Limits...
Genome-wide analysis reveals a major role in cell fate maintenance and an unexpected role in endoreduplication for the Drosophila FoxA gene Fork head.

Maruyama R, Grevengoed E, Stempniewicz P, Andrew DJ - PLoS ONE (2011)

Bottom Line: Transcription factors drive organogenesis, from the initiation of cell fate decisions to the maintenance and implementation of these decisions.Thus, unlike the worm FoxA protein PHA-4, Fkh does not function to specify cell fate.Overall, this study demonstrates an important role for Fkh in determining how an organ preserves its identity throughout development and provides an alternative paradigm for how FoxA proteins function in organogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, The Johns Hopkins University, School of Medicine, Baltimore, Maryland, United States of America.

ABSTRACT
Transcription factors drive organogenesis, from the initiation of cell fate decisions to the maintenance and implementation of these decisions. The Drosophila embryonic salivary gland provides an excellent platform for unraveling the underlying transcriptional networks of organ development because Drosophila is relatively unencumbered by significant genetic redundancy. The highly conserved FoxA family transcription factors are essential for various aspects of organogenesis in all animals that have been studied. Here, we explore the role of the single Drosophila FoxA protein Fork head (Fkh) in salivary gland organogenesis using two genome-wide strategies. A large-scale in situ hybridization analysis reveals a major role for Fkh in maintaining the salivary gland fate decision and controlling salivary gland physiological activity, in addition to its previously known roles in morphogenesis and survival. The majority of salivary gland genes (59%) are affected by fkh loss, mainly at later stages of salivary gland development. We show that global expression of Fkh cannot drive ectopic salivary gland formation. Thus, unlike the worm FoxA protein PHA-4, Fkh does not function to specify cell fate. In addition, Fkh only indirectly regulates many salivary gland genes, which is also distinct from the role of PHA-4 in organogenesis. Our microarray analyses reveal unexpected roles for Fkh in blocking terminal differentiation and in endoreduplication in the salivary gland and in other Fkh-expressing embryonic tissues. Overall, this study demonstrates an important role for Fkh in determining how an organ preserves its identity throughout development and provides an alternative paradigm for how FoxA proteins function in organogenesis.

Show MeSH

Related in: MedlinePlus

Microarray analysis suggests a role for Fkh in terminal differentiation and endoreplication.(A) Genes identified as both downregulated and upregulated by microarray analysis show the expected expression changes in WT versus fkh mutant embryos when examined by whole mount in situ hybridization. CG11275, CG7637 and dro5 all had notably reduced expression in fkh mutant embryos, whereas rpr expression was notably higher in fkh mutants. The numbers indicate the fold-change of each gene. Red arrowheads: SGs. (B) Volcano plot shows genes whose expression is significantly downregulated (blue filled circles) and upregulated (red filled circles) in fkh mutant embryos. Genes whose expression changed but with P-values greater than 0.05 are shown with open circles. Highlighted in green are examples of upregulated genes in fkh mutants. Chitin related: Cht3; cell junction and synapse: Syt4, Fas3, cora, and vari; cuticle: dy, Lcp65Ag3 and Cpr49Ac, ECM: Cg25C, vkg, mmy, and Mmp2; muscle: mbl, Calcium: CalpA and TpnC41C. (C) The same volcano plot is shown in B with downregulated genes involved in chromosome metabolism and cell cycle progression highlighted in green.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3116861&req=5

pone-0020901-g006: Microarray analysis suggests a role for Fkh in terminal differentiation and endoreplication.(A) Genes identified as both downregulated and upregulated by microarray analysis show the expected expression changes in WT versus fkh mutant embryos when examined by whole mount in situ hybridization. CG11275, CG7637 and dro5 all had notably reduced expression in fkh mutant embryos, whereas rpr expression was notably higher in fkh mutants. The numbers indicate the fold-change of each gene. Red arrowheads: SGs. (B) Volcano plot shows genes whose expression is significantly downregulated (blue filled circles) and upregulated (red filled circles) in fkh mutant embryos. Genes whose expression changed but with P-values greater than 0.05 are shown with open circles. Highlighted in green are examples of upregulated genes in fkh mutants. Chitin related: Cht3; cell junction and synapse: Syt4, Fas3, cora, and vari; cuticle: dy, Lcp65Ag3 and Cpr49Ac, ECM: Cg25C, vkg, mmy, and Mmp2; muscle: mbl, Calcium: CalpA and TpnC41C. (C) The same volcano plot is shown in B with downregulated genes involved in chromosome metabolism and cell cycle progression highlighted in green.

Mentions: Our in situ hybridization analysis identified a large number of Fkh-dependent SG genes; this analysis will have missed many Fkh targets, however, since the BDGP expression datasets are incomplete (only about 1/3 of the genome is represented so far) and because the in situ hybridization analysis cannot identify genes that might normally be repressed in the early SG by Fkh. Thus, to identify additional fkh downstream target genes, we performed a microarray analysis comparing the expression profiles of WT and fkh stage 11 embryos. From the microarrays, we discovered 1102 down-regulated genes (with a fold change <−1.4, P<0.05) and 1087 up-regulated genes (with a fold change >1.4, P<0.05) in fkh mutants compared to WT (Table S2). To validate the microarray data, we selected a set of both down-regulated and up-regulated genes from the microarray data and performed in situ hybridization analysis of these genes in WT and fkh mutant embryos. Five of the seven down-regulated genes we tested had notably reduced expression and five of the nine upregulated genes we tested had notably higher expression in fkh mutant embryos (examples are shown in Figure 6A). These findings indicate that the microarray approach can identify new Fkh dependent genes in the SG and many additional cell types in which this transcription factor is expressed.


