DmiR-1 is detected initially in the presumptive mesoderm and later in the pharyngeal, somatic, and visceral musculature as well as the dorsal vessel. Wild-type (A-K) or DmiR-1^KO mutant (L) embryos were hybridized with a 1.1-kb riboprobe to detect the full-length pri-DmiR-1 transcript (A-F) or an LNA oligo to detect the processed DmiR-1 21mer (G-L). In all figures, embryos are oriented with anterior to the left and dorsal up. (A) Lateral view, stage 5 embryo with DmiR-1 expression in the presumptive mesoderm. Anterior and posterior limits of expression are indicated (arrowheads). (B) Lateral view, gastrulating stage 6 embryo with DmiR-1 expression in most of the invaginating mesoderm except in a portion of the head mesoderm (aster isked bracket). (C) Lateral view, stage 11 embryo with DmiR-1 expression restricted to the cephalic (cm), somatic (sm), and visceral (vm) mesoderm. (D) Lateral view, stage 13 embryo with punctate, segmental repeated expression of DmiR-1 in the forming somatic musculature. (E) Dorsal view of the same embryo presented in D showing expression in the forming pharyngeal (phm), somatic (sm), and visceral (vm) musculature. (F) Dorsal view, stage 15 embryo with DmiR-1 expression in clusters of fused somatic mesodermal cells as well as in the two rows of heart precursor cells comprising the forming dorsal vessel (dv). (G) Ventral view, gastrulating stage 6 embryo with processed DmiR-1 expression faintly detected in the invaginating presumptive mesodermal layer. (H) Lateral view, stage 10 embryo with DmiR-1 expression throughout the mesoderm (m). (I) Lateral view, stage 11 embryo with processed DmiR-1 expression restricted to the cephalic (cm), somatic (sm), and visceral (vm) mesoderm. (J) Dorsolateral view, stage 15 embryo with processed DmiR-1 expression in the somatic musculature and dorsal vessel (dv). (K) Lateral view, stage 16 embryo with cytoplasmic staining of processed DmiR-1 in syncytial fibers of differentiating somatic muscles. (L) Lateral view, stage 10 DmiR-1^KO mutant embryo in which processed DmiR-1 expression is absent, confirming the specificity of the DmiR-1 LNA oligo.

Twist is necessary and sufficient for DmiR-1 expression. Embryos were hybridized with a digoxigenin-labeled 1.1-kb riboprobe to detect pri-DmiR-1. All panels are lateral views, anterior to the left and dorsal up. (A) Stage 10 wild-type embryo with DmiR-1 expression in the invaginated mesodermal layer. (B) Stage 10 twi¹ mutant embryo with absence of DmiR-1 expression. (C) Stage 10 sna¹⁸ mutant embryo with DmiR-1 expression. (D) Stage 10 sna¹⁸ twi³ mutant embryo with absence of DmiR-1. (E) Stage 5 wild-type embryo with DmiR-1 expression in the presumptive mesoderm. (F) Stage 5 embryo that contains a twist-bcd transgene and consequently expresses ectopic Twist at the anterior pole of the embryo (Stathopoulos and Levine 2002b). DmiR-1 expression is detected in the presumptive mesoderm as well as at the anterior pole. (G) Stage 5 gd⁷ embryo that completely lacks Dorsal nuclear protein. DmiR-1 is not detected. (H) Stage 5 gd⁷ embryo containing a twist-bcd transgene. DmiR-1 expression is only detected at the anterior pole.

