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Madm (Mlf1 adapter molecule) cooperates with Bunched A to promote growth in Drosophila.

Gluderer S, Brunner E, Germann M, Jovaisaite V, Li C, Rentsch CA, Hafen E, Stocker H - J. Biol. (2010)

Bottom Line: In order to test for functional conservation among TSC22DF members, we expressed the human TSC22DF proteins in the fly and found that all long isoforms can replace BunA function.The growth-promoting potential of long TSC22DF proteins is evolutionarily conserved.Furthermore, we provide biochemical and genetic evidence for a growth-regulating complex involving the long TSC22DF protein BunA and the adapter molecule Madm.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Molecular Systems Biology, ETH Zurich, Wolfgang-Pauli-Strasse 16, 8093 Zurich, Switzerland.

ABSTRACT

Background: The TSC-22 domain family (TSC22DF) consists of putative transcription factors harboring a DNA-binding TSC-box and an adjacent leucine zipper at their carboxyl termini. Both short and long TSC22DF isoforms are conserved from flies to humans. Whereas the short isoforms include the tumor suppressor TSC-22 (Transforming growth factor-beta1 stimulated clone-22), the long isoforms are largely uncharacterized. In Drosophila, the long isoform Bunched A (BunA) acts as a growth promoter, but how BunA controls growth has remained obscure.

Results: In order to test for functional conservation among TSC22DF members, we expressed the human TSC22DF proteins in the fly and found that all long isoforms can replace BunA function. Furthermore, we combined a proteomics-based approach with a genetic screen to identify proteins that interact with BunA. Madm (Mlf1 adapter molecule) physically associates with BunA via a conserved motif that is only contained in long TSC22DF proteins. Moreover, Drosophila Madm acts as a growth-promoting gene that displays growth phenotypes strikingly similar to bunA phenotypes. When overexpressed, Madm and BunA synergize to increase organ growth.

Conclusions: The growth-promoting potential of long TSC22DF proteins is evolutionarily conserved. Furthermore, we provide biochemical and genetic evidence for a growth-regulating complex involving the long TSC22DF protein BunA and the adapter molecule Madm.