Genome-wide analysis reveals a major role in cell fate maintenance and an unexpected role in endoreduplication for the Drosophila FoxA gene Fork head.

Maruyama R, Grevengoed E, Stempniewicz P, Andrew DJ - PLoS ONE (2011)

Microarray analysis suggests a role for Fkh in terminal differentiation and endoreplication.(A) Genes identified as both downregulated and upregulated by microarray analysis show the expected expression changes in WT versus fkh mutant embryos when examined by whole mount in situ hybridization. CG11275, CG7637 and dro5 all had notably reduced expression in fkh mutant embryos, whereas rpr expression was notably higher in fkh mutants. The numbers indicate the fold-change of each gene. Red arrowheads: SGs. (B) Volcano plot shows genes whose expression is significantly downregulated (blue filled circles) and upregulated (red filled circles) in fkh mutant embryos. Genes whose expression changed but with P-values greater than 0.05 are shown with open circles. Highlighted in green are examples of upregulated genes in fkh mutants. Chitin related: Cht3; cell junction and synapse: Syt4, Fas3, cora, and vari; cuticle: dy, Lcp65Ag3 and Cpr49Ac, ECM: Cg25C, vkg, mmy, and Mmp2; muscle: mbl, Calcium: CalpA and TpnC41C. (C) The same volcano plot is shown in B with downregulated genes involved in chromosome metabolism and cell cycle progression highlighted in green.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3116861&req=5

pone-0020901-g006: Microarray analysis suggests a role for Fkh in terminal differentiation and endoreplication.(A) Genes identified as both downregulated and upregulated by microarray analysis show the expected expression changes in WT versus fkh mutant embryos when examined by whole mount in situ hybridization. CG11275, CG7637 and dro5 all had notably reduced expression in fkh mutant embryos, whereas rpr expression was notably higher in fkh mutants. The numbers indicate the fold-change of each gene. Red arrowheads: SGs. (B) Volcano plot shows genes whose expression is significantly downregulated (blue filled circles) and upregulated (red filled circles) in fkh mutant embryos. Genes whose expression changed but with P-values greater than 0.05 are shown with open circles. Highlighted in green are examples of upregulated genes in fkh mutants. Chitin related: Cht3; cell junction and synapse: Syt4, Fas3, cora, and vari; cuticle: dy, Lcp65Ag3 and Cpr49Ac, ECM: Cg25C, vkg, mmy, and Mmp2; muscle: mbl, Calcium: CalpA and TpnC41C. (C) The same volcano plot is shown in B with downregulated genes involved in chromosome metabolism and cell cycle progression highlighted in green.
Mentions: Our in situ hybridization analysis identified a large number of Fkh-dependent SG genes; this analysis will have missed many Fkh targets, however, since the BDGP expression datasets are incomplete (only about 1/3 of the genome is represented so far) and because the in situ hybridization analysis cannot identify genes that might normally be repressed in the early SG by Fkh. Thus, to identify additional fkh downstream target genes, we performed a microarray analysis comparing the expression profiles of WT and fkh stage 11 embryos. From the microarrays, we discovered 1102 down-regulated genes (with a fold change <−1.4, P<0.05) and 1087 up-regulated genes (with a fold change >1.4, P<0.05) in fkh mutants compared to WT (Table S2). To validate the microarray data, we selected a set of both down-regulated and up-regulated genes from the microarray data and performed in situ hybridization analysis of these genes in WT and fkh mutant embryos. Five of the seven down-regulated genes we tested had notably reduced expression and five of the nine upregulated genes we tested had notably higher expression in fkh mutant embryos (examples are shown in Figure 6A). These findings indicate that the microarray approach can identify new Fkh dependent genes in the SG and many additional cell types in which this transcription factor is expressed.

Bottom Line: Transcription factors drive organogenesis, from the initiation of cell fate decisions to the maintenance and implementation of these decisions.Thus, unlike the worm FoxA protein PHA-4, Fkh does not function to specify cell fate.Overall, this study demonstrates an important role for Fkh in determining how an organ preserves its identity throughout development and provides an alternative paradigm for how FoxA proteins function in organogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, The Johns Hopkins University, School of Medicine, Baltimore, Maryland, United States of America.

ABSTRACT
Transcription factors drive organogenesis, from the initiation of cell fate decisions to the maintenance and implementation of these decisions. The Drosophila embryonic salivary gland provides an excellent platform for unraveling the underlying transcriptional networks of organ development because Drosophila is relatively unencumbered by significant genetic redundancy. The highly conserved FoxA family transcription factors are essential for various aspects of organogenesis in all animals that have been studied. Here, we explore the role of the single Drosophila FoxA protein Fork head (Fkh) in salivary gland organogenesis using two genome-wide strategies. A large-scale in situ hybridization analysis reveals a major role for Fkh in maintaining the salivary gland fate decision and controlling salivary gland physiological activity, in addition to its previously known roles in morphogenesis and survival. The majority of salivary gland genes (59%) are affected by fkh loss, mainly at later stages of salivary gland development. We show that global expression of Fkh cannot drive ectopic salivary gland formation. Thus, unlike the worm FoxA protein PHA-4, Fkh does not function to specify cell fate. In addition, Fkh only indirectly regulates many salivary gland genes, which is also distinct from the role of PHA-4 in organogenesis. Our microarray analyses reveal unexpected roles for Fkh in blocking terminal differentiation and in endoreduplication in the salivary gland and in other Fkh-expressing embryonic tissues. Overall, this study demonstrates an important role for Fkh in determining how an organ preserves its identity throughout development and provides an alternative paradigm for how FoxA proteins function in organogenesis.

Show MeSH
Related in: MedlinePlus