PMC

Mid, H15 and Elav are co-expressed in photoreceptor neurons of third-instar larval eye imaginal discs. Confocal images of WT third-instar larval eye discs co-immunolabeled with (A) Mid (green), (B) H15 (red) and (C) Elav (blue) antisera show that all proteins are expressed within photoreceptor neurons. (D) The merged image demonstrates that Mid, H15 and Elav are also co-expressed within photoreceptor neurons (yellow). The white arrowhead points to the MF. Posterior is right.

A mid gain-of-function mutation exhibits features that are similar to a Notch LOF phenotype. (A–C) Scanning electron micrographs of 1-day old adult eyes depicting (A) a WT compound eye (B) a mid-RNAi eye and (C) a UAS-mid/CyO;GMR-Gal4/TM3 mutant eye. Overexpressing mid causes bristle defects, a smoothened surface and IOB loss. (C′) An enlarged image of panel C depicts an excess of socketless, small shaft cells and duplicated IOB complexes.

Mid and H15 are co-expressed in photoreceptor neurons and sensory organ precursor cells of early-staged pupal eye imaginal discs. Confocal images of WT P2-staged pupal eye discs immunolabeled with (A) Mid (green), (B) H15 (red) and (C) Elav (blue) antisera detecting these proteins within photoreceptor neurons during the P2 stage of development. (D) The merged image shows that Mid, H15 and Elav exhibit an overlapping expression pattern within photoreceptor neurons (magenta). (A′–D′) Higher magnification images of panels A–D represent the area enclosed by the dashed box in panel D. (E–H) WT P3-staged pupal eye discs are immunolabeled with (E) Mid (green), (F) H15 (red) and (G) Elav (blue) antisera. (H) The orientation of the disc as mounted reveals two unique populations of cells in one focal plane represented by the merged image. Mid, H15 and Elav expression is diminishing in photoreceptor neurons (H, merge, magenta) while a Mid- and H15-expressing population of SOP cells is also detected (arrow). (E′–H′) Higher magnification images of panels E–H represent the area enclosed by the dashed boxed region shown in panel H. Posterior is to the right.

A LOF H15/mid null mitotic clone generated in developing eye tissues closely resembles a Notch gain-of-function phenotype. Scanning electron micrographs depict adult compound eyes of (A) a wild-type, (B) mid-RNAi and (C) a LOF H15/mid null mitotic clone generated during the P0–P2 pupal stages. The LOF H15/mid null tissue exhibits similar yet more severe mutant features observed in mid-RNAi eyes including the loss of IOBs and ommatidial fusion. (D) Overexpressing Notch using the GMR-Gal4 line (+/CyO;UAS-N^full/GMR-Gal4) resulted in a mutant phenotype resembling tissues generated from H15/mid LOF clones. (E) A light microscope image at 120× of an eye from a sca-Gal4;pros-Gal4 transgenic line showing a normal pattern of bristle loss in ommatidial columns in the posterior-most region of the eye. (F) An eye from a UAS-mid-RNAi/sca-Gal4;pros-Gal4/TM3 fly exhibits expanded IOB loss in ommatidial columns in the posterior area (thick arrow) and IOBs that have shifted in polarity (thin arrow). The inset details these changes at a higher magnification.

mid regulates the expression of the proneural genes achaetae and senseless. Confocal image of a P2-staged WT pupal eye disc co-immunolabeled with (A) anti-Mid (green) and (B) anti-Ac (red) antisera shows that Mid and Ac are (C) co-expressed in SOP cells (merge). (D) A P3-staged mid-RNAi pupal eye disc also co-immunolabeled with (D) anti-Mid (green) and (E) anti-Ac antisera (red) depicting reduced Mid and Ac expression (merge) (F). (G–I) H15/mid LOF clones generated within the P3-staged eye disc are numerous. (G) 15/mid clones (–GFP) immunolabeled with (H) anti-Ac antibody exhibit reduced expression of Ac (red). (I) A merge of panels (G) and (H) depicts extensive