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Dimerization partners determine the activity of the Twist bHLH protein during Drosophila mesoderm development

Irinka Castanon, Stephen Von Stetina*, Jason Kass and Mary K. Baylies{ddagger}

Program in Molecular Biology, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA
* Present address: Graduate Program at Vanderbilt University, Cell Biology Department, Vanderbilt University, Nashville, TN 37232-2175
{ddagger} Author for correspondence (e-mail: m-baylies{at}ski.mskcc.org )



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  		Fig. 1. Twist binds DNA and activates gene expression as a homodimer. (A)Mobility shift assays with rho E box (CATATG; Ip et al., 1992Go) were performed with full-length Twist (lanes 2, 3, 4), truncated Twist (bHLHTwist; lanes 8, 9, 10) or both (lanes 5, 6, 7). An * marks the full-length homodimer (lane 2) and the truncated Twist homodimer (lane 8). A mixture of full-length and the bHLH region of Twist produced an intermediate band shift (lanes 5, 7 *), corresponding to a full-length and bHLH Twist heterodimer. These bands are competed away with 200x unlabeled rho E box (lanes 3, 6, 9), but not with a mutated (mt) E box (AGTGTG) (lanes 4, 7, 10). The unprogrammed lysate is included in lane 1. Probe is in excess in all lanes in all shifts shown. (B) Schematic representation of two Twist proteins joined in-frame by a flexible 16 amino acid Gly/Ser rich linker (Markus, 2000Go; Neuhold and Wold, 1993Go). The boxes signify the basic, DNA-binding domain and the HLH dimerization domain. The linker sequence is given in single letter amino acid code. Numbers below the line refer to the nucleotide sequence of the twist cDNA (Thisse et al., 1988Go). (C) DNA binding properties of tethered Twist-Twist. In vitro translated products are assayed for binding to rho E box (CATATG). Twist-Twist linked dimers (*, lane 3 and 5) have the same DNA binding specificity as Twist alone (lane 2). The upper band in lanes 3 and 5 is a dimer of the linked dimers. These higher order complexes can be competed away easily upon addition of a bHLH monomer, leaving only the forced homodimer (data not shown). We have no evidence that this complex nor other complexes (the tethered dimer and another bHLH) are found in tissue culture or in vivo, yet this possibility does exist. (D) Twist and Twist-Twist linked dimers activate a 175 bp Mef2 enhancer in Drosophila SL2 cells (Cripps et al., 1998Go). SL2 cells are transfected with indicated amounts of actin-lacZ plasmid, 175bp Mef2 enhancer-luciferase reporter plasmid, equimolar amounts of Twist or Twist-Twist expression vector and carrier DNA to equalize total DNA/transfection. Activation values are expressed relative to controls (see Materials and Methods). Error bars represent the standard error of means of triplicated experiments. Transfection of Twist-Twist linked dimers results in slightly better reporter gene transactivation as compared to Twist monomers alone.

 


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  		Fig. 7. Twist dimerizes with Da and represses muscle transcription in vitro. (A) Mobility shift analysis using rho CATATG (Ip et al., 1992Go) (lanes 2, 3, 4) and CACCTG E boxes (lanes 5, 6, 7) performed with Twist (lanes 2, 5), Da (lanes 4, 7), and Twist + Da (lanes 3, 6). Equal amounts of protein are used in each lane. Heterodimer shifts are marked with an asterisk. The Da homodimer shows binding to class A sites (CACCTG), whereas the Twist homodimer shows a marked affinity for a class B site (CATATG) (Ohsako et al., 1994Go). Cotranslation of both proteins and incubation with class A or B DNA sites results in formation of an intermediate shift between the Da homodimer and the Twist homodimer, which corresponds to the Twist/Da heterodimer (*). The apparent Kds for Twi/Da and Da/Da on the rho E box are 1.6x10-6 and 3.2x10-6, respectively. (B) Da represses Twist activation of the 175 bp Mef2 enhancer in SL2 cells. SL2 cells were transfected with indicated amounts of actin-lacZ plasmid, the 175 bp Mef2 enhancer-luciferase reporter plasmid, equimolar amounts of either Twist, Da, Twi+Da, or Twist-Da linked dimer expression vectors and carrier DNA. The values are expressed relative to controls (see Methods). Error bars represent the standard error of means of triplicated experiments. Transfection of Twist alone results in reporter gene activation. The same molar amount of transfected Da results in little activation. Cotransfection of equimolar amounts of Twist and Da results in reduction of reporter gene activity. Transfection of Twist-Da linked dimers led to little reporter gene activity. (C) Schematic representation of the tethered Twist-Da protein. DNA segments encoding Twist and Da are joined in frame via a flexible linker. The boxes denote the basic helix-loop-helix domain. Linker sequence is given in the single letter amino acid code. Numbers below the line represent the nucleotide sequence of twist and da cDNAs (Caudy et al., 1988; Cronmiller and Cline, 1988). (D) Mobility shift analysis with the tethered Twi-Da heterodimer. In vitro translated products are assayed for binding to class A E box. Twist-Daughterless linked dimers (lane 2 and 4) have the same DNA binding specificity as Twist/Da unlinked heterodimers (*, lane 1).

