The RNF220 domain nuclear factor Teyrha-Meyrha (Tey) regulates the migration and differentiation of specific visceral and somatic muscles in Drosophila

ABSTRACT Development of the visceral musculature of the Drosophila midgut encompasses a closely coordinated sequence of migration events of cells from the trunk and caudal visceral mesoderm that underlies the formation of the stereotypic orthogonal pattern of circular and longitudinal midgut muscles. Our study focuses on the last step of migration and morphogenesis of longitudinal visceral muscle precursors and shows that these multinucleated precursors utilize dynamic filopodial extensions to migrate in dorsal and ventral directions over the forming midgut tube. The establishment of maximal dorsoventral distances from one another, and anteroposterior alignments, lead to the equidistant coverage of the midgut with longitudinal muscle fibers. We identify Teyrha-Meyhra (Tey), a tissue-specific nuclear factor related to the RNF220 domain protein family, as a crucial regulator of this process of muscle migration and morphogenesis that is further required for proper differentiation of longitudinal visceral muscles. In addition, Tey is expressed in a single somatic muscle founder cell in each hemisegment, regulates the migration of this founder cell, and is required for proper pathfinding of its developing myotube to specific myotendinous attachment sites.


Fig. S1 .
Fig. S1.Sequence alignments of wildtype and mutant versions of Tey from Drosophila melanogaster with its orthologous proteins from Aedes egypti, Tenebrio molitor, mus musculus, and its D. mel.paralog CG4813.The highly conserved RNF220 domains and RING domains are boxed in magenta and green, respectively.The bars on top of the sequences mark stretches with predicted a-helical (light blue), looped (dark blue) and b-sheet (green) conformations in fly Tey and mouse RNF220, respectively, as derived from the AlphaFold Protein Structure Database.(A stretch indicated by the thin line between brackets is absent in the mouse isoform used in AlphaFold).The C-termini of the mutant Tey D.M2-1 and Tey D.M1-11 proteins are depicted in red on top of the Tey (D. mel.) sequence.The amino acids preceding the vertical bars represent the last residues of native Tey present in the respective mutant versions, which in Tey D.M2-1 is followed by a stop codon and in Tey D.M1-11 by a short out-of-frame peptide sequence.Likewise for Tey DRNF.sfGFP the transition between the N-terminal Tey sequences and a linker plus sfGFP is indicated.

Fig. S2 .
Fig. S2.Lack of Tey protein expression in tey 5053A homozygous embryos and congruence of tey-GAL4 activity with Tey expression in heterozygotes.(A, A') Homozygous st. 14 tey 5053A UAS-lacZ embryo showing reporter expression in longitudinal visceral and somatic M12 muscle precursors (A) but no Tey protein in these or any other cells.(B, B') Embryo at early st.14 tey 5053A UAS-lacZ in trans to TM3 eve-LacZ balancer showing spatial congruence of tey-driven reporter gene activity and nuclear Tey protein in longitudinal visceral and somatic M12 muscle precursors (as well as striped balancer-derived bGal expression).

Fig. S3 .
Fig. S3.Longitudinal visceral muscle phenotypes in embryos with various tey allelic combinations.Left hand column shows controls and right hand column tey mutants.(A -D) Stage 14 and stage 16 embryos, respectively, carrying HLH54F-GFP together with tey D.M1-11 in trans to TM6 Dfd-EYFP (left) or Df(3L)ED228 (right).Anti-GFP is shown in green and anti-Fas3 in magenta.(E -H) Stage 15 and stage 16 embryos, respectively, carrying HLH54F-cyto-RFP together with teyDRNF.sfGFP in trans to TM6 Dfd-EYFP (left) or Df(3L)ED228 (right).Anti-GFP is shown in green and anti-RFP in magenta.(I, J) Stage 16 embryos carrying UAS-lifeact-GFP in trans to bap3-RFP on the second chromosome together with tey 5053A in trans to TM6 Dfd-EYFP (left) or Df(3L)ED228 (right).Anti-GFP is shown in green and anti-RFP in magenta.(K, L) Stage 15 embryos carrying UAS-apoliner9 on the second chromosome together with tey 5053A UAS-lacZ in trans to TM6 Dfd-EYFP (left) or Df(3L)ED228 (right).Anti-RFP is shown in red, anti-bGal in green, and Hoechst-stained DNA in blue.As in the control (K), no nuclear RFP is detectable in the mutant (L), which argues against increased apoptosis in longitudinal visceral muscle precursors lacking tey function (a and p denote anterior and posterior, respectively).(M -N') Stage 15 embryos with tey DRNF.sfGFP in trans to TM6 Dfd-EYFP (left) or Df(3L)ED228 (right) were stained with anti-GFP for visualizingTeyDRNF::sfGFP and with anti-Tey (magenta; ca.50 % nuclear in the heterozygous control and largely cytoplasmatic in the mutant).For better clarity, the phenotypes in the longitudinal visceral muscle precursors were separated from those of the somatic M12 muscle precursors in the same embryos by showing the Z-planes of the former in (M, N) and the Z-planes of the latter in (M', N').Scale bars provided in control panels also apply to the other panels with controls or mutants from the respective series.