E-Box fragments from the DmiR-1 genomic locus are sufficient to recapitulate the mesodermal expression pattern of DmiR-1. (A) Schematic representation of a 4-kb region of the DmiR-1 genomic locus. The transcription start site of pri-DmiR-1 as determined by 5′ RACE is indicated with an arrow. Two evolutionarily conserved TATA boxes (T) and polyadenylation site (A) are also indicated. Thirteen Twist-binding sites (CANNTG), termed E-boxes, were identified (boxes numbered 1-13) and the evolutionary conservation of nine E-boxes, from Drosophila melanogaster (mel) to either Drosophila virilis (vir) or Drosophila pseudoobscura (pse), are shown. (B,E,H) Schematic representations of enhancer-LacZ constructs containing either seven E-boxes (E1-7 shown in B), four E-boxes (E2-5 shown in E), or three E-boxes (E3-5 shown in H). (C,D,F,G,I,J) Embryos were hybridized with a LacZ RNA probe and are oriented with anterior to the left and dorsal up. (C) Stage 10 embryo containing the E1-7 transgene with LacZ expression in the mesoderm. (F) Stage 10 embryo containing the E2-5 transgene with LacZ expression in the mesoderm. (I) Stage 10 embryo containing the E3-5 transgene with LacZ expression in the ectoderm but not the mesoderm. (D,G,J) daughterless-Gal4 driven expression of UAS-Twist (Baylies and Bate 1996) leads to ectopic expression of the E1-7 transgene (D, arrowhead), the E2-5 transgene (G, arrowhead), and the E3-5 transgene (J, arrowhead).

DMef2 is required for DmiR-1 expression. (A,B) Stage 15 wild-type (A) and Dmef2^22-21 mutant (B) embryos were hybridized with a digoxigenin-labeled 1.1-kb riboprobe to detect pri-DmiR-1. Expression is detected in the wild-type embryo (A) but not in the Dmef2^22-21 mutant embryo (B). (C) A DMef2-binding site (YTAWWWWTAR) was identified next to E-box 7 in the DmiR-1 genomic locus and is evolutionarily conserved between D. melanogaster (mel) and D. virilis (vir). This DMef2-binding site is contained in the E1-7-LacZ enhancer construct but not the E1-5-LacZ enhancer construct. (D,E) Stage 13 embryos containing either the E1-7 transgene (D) or the E1-5 transgene (E) were stained with a LacZ RNA probe. (D) Embryo containing the E1-7 transgene with LacZ expression in all somatic mesodermal cells. (E) Embryo containing the E1-5 transgene with LacZ expression in just a few somatic mesodermal cells. The remaining lacZ-positive cells are presumably cells with persistent Twist expression (Bate et al. 1991).

DmiR-1 is required in the mesoderm for viability. (A) Schematic representations of the wild-type DmiR-1 genomic locus; the ends-in targeting fragment in which 57 bp of the DmiR-1 genomic locus, including the sequence for the entire DmiR-1 21mer, was replaced with a XhoI site; and the mutated DmiR-1 genomic locus after targeting. Locations of the probes and restriction enzyme sites used in the Southern blots in B are also shown. (B) Southern blot analysis indicates successful targeting of the DmiR-1 genomic locus and the replacement of DmiR-1 with a XhoI Site. Genomic DNA from wild-type (+/+), heterozygous DmiR-1^KO (DmiR-1^KO/+), or homozygous DmiR-1^KO (DmiR-1^KO/DmiR-1^KO; P[W8-DmiR-1^8.6kb]) flies was hybridized with either a distal (Probe 1, left panel) or proximal (Probe 2, right panel) probe. Genomic DNA in both panels was double-digested with restriction enzymes NaeI and XhoI. Both probes detected a 13.5-kb XhoI → NaeI fragment in wild type (lane 1, left and right panels). Insertion of the XhoI site of the targeting vector into the DmiR-1 locus was predicted to cleave the 13.5-kb XhoI → NaeI into a 10-kb XhoI → XhoI fragment and a 3.5-kb XhoI → NaeI fragment. In addition to the wild-type 13.5-kb fragment, Probe 1 detected the 10-kb fragment in heterozygous DmiR-1^KO genomic DNA (lane 2, left panel) and likewise Probe 2 detected the 3.5-kb fragment in heterozygous DmiR-1^KO genomic DNA (lane 2, right panel). Since both Probes 1 and 2 lie outside of the DNA encoded by the P(W8-DmiR-1^8.6kb)-rescuing transgene (cf. probe locations in A and rescuing transgene in C), Probe 1 detected only the 10-kb fragment in homozygous DmiR-1^KO genomic DNA (lane 3, left panel), and likewise Probe 2 detected only the 3.5-kb fragment in homozygous DmiR-1^KO genomic DNA (lane 3, right panel). (C) Flies homozygous for the mutated DmiR-1^KO locus were inviable. This lethality was rescued with an 8.6-kb fragment containing the wild-type DmiR-1 genomic locus (P[W8-DmiR-1^8.6kb]) but was not rescued with the same 8.6-kb fragment in which DmiR-1 was replaced with a XhoI site (P[W8-DmiR-1^8.6kb → XhoI]). Finally, DmiR-1^KO-associated lethality was also rescued when DmiR-1 was expressed specifically in the mesoderm and its muscle cell derivatives using the panmesodermal how^24B Gal4 line to drive expression of a UAS-DmiR-1 transgene.