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The Madm loss- or reduction-of-function phenotypes strongly resemble bunA phenotypes. (a-c) Scanning electron micrographs of eyFLP/FRT mosaic eyes. (d) Madm mosaic heads (b, c) contain significantly fewer ommatidia than control mosaic heads (a) (n = 6). (a'-c') Images of tangential eye sections showing that Madm mutant (unpigmented) ommatidia (b', c') display an autonomous reduction in rhabdomere size relative to wild-type sized (pigmented) ommatidia. Furthermore, differentiation defects such as misrotation and missing photoreceptors are observed in Madm mutant ommatidia. Clones were induced 24-48 h after egg deposition using the hsFLP/FRT technique. (e) Rhabdomere size of Madm-mutant ommatidia is significantly reduced (by 29-56%). The area enclosed by rhabdomeres of photoreceptors R1-R6 in unpigmented mutant ommatidia was compared to the area measured in pigmented wild-type sized ommatidia. For each genotype, three pairs of ommatidia without differentiation defects from three different eye sections were measured (n = 9). Significant changes are marked by asterisks, **p < 0.01 and ***p < 0.001 (Student's t-test) in (d) and (e). (f) Heteroallelic combinations of the hypomorphic Madm allele 3T4 produce few viable small flies (<10% of the expected Mendelian ratio) that can be rescued by one copy of a genomic Madm rescue construct. (g) The dry weight of Madm hypomorphic females is reduced by 37% compared to control flies (Df/+). One copy of a genomic rescue construct restores normal weight. The genomic rescue construct has no significant dominant effect on dry weight ('rescue Df/+' females do not significantly differ from 'Df/+' females). n = 15, except for Df/3T4 (n = 9). (h) Tangential section of an eye from a Madm hypomorphic mutant female displaying rotation defects (yellow asterisk), missing rhabdomeres (green asterisk), and cell-fate transformations (red asterisk). (i) Wings of hypomorphic Madm males exhibiting wing notches and an incomplete wing vein V (arrows). Genotypes are: (a, a') y, w, eyFlp or hsFlp/y, w; FRT82B/FRT82B, w+, cl3R3 or M. (b, b', c, c') y, w, eyFlp or hsFlp/y, w; FRT82B, Madm7L2 or 3G5/FRT82B, w+, cl3R3 or M; (Df/+) y, w; FRT82B/Df(3R)Exel7283; (Df/3T4) y, w; FRT82B, Madm3T4/Df(3R)Exel7283; (rescue Df/3T4) y, w; gen.Madm(LCQ139)/+; FRT82B, Madm3T4/Df(3R)Exel7283; (rescue Df/+) y, w; gen.Madm(LCQ139)/+; FRT82B/Df(3R)Exel7283.
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Figure 4: The Madm loss- or reduction-of-function phenotypes strongly resemble bunA phenotypes. (a-c) Scanning electron micrographs of eyFLP/FRT mosaic eyes. (d) Madm mosaic heads (b, c) contain significantly fewer ommatidia than control mosaic heads (a) (n = 6). (a'-c') Images of tangential eye sections showing that Madm mutant (unpigmented) ommatidia (b', c') display an autonomous reduction in rhabdomere size relative to wild-type sized (pigmented) ommatidia. Furthermore, differentiation defects such as misrotation and missing photoreceptors are observed in Madm mutant ommatidia. Clones were induced 24-48 h after egg deposition using the hsFLP/FRT technique. (e) Rhabdomere size of Madm-mutant ommatidia is significantly reduced (by 29-56%). The area enclosed by rhabdomeres of photoreceptors R1-R6 in unpigmented mutant ommatidia was compared to the area measured in pigmented wild-type sized ommatidia. For each genotype, three pairs of ommatidia without differentiation defects from three different eye sections were measured (n = 9). Significant changes are marked by asterisks, **p < 0.01 and ***p < 0.001 (Student's t-test) in (d) and (e). (f) Heteroallelic combinations of the hypomorphic Madm allele 3T4 produce few viable small flies (<10% of the expected Mendelian ratio) that can be rescued by one copy of a genomic Madm rescue construct. (g) The dry weight of Madm hypomorphic females is reduced by 37% compared to control flies (Df/+). One copy of a genomic rescue construct restores normal weight. The genomic rescue construct has no significant dominant effect on dry weight ('rescue Df/+' females do not significantly differ from 'Df/+' females). n = 15, except for Df/3T4 (n = 9). (h) Tangential section of an eye from a Madm hypomorphic mutant female displaying rotation defects (yellow asterisk), missing rhabdomeres (green asterisk), and cell-fate transformations (red asterisk). (i) Wings of hypomorphic Madm males exhibiting wing notches and an incomplete wing vein V (arrows). Genotypes are: (a, a') y, w, eyFlp or hsFlp/y, w; FRT82B/FRT82B, w+, cl3R3 or M. (b, b', c, c') y, w, eyFlp or hsFlp/y, w; FRT82B, Madm7L2 or 3G5/FRT82B, w+, cl3R3 or M; (Df/+) y, w; FRT82B/Df(3R)Exel7283; (Df/3T4) y, w; FRT82B, Madm3T4/Df(3R)Exel7283; (rescue Df/3T4) y, w; gen.Madm(LCQ139)/+; FRT82B, Madm3T4/Df(3R)Exel7283; (rescue Df/+) y, w; gen.Madm(LCQ139)/+; FRT82B/Df(3R)Exel7283.

Mentions: We further characterized the Madm growth phenotype by testing effects on cell number and cell size. To assess cell number, ommatidia were counted in scanning electron microscope (SEM) pictures taken of mosaic eyes largely homozygous mutant for Madm. Compared to control mosaic eyes (Figure 4a), Madm mutant eyes (Figure 4b, c) had significantly fewer ommatidia (Figure 4d). To detect changes in cell size, we determined the size of rhabdomeres - the light-sensing organelles of the photoreceptors - in tangential eye sections containing homozy-gous mutant clones (Figure 4a'-c'). In addition, we measured the entire cell bodies of photoreceptor cells. Madm mutant rhabdomeres and photoreceptor cell bodies were smaller than the controls (by 29-56%; Figure 4e, and data not shown). The reduction was cell-autonomous because only homozygous mutant photoreceptor cells (marked by the absence of pigmentation) were affected.


