; arrowhead). Anterior is always to the left. (J) The lab regulatory region contains multiple consensus FH-binding sites (open circles), five verified FoxK-binding sites (black circles), a cluster of Smad/Mad-binding sites (diamonds), and Dfos/AP1-binding sites (open squares). The lab550 regulatory element and a 678-bp element containing five FH-binding sites are indicated. The coordinates with respect to lab ATG are shown in red. (K) EMSA performed with an oligonucleotide containing the ATAAATA sequence and GST-FoxK[414–654]. FoxK strongly and specifically binds to this sequence (arrow) as indicated by the effective competition of the cold probe. (L) Transactivation assays in cell extracts expressing FoxK-L and FoxK-S show that a single copy of the lab678 element robustly responds to FoxK in vitro. The error bars correspond to the standard deviation of three independent experiments. This experiment was conducted as described in Fig. 2.
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fig6: FoxK regulates Lab expression in midgut endoderm. (A and B) Lab (red; arrowhead) and FoxK (green; arrow) partially colocalize in midgut endoderm in a wild-type embryo. (C) Lab does not accumulate in the endoderm in FoxK16 homozygous embryos (arrowhead). (D) GFP accumulates in the endoderm under the control of 48Y-Gal4 (arrow). (E and F) Silencing of FoxK transcripts in the endoderm with an RNA interference construct (48Y-Gal4/UAS-FoxKi) also eliminates Lab expression (E, arrowhead). (G–I) Overexpression of FoxK in the endoderm (arrow) does not induce ectopic Lab accumulation (red; arrowhead). Anterior is always to the left. (J) The lab regulatory region contains multiple consensus FH-binding sites (open circles), five verified FoxK-binding sites (black circles), a cluster of Smad/Mad-binding sites (diamonds), and Dfos/AP1-binding sites (open squares). The lab550 regulatory element and a 678-bp element containing five FH-binding sites are indicated. The coordinates with respect to lab ATG are shown in red. (K) EMSA performed with an oligonucleotide containing the ATAAATA sequence and GST-FoxK[414–654]. FoxK strongly and specifically binds to this sequence (arrow) as indicated by the effective competition of the cold probe. (L) Transactivation assays in cell extracts expressing FoxK-L and FoxK-S show that a single copy of the lab678 element robustly responds to FoxK in vitro. The error bars correspond to the standard deviation of three independent experiments. This experiment was conducted as described in Fig. 2.

Mentions: Previous studies demonstrated the importance of lab in midgut endoderm: lab is expressed in the endoderm under the control of Dpp signaling and is required for copper cell identity and function (Immergluck et al., 1990; Panganiban et al., 1990; Reuter et al., 1990). The distribution of Lab in the midgut endoderm overlaps with FoxK in parasegment 7 (Fig. 6, A and B), suggesting a potential functional relationship between these two proteins. We found that FoxK mutant embryos lacked Lab in the endoderm (Fig. 6 C), suggesting that lab expression depends on FoxK activity in the midgut endoderm. To confirm this result, we specifically eliminated FoxK activity in the endoderm by expressing the FoxKi silencing construct. These embryos also exhibited incomplete midgut development and loss of Lab expression (Fig. 6, E and F). These results confirmed that FoxK activity is essential for lab expression in the endoderm. Next, we examined whether FoxK overexpression in the endoderm could induce ectopic Lab accumulation; however, Lab expression was normal in these embryos (Fig. 6, G–I). These observations argue that FoxK is required, but not sufficient, to specifically activate lab in the endoderm. Moreover, we found no changes in Tsh expression in embryos carrying FoxK mutant alleles or FoxK overexpression (unpublished data).