Feeding triggers DmiR-1^KO-associated lethality. (A) DmiR-1^KO mutants arrest and die as second instar larvae. Compare a 4-d-old wild-type (wt) larva and a 6-d-old DmiR-1^KO mutant larva (DmiR-1). (B) DmiR-1^KO mutant larvae displayed delayed solid food uptake. Note that the blue food in the gut of the 4-h-old wild-type (wt) larva (arrowhead) and its absence in the gut of an identically aged DmiR-1^KO mutant larva. (C) DmiR-1^KO mutant larvae displayed normal liquid food uptake. Note that the blue food in the gut of the 1-h-old wild-type (wt) and DmiR-1^KO mutant (DmiR-1) larvae (arrowheads). (D) Solid and liquid food uptake rates of newly hatched wild-type larvae (n = 50 per media) and DmiR-1^KO mutant larvae (n = 50 per media). DmiR-1^KO mutant larvae displayed delayed solid food uptake rates but normal liquid food uptake rates. (E) Lethality rates of yeast-fed wild-type larvae (▪, n = 50), sucrose-fed wild-type larvae (□, n = 50), yeast-fed DmiR-1^KO mutant larvae (•, n = 50), and sucrose-fed DmiR-1^KO mutant larvae (○, n = 50). Time of puparium formation indicated by ×. (F) Body wall contraction rates (BWC/minute) over time of yeast-fed wild-type larvae (▪, n = 10), sucrose-fed wild-type larvae (□, n = 10), yeast-fed DmiR-1^KO mutant larvae (•, n = 10), and sucrose-fed DmiR-1^KO mutant larvae (○, n = 10). Time of puparium formation indicated by ×. Vertical lines indicate standard deviations.

Body wall musculature of second instar DmiR-1^KO mutant larvae is severely disrupted. Dorsal views of the expression pattern of UAS-GFP driven by the muscle-cell-specific how^24B Gal4 driver in first instar (A,D) and second instar (B,E), wild-type (A,B) or DmiR-1^KO mutant (D,E) larvae. (A) Muscle pattern of a wild-type first instar larva. (B) Well-organized network of the somatic musculature of a second instar wild-type larva. Interweaving dorsal muscles are clearly seen in the center of the animal as well as the bright, punctate spots of the lateral transverse muscle down each side of the animal. (C) Close-up (rotated 90° clockwise), indicated by the white box in B, of the lateral transverse muscles of a single hemisegment. (D) Muscle pattern of DmiR-1^KO mutant larva. Interweaving dorsal as well as the punctate lateral transverse muscle are clearly essentially normal. (E) Severely disrupted musculature of a second instar DmiR-1^KO mutant larvae. The GFP pattern appears as amorphous, irregular condensations. (F) Closeup (rotated 90° clockwise), indicated by the white box in E, of the disorganized muscles of a single hemisegment. Bars: A,D, 100 μm; B,E, 200 μm; C,F, 30 μm.