 


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  		Fig. 2. Overexpression of Twist or Twist-Twist in vivo leads to ectopic muscle formation. (A-F) Wild-type embryos; (G-L) ectopic expression of UAS-twist; (M-R) ectopic expression of UAS-twi-twi. In this and the following figures, dorsal is up and anterior to the left, unless noted. Expression of Twist (Twi; G) or Twi-Twi (M) using one copy of da-GAL4 driver led to conversion of all ectoderm into somatic muscle (cf. Baylies and Bate, 1996Go). G and M show high magnification of multinucleated, Myosin heavy chain (Mhc)-positive external cells (arrows). Insets show a lateral view of whole embryos. Fused di- and tri-nucleated cells, which are characteristic of somatic myogenesis, were found. Since no ectodermal derivatives were detected, these muscles did not spread out to form a pattern owing to lack of epidermis. Expression of Twist (H) or Twi-Twi (N) in the mesoderm using twist-GAL4 driver led to ectopic formation of Mhc-positive muscle cells, shown here ventrally (arrowheads). Note that Mhc is found in cells where it is never usually expressed. Extra muscle formation was supported by the ectopic expression of founder cell markers, such as Kr (I and O; white arrow; asterisks are shown as a reference). Zfh1 expression in pericardial and cardial cells was lost in embryos overexpressing Twist (J) and Twi-Twi (P). Fas III expression in visceral muscle progenitors was also disrupted in stage 12 embryos that ectopically expressed Twist (K) and Twi-Twi (Q). At stage 10, Bap is expressed in a subset of mesodermal cells that are progenitors for visceral mesoderm and fat body. Bap is highly reduced in embryos that overexpress Twist (L) or Twi-Twi (R). Despite greatly reduced Bap, a complete loss of Fas III under these conditions was not found. Azpiazu and Frasch reported similar effects for bap hypomorphs (Azpiazu and Frasch, 1998).

 


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  		Fig. 3. Overexpression of the tethered Twist-Twist dimer in the mesoderm leads to ectopic expression of Kr in the visceral mesoderm. (A,B) Lateral view of stage 15 embryos stained for Kr. (A) Wild-type visceral muscle showing no Kr staining surrounding the gut. (B) Ectopic expression of UAS-Twi-Twi in the mesoderm induces expression of the founder cell marker Kr (arrowhead) in the mesoderm surrounding the gut, suggesting a conversion of visceral mesoderm into somatic muscles.

 


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  		Fig. 4. Tethered Twist homodimers rescue some mesodermal defects associated with loss of Twist. (A-E) Wild-type embryos; (F-J) twiID96 null mutant embryos; (K-O) twiID96 embryos expressing UAS-Twist-Twist. twist null mutant embryos completely lack somatic musculature as shown by Mhc expression (F,G, arrowhead in G) as well as Zfh1-positive pericardial and cardial cells (H; arrowhead), Fas III-positive visceral muscle progenitors (I; arrowhead), and Htl-positive migrating mesoderm (J). Twist-Twist expression in mutant embryos rescued Htl expression during mesoderm induction (O), and somatic muscle formation, although muscle patterning is disrupted (K). Twist-Twist, however, did not rescue either the visceral mesoderm (N; arrowhead) or the heart (M; arrowhead). Instead, multinucleated, Mhc-positive cells appeared dorsally where the heart usually develops (L; arrowhead). Rescue with the Twist monomer or the Twist-Twist homodimer was 100% or 96% respectively.

 


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  		Fig. 5. Absence of both maternal and zygotic Da function leads to loss of mesodermal tissues. (A-E) Wild-type embryos; (F-J) da maternal and zygotic mutant embryos. The somatic musculature failed to form in da holonull embryos. Most somatic muscles were missing, although oddly patterned multinucleated fibres were detected (F). Absence of Fas III positive visceral muscle progenitors and Zfh1 positive pericardial and cardial cells was observed in mutant embryos (G,H; arrowheads). Although the mesoderm does migrate, Twist levels were reduced or absent, shown here in stage 8 embryos (J) and in stage 10/11 embryos when subdivision of the mesoderm occurs (I; arrowhead).