Madm (Mlf1 adapter molecule) cooperates with Bunched A to promote growth in Drosophila.

Gluderer S, Brunner E, Germann M, Jovaisaite V, Li C, Rentsch CA, Hafen E, Stocker H - J. Biol. (2010)

The Madm loss- or reduction-of-function phenotypes strongly resemble bunA phenotypes. (a-c) Scanning electron micrographs of eyFLP/FRT mosaic eyes. (d) Madm mosaic heads (b, c) contain significantly fewer ommatidia than control mosaic heads (a) (n = 6). (a'-c') Images of tangential eye sections showing that Madm mutant (unpigmented) ommatidia (b', c') display an autonomous reduction in rhabdomere size relative to wild-type sized (pigmented) ommatidia. Furthermore, differentiation defects such as misrotation and missing photoreceptors are observed in Madm mutant ommatidia. Clones were induced 24-48 h after egg deposition using the hsFLP/FRT technique. (e) Rhabdomere size of Madm-mutant ommatidia is significantly reduced (by 29-56%). The area enclosed by rhabdomeres of photoreceptors R1-R6 in unpigmented mutant ommatidia was compared to the area measured in pigmented wild-type sized ommatidia. For each genotype, three pairs of ommatidia without differentiation defects from three different eye sections were measured (n = 9). Significant changes are marked by asterisks, **p < 0.01 and ***p < 0.001 (Student's t-test) in (d) and (e). (f) Heteroallelic combinations of the hypomorphic Madm allele 3T4 produce few viable small flies (<10% of the expected Mendelian ratio) that can be rescued by one copy of a genomic Madm rescue construct. (g) The dry weight of Madm hypomorphic females is reduced by 37% compared to control flies (Df/+). One copy of a genomic rescue construct restores normal weight. The genomic rescue construct has no significant dominant effect on dry weight ('rescue Df/+' females do not significantly differ from 'Df/+' females). n = 15, except for Df/3T4 (n = 9). (h) Tangential section of an eye from a Madm hypomorphic mutant female displaying rotation defects (yellow asterisk), missing rhabdomeres (green asterisk), and cell-fate transformations (red asterisk). (i) Wings of hypomorphic Madm males exhibiting wing notches and an incomplete wing vein V (arrows). Genotypes are: (a, a') y, w, eyFlp or hsFlp/y, w; FRT82B/FRT82B, w+, cl3R3 or M. (b, b', c, c') y, w, eyFlp or hsFlp/y, w; FRT82B, Madm7L2 or 3G5/FRT82B, w+, cl3R3 or M; (Df/+) y, w; FRT82B/Df(3R)Exel7283; (Df/3T4) y, w; FRT82B, Madm3T4/Df(3R)Exel7283; (rescue Df/3T4) y, w; gen.Madm(LCQ139)/+; FRT82B, Madm3T4/Df(3R)Exel7283; (rescue Df/+) y, w; gen.Madm(LCQ139)/+; FRT82B/Df(3R)Exel7283.
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Related In: Results  -  Collection