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 regulates Lab expression in midgut endoderm. (A and B) Lab (red; arrowhead) and FoxK (green; arrow) partially colocalize in midgut endoderm in a wild-type embryo. (C) Lab does not accumulate in the endoderm in FoxK16 homozygous embryos (arrowhead). (D) GFP accumulates in the endoderm under the control of 48Y-Gal4 (arrow). (E and F) Silencing of FoxK transcripts in the endoderm with an RNA interference construct (48Y-Gal4/UAS-FoxKi) also eliminates Lab expression (E, arrowhead). (G–I) Overexpression of FoxK in the endoderm (arrow) does not induce ectopic Lab accumulation (red; arrowhead). Anterior is always to the left. (J) The lab regulatory region contains multiple consensus FH-binding sites (open circles), five verified FoxK-binding sites (black circles), a cluster of Smad/Mad-binding sites (diamonds), and Dfos/AP1-binding sites (open squares). The lab550 regulatory element and a 678-bp element containing five FH-binding sites are indicated. The coordinates with respect to lab ATG are shown in red. (K) EMSA performed with an oligonucleotide containing the ATAAATA sequence and GST-FoxK[414–654]. FoxK strongly and specifically binds to this sequence (arrow) as indicated by the effective competition of the cold probe. (L) Transactivation assays in cell extracts expressing FoxK-L and FoxK-S show that a single copy of the lab678 element robustly responds to FoxK in vitro. The error bars correspond to the standard deviation of three independent experiments. This experiment was conducted as described in Fig. 2.
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Related In: Results  -  Collection

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fig6: FoxK regulates Lab expression in midgut endoderm. (A and B) Lab (red; arrowhead) and FoxK (green; arrow) partially colocalize in midgut endoderm in a wild-type embryo. (C) Lab does not accumulate in the endoderm in FoxK16 homozygous embryos (arrowhead). (D) GFP accumulates in the endoderm under the control of 48Y-Gal4 (arrow). (E and F) Silencing of FoxK transcripts in the endoderm with an RNA interference construct (48Y-Gal4/UAS-FoxKi) also eliminates Lab expression (E, arrowhead). (G–I) Overexpression of FoxK in the endoderm (arrow) does not induce ectopic Lab accumulation (red; arrowhead). Anterior is always to the left. (J) The lab regulatory region contains multiple consensus FH-binding sites (open circles), five verified FoxK-binding sites (black circles), a cluster of Smad/Mad-binding sites (diamonds), and Dfos/AP1-binding sites (open squares). The lab550 regulatory element and a 678-bp element containing five FH-binding sites are indicated. The coordinates with respect to lab ATG are shown in red. (K) EMSA performed with an oligonucleotide containing the ATAAATA sequence and GST-FoxK[414–654]. FoxK strongly and specifically binds to this sequence (arrow) as indicated by the effective competition of the cold probe. (L) Transactivation assays in cell extracts expressing FoxK-L and FoxK-S show that a single copy of the lab678 element robustly responds to FoxK in vitro. The error bars correspond to the standard deviation of three independent experiments. This experiment was conducted as described in Fig. 2.
Mentions: Previous studies demonstrated the importance of lab in midgut endoderm: lab is expressed in the endoderm under the control of Dpp signaling and is required for copper cell identity and function (Immergluck et al., 1990; Panganiban et al., 1990; Reuter et al., 1990). The distribution of Lab in the midgut endoderm overlaps with FoxK in parasegment 7 (Fig. 6, A and B), suggesting a potential functional relationship between these two proteins. We found that FoxK mutant embryos lacked Lab in the endoderm (Fig. 6 C), suggesting that lab expression depends on FoxK activity in the midgut endoderm. To confirm this result, we specifically eliminated FoxK activity in the endoderm by expressing the FoxKi silencing construct. These embryos also exhibited incomplete midgut development and loss of Lab expression (Fig. 6, E and F). These results confirmed that FoxK activity is essential for lab expression in the endoderm. Next, we examined whether FoxK overexpression in the endoderm could induce ectopic Lab accumulation; however, Lab expression was normal in these embryos (Fig. 6, G–I). These observations argue that FoxK is required, but not sufficient, to specifically activate lab in the endoderm. Moreover, we found no changes in Tsh expression in embryos carrying FoxK mutant alleles or FoxK overexpression (unpublished data).

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