 


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  		Fig. 6. Loss of zygotic Da leads to defects in allocation and patterning of heart and somatic muscle. (A-F) Wild-type embryos; (G-L) da zygotic mutant embryos. (A,B,G and H) show somatic muscles in embryos stained for Mhc. (G) An example of more muscle in da zygotic mutant embryos: Lateral muscles were duplicated as compared to wild-type. (H) Example of muscle loss. Muscle VT1 is absent is some abdominal segments (white arrowhead), but is present in others (black arrowhead). VT1 loss can be tied to loss of founder gene expression. da embryos lack S59 (slouch) expression in the cluster, which gives rise to this muscle (cluster I). Despite losses in S59 cluster I staining in many segments, not all VT1 muscles are absent. We attribute this effect to low levels of S59 expression and/or expression of other factors that allow formation of this muscle (J; arrowhead). (I) Ectopic Eve expression in the anterior region of the mesodermal segment (arrowhead) suggests a role for da in the allocation of somatic mesoderm. Zfh1-positive pericardial and cardial cells and Fas III-expressing visceral muscle progenitors were present in normal positions in the majority of analyzed embryos (K,L), however absence of some Zfh1-positive cells can be observed in some mutant embryos (arrowhead, K).

 


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  		Fig. 8. Genetic interactions between da and twist. (A-C) Wild-type embryos; (D-F) Embryos expressing one copy of UAS-twist (G-I) or embryos expressing two copies of UAS-twist (2X UAS-twist) in a wild-type background; (J-L) Embryos expressing one copy of UAS-twist or (M-O) embryos expressing two copies of UAS-twist in a da heterozygous background. Increasing Twist levels in the mesoderm while decreasing Da levels by 50% led to an increase of ectopic somatic muscle throughout the embryo measured by Mhc staining, shown here in ventral view (A,D,G,J,M), as well as a decrease in Fas III0-positive visceral muscle progenitors (B,E,H,K,N) and Zfh1-positive pericardial and cardial cells (C,F,I,L,O).

 


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  		Fig. 9. Twist/Da heterodimers repress somatic muscle formation in vivo. (A-E) Wild-type embryos; (F-J) embryos expressing Da in a heterozygous twist background; (K-O) embryos expressing Twist-Da linked dimer in a wild-type background. Overexpression is achieved using twist-GAL4. (A,F,K) Mhc expression in stage 16 embryos. Embryos expressing Da or Twist-Da in mesoderm show severely reduced numbers of muscle cells. (B,G,L) Kr protein in stage 12 embryos. Kr expression in muscle precursors is highly reduced in embryos overexpressing Da (arrow). This phenotype is more severe in embryos expressing Twist-Da (L, arrow). (C,H,M) Fas III expression in stage 12 embryos. Fas III mesodermal expression is normal in embryos expressing Da protein (H), however some subtle defects can be observed in embryos expressing Twi-Da linked dimer (M; arrowhead). (D,I,N) Bap expression in stage 11 embryos. Progenitor cells of the visceral mesoderm and fat body show normal levels of Bap. (E,J,O) ß-galactosidase expression in stage 10 embryos. Transgenic flies carrying a 175 bp Mef2-lacZ enhancer (the same enhancer used in transient transfection assays) were stained for ß-galactosidase in the absence of ectopically expressed protein (E), in the presence of Da expression in a twist heterozygous background (J), or in the presence of Twist-Da expression in a wild-type background (O). The 175 bp enhancer was active in wild-type embryos, however, expression of Da or Twist-Da caused reduction of enhancer activity as seen in tissue culture cells. Using other GAL4s (i.e., 24BGAL4; Brand and Perrimon, 1993Go; Baylies et al., 1995Go) to drive expression of the tethered Twist-Da heterodimer in a wild-type background or Da in the twist heterozygous background resulted in similar defects.

 


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  		Fig. 10. The role of Twist homodimers and Twist/Da heterodimers during the subdivision of the mesoderm. (A) Lateral view of a stage 10 embryo showing modulated expression of Twist. Each segment has an anterior domain that expresses lower Twist levels and a posterior domain that expresses higher levels of Twist. (B) Cartoon of similar staged embryo. High Twist levels are indicated by dark blue, lower levels by light blue. Da is expressed uniformly in the mesoderm at this stage. (C) Close up of a mesodermal segment at stage 10. Cells that express high Twist levels favor formation of Twist homodimers relative to that of Twist/Da heterodimers. Twist homodimers in these cells promote the somatic muscle program. By increasing Twist/Da heterodimers levels either by reducing Twist levels or by overexpressing the linked Twist-Da dimer, the somatic muscle program is inhibited. Formation of Twist/Da heterodimers are favored relative to Twist/Twist homodimers in domains that express low Twist levels. Higher Twist/Da heterodimers levels lead to repression of somatic myogenesis in these cells, which are destined form tissues such as visceral muscle, fat body and gonadal mesoderm.

 

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