Fig. S5 .
Fig. S5.tey phenotypes in midguts of 3 rd instar larvae (high magnifications) and adult flies.(A, B) 3 rd instar larval midguts with tey 5053A UAS-lacZ in trans to TM6 Dfd-EYFP (A) or Df(3L)A23 (B) and stained with anti-bGal for visualizing the longitudinal visceral muscles and for F-actin (Alexa Fluor™ 555 Phalloidin) in all gut muscles.Examples of nuclei within individual longitudinal visceral muscle fibers (visible due to partially nuclear GFP) are marked by arrow heads,

Fig. S6 .
Fig. S6.Disrupted muscle migration and morphogenesis upon forced tey expression in developing longitudinal visceral muscles and somatic muscles.Left hand column shows control embryos and right hand columns embryos containing both UAS-tey and various GAL4 drivers (denoted as X-GAL4, where the genotypes of X are provided on the respective panels).(A, B) As compared to the control (HLH54Fb-lacZ UAS-tey, stained for bGal (A)), in stage 13 embryo with tey overexpression within LVMp's via tey-GAL4 (from tey 5053A ) these cells prematurely spread over the entire width of the TVM.(C, D) Genotypes and staining as in (A, B).As compared to the control, stage 14 embryo with overexpression of tey shows abnormal orientations and shapes of LVMp's.(E, F) Somatic muscle patterns in stage 16 embryos visualized with anti-tropomyosin I show severely disrupted muscle morphologies when twi-GAL4 is used to drive ectopic tey expression in the mesoderm.(G, H) Forced expression of tey in a homozygous tey mutant background (H; UAS-tey/ +; tey 5053A UAS-lacZ hom.) rescues the zig-zag pattern seen in tey mutants (G; tey 5053A UAS-lacZ hom.) and produces almost normal M12 morphologies.(I) Stage 16 control embryo (RRHS59-lacZ/+; HN39org-1-GAL4 S18org-1-RFP/+) showing the normal somatic muscle patterns of the org-1 expressing muscles M5, M25, SBM and alary muscles (AM) (anti-RFP, red) and the slou expressing muscles M5, M25, SBM, M18, M27 (anti-bGal, green).(J) In stage 16 embryo with ectopic expression of tey via org-1-GAL4(RRHS59-lacZ UAS-tey/+; HN39org-1-GAL4 S18org-1-RFP/+), the muscles with ectopic tey (particularly M5, M25, and SBM) display severely abnormal orientations and shapes.By contrast, the slou-specific muscles M18 and M27 lacking GAL4 activity are normal and can serve as landmarks.Continued expression of the org-1-RFP and slou-LacZ markers in the muscles with ectopic tey supports our interpretation that ectopic expression of tey leads to migration and morphogenesis defects in somatic muscles rather than to cell fate transformations.
Development: doi:10.1242/dev.201457:Supplementary information Development • Supplementary information Movie 3. Time lapse movie of LVMp migration in homozygous tey 5053A mutant embryo from early stage 14 to stage 16 (marked and labeled as in Movie S1).Movie 4. Time lapse movie of LVMp and LVMu migration in homozygous tey 5053A mutant embryo from mid stage 14 to stage 17 (mainly focusing on late events; marked and labeled as in Movie S1).