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Figure 4: The Madm loss- or reduction-of-function phenotypes strongly resemble bunA phenotypes. (a-c) Scanning electron micrographs of eyFLP/FRT mosaic eyes. (d) Madm mosaic heads (b, c) contain significantly fewer ommatidia than control mosaic heads (a) (n = 6). (a'-c') Images of tangential eye sections showing that Madm mutant (unpigmented) ommatidia (b', c') display an autonomous reduction in rhabdomere size relative to wild-type sized (pigmented) ommatidia. Furthermore, differentiation defects such as misrotation and missing photoreceptors are observed in Madm mutant ommatidia. Clones were induced 24-48 h after egg deposition using the hsFLP/FRT technique. (e) Rhabdomere size of Madm-mutant ommatidia is significantly reduced (by 29-56%). The area enclosed by rhabdomeres of photoreceptors R1-R6 in unpigmented mutant ommatidia was compared to the area measured in pigmented wild-type sized ommatidia. For each genotype, three pairs of ommatidia without differentiation defects from three different eye sections were measured (n = 9). Significant changes are marked by asterisks, **p < 0.01 and ***p < 0.001 (Student's t-test) in (d) and (e). (f) Heteroallelic combinations of the hypomorphic Madm allele 3T4 produce few viable small flies (<10% of the expected Mendelian ratio) that can be rescued by one copy of a genomic Madm rescue construct. (g) The dry weight of Madm hypomorphic females is reduced by 37% compared to control flies (Df/+). One copy of a genomic rescue construct restores normal weight. The genomic rescue construct has no significant dominant effect on dry weight ('rescue Df/+' females do not significantly differ from 'Df/+' females). n = 15, except for Df/3T4 (n = 9). (h) Tangential section of an eye from a Madm hypomorphic mutant female displaying rotation defects (yellow asterisk), missing rhabdomeres (green asterisk), and cell-fate transformations (red asterisk). (i) Wings of hypomorphic Madm males exhibiting wing notches and an incomplete wing vein V (arrows). Genotypes are: (a, a') y, w, eyFlp or hsFlp/y, w; FRT82B/FRT82B, w+, cl3R3 or M. (b, b', c, c') y, w, eyFlp or hsFlp/y, w; FRT82B, Madm7L2 or 3G5/FRT82B, w+, cl3R3 or M; (Df/+) y, w; FRT82B/Df(3R)Exel7283; (Df/3T4) y, w; FRT82B, Madm3T4/Df(3R)Exel7283; (rescue Df/3T4) y, w; gen.Madm(LCQ139)/+; FRT82B, Madm3T4/Df(3R)Exel7283; (rescue Df/+) y, w; gen.Madm(LCQ139)/+; FRT82B/Df(3R)Exel7283.
Mentions: We further characterized the Madm growth phenotype by testing effects on cell number and cell size. To assess cell number, ommatidia were counted in scanning electron microscope (SEM) pictures taken of mosaic eyes largely homozygous mutant for Madm. Compared to control mosaic eyes (Figure 4a), Madm mutant eyes (Figure 4b, c) had significantly fewer ommatidia (Figure 4d). To detect changes in cell size, we determined the size of rhabdomeres - the light-sensing organelles of the photoreceptors - in tangential eye sections containing homozy-gous mutant clones (Figure 4a'-c'). In addition, we measured the entire cell bodies of photoreceptor cells. Madm mutant rhabdomeres and photoreceptor cell bodies were smaller than the controls (by 29-56%; Figure 4e, and data not shown). The reduction was cell-autonomous because only homozygous mutant photoreceptor cells (marked by the absence of pigmentation) were affected.

Bottom Line: In order to test for functional conservation among TSC22DF members, we expressed the human TSC22DF proteins in the fly and found that all long isoforms can replace BunA function.The growth-promoting potential of long TSC22DF proteins is evolutionarily conserved.Furthermore, we provide biochemical and genetic evidence for a growth-regulating complex involving the long TSC22DF protein BunA and the adapter molecule Madm.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Molecular Systems Biology, ETH Zurich, Wolfgang-Pauli-Strasse 16, 8093 Zurich, Switzerland.

ABSTRACT

Background: The TSC-22 domain family (TSC22DF) consists of putative transcription factors harboring a DNA-binding TSC-box and an adjacent leucine zipper at their carboxyl termini. Both short and long TSC22DF isoforms are conserved from flies to humans. Whereas the short isoforms include the tumor suppressor TSC-22 (Transforming growth factor-beta1 stimulated clone-22), the long isoforms are largely uncharacterized. In Drosophila, the long isoform Bunched A (BunA) acts as a growth promoter, but how BunA controls growth has remained obscure.

Results: In order to test for functional conservation among TSC22DF members, we expressed the human TSC22DF proteins in the fly and found that all long isoforms can replace BunA function. Furthermore, we combined a proteomics-based approach with a genetic screen to identify proteins that interact with BunA. Madm (Mlf1 adapter molecule) physically associates with BunA via a conserved motif that is only contained in long TSC22DF proteins. Moreover, Drosophila Madm acts as a growth-promoting gene that displays growth phenotypes strikingly similar to bunA phenotypes. When overexpressed, Madm and BunA synergize to increase organ growth.

Conclusions: The growth-promoting potential of long TSC22DF proteins is evolutionarily conserved. Furthermore, we provide biochemical and genetic evidence for a growth-regulating complex involving the long TSC22DF protein BunA and the adapter molecule Madm.

Show MeSH
Related in: MedlinePlus