The Him gene reveals a balance of inputs controlling muscle differentiation in Drosophila

Abstract

Tissue development requires the controlled regulation of cell-differentiation programs. In muscle, the Mef2 transcription factor binds to and activates the expression of many genes and has a major positive role in the orchestration of differentiation. However, little is known about how Mef2 activity is regulated in vivo during development. Here, we characterize a gene, Holes in muscle (Him), which our results indicate is part of this control in Drosophila. Him expression rapidly declines as embryonic muscle differentiates, and consistent with this, Him overexpression inhibits muscle differentiation. This inhibitory effect is suppressed by mef2, implicating Him in the mef2 pathway. We then found that Him downregulates the transcriptional activity of Mef2 in both cell culture and in vivo. Furthermore, Him protein binds Groucho, a conserved, transcriptional corepressor, through a WRPW motif and requires this motif and groucho function to inhibit both muscle differentiation and Mef2 activity during development. Together, our results identify a mechanism that can inhibit muscle differentiation in vivo. We conclude that a balance of positive and negative inputs, including Mef2, Him, and Groucho, controls muscle differentiation during Drosophila development and suggest that one outcome is to hold developing muscle cells in a state with differentiation genes poised to be expressed.

Figure 1

Him Expression and Function in Muscle Development (A–H) Him expression decreases as muscle differentiates. In situ hybridization for Him RNA (A–D), immunostaining for Him protein (E and F), and double immunostaining for a Him-GFP fusion protein (green) and Myosin heavy chain (Mhc) protein (red) (G and H) are shown. The anterior is shown to the left, and dorsal side is shown uppermost, here and in all other figures. Stage 9 and 11 embryos showing that Him is initially expressed widely in the mesoderm (arrow) are shown in (A) and (B), respectively. Stage 12 embryos (∼8 hr 20 AEL) showing Him expression in somatic muscle precursors (arrow) and heart precursors (arrowhead) are shown in (C), (E), and (G). Stage 13 embryos (∼9 hr 50 AEL) showing absence of Him expression in developing somatic muscle and continued expression in adult muscle precursors (arrow) and developing heart (arrowhead) are shown in (D), (F), and (H). (I and J) Him inhibits muscle differentiation in vivo. An immunostain for Mhc of stage 17 embryos with UAS-Him expression driven in the developing mesoderm by twi-Gal4;twi-Gal4 at 25°C shows that Him inhibits the terminal differentiated muscle phenotype. (I) shows the wild-type, and (J) shows Him overexpression. A representative example of the phenotype is shown. (K and L) Him knockdown embryos have abnormal muscle differentiation. An immunostain for Mhc of stage 17 embryos with UAS-Him RNAi driven by twi-Gal4;twi-Gal4 at 25°C shows that Him is required for correct muscle differentiation. (K) shows the wild-type, and (L) shows Him knockdown. The Him knockdown muscle phenotype was assayed as described in Experimental Procedures. Approximately one-third of the embryos had six or more muscles per hemisegment with abnormal morphology. (L) shows a representative example of this phenotype in which most of the dorsal muscles are misshapen and are frequently thinner than the wild-type. Approximately one-third had a weaker phenotype, and approximately one-third had no apparent phenotype.

Figure 2

Him Genetically Interacts with mef2 (A–D) An immunostain for Mhc at stage 17 shows that overexpression of Him, from 24B-Gal4 × UAS-Him at 25°C (C and D), phenocopies the mef2¹¹³ hypomorph (A and B). (E–G) Him is expressed in somatic muscle precursors with Mef2. Confocal microscopy of developing somatic muscle in two hemisegments at stage 12 showing Him-GFP (E), Mef2 (F), and merge (G) is shown. (H–J) mef2 suppresses the muscle phenotype and lethality induced by Him overexpression from twi-Gal4;twi-Gal4 × UAS-Him at 18°C. An immunostain for Mhc at stage 17 shows the wild-type differentiated muscle pattern (H), the inhibited muscle development from Him overexpression (I), and the suppression of this phenotype by coexpression of UAS-mef2 with UAS-Him (J). Each panel shows a representative phenotype for each condition and the percentage of survival until adulthood. The number of wild-type muscles per three hemisegments in UAS-Him with UAS-mef2 was 61.0 ± 19.0 (mean ± SD, n = 51 embryos) and significantly higher than in UAS-Him alone, 39.1 ± 20.3 (mean ± SD, n = 50 embryos) (p < 0.001, two-sample t test).

Figure 3

Him Requires gro to Mediate Its Action (A) Him predicted protein with a putative NLS (see Experimental Procedures) and a C-terminal WRPW motif, highlighted in blue and red, respectively. (B–D) Him is a predominantly nuclear protein. Confocal microscopy of two AMPs showing Him-GFP (B), the nuclear Twist protein (C), and merge (D) is shown. (E) Autoradiograph of a protein gel of a “pulldown” assay showing that GST-Him (lane 2), but not GST-HimΔWRPW (lane 3) or GST alone (lane 1), binds to the input radiolabelled Groucho (lane 4). (F and G) Him requires its WRPW motif to inhibit muscle differentiation. An immunostain for Mhc at stage 17 shows that expression of UAS-HimΔWRPW driven in the developing mesoderm by twi-Gal4;twi-Gal4 at 25°C produces no dramatic loss of muscles (G), in contrast to full-length UAS-Him (F). (H–J) Him requires gro function to inhibit muscle differentiation. gro suppresses the muscle phenotype induced by Him overexpression from twist-Gal4 × UAS-Him at 25°C. An immunostain for Mhc at stage 17 shows the wild-type differentiated muscle pattern (H), the inhibited muscle development from Him overexpression (I), and the suppression of this phenotype in a gro^E48/gro^BX22 mutant background (J). Each panel shows a representative phenotype for each condition. The number of wild-type muscles per three hemisegments in UAS-Him in a groucho background was 61.0 ± 16.6 (mean ± SD, n = 45 embryos) and significantly higher than in UAS-Him alone, 35.3 ± 16.7 (mean ± SD, n = 52 embryos) (p < 0.001, two-sample t test). (K–M) Schematic representation of the balance between promoting and restraining influences controlling muscle differentiation illustrated by these experiments.

Figure 4

Him Downregulates Mef2 Activity (A) Him inhibits Mef2 activity in cell culture. In transfected S2 cells, which contain significant levels of Gro , a Mef2-responsive miR1-luciferase reporter is activated by Mef2. This effect is inhibited by cotransfection with Him. (B–I) Him inhibits Mef2 activity in vivo during muscle development. Mef2 protein is visualized by an immunostain of stage 12 embryos (B, D, F, and H). Somatic mesoderm expression (arrowhead) is indicated. Expression of a Mef2 target in the developing somatic muscle, β3-tubulin, is visualized by in situ hybridization of stage 12 embryos (C, E, G, and I). Somatic mesoderm (arrowhead) and visceral mesoderm (arrow) expression are indicated. (B) and (C) show the wild-type; (D) and (E) show that expression of UAS-Him driven by twi-Gal4; twi-Gal4 at 25°C does not affect Mef2 protein expression, but dramatically downregulates β3-tubulin expression in the somatic mesoderm. Expression of β3-tubulin is unaffected in the visceral mesoderm, where it is not a Mef2 target , but where twi-Gal4; twi-Gal4 drives Him expression at this stage (data not shown). Downregulation of β3-tubulin expression is not seen if either the Him C-terminal WRPW motif is deleted (F and G), or the experiment is in a gro^E48/gro^BX22 mutant background (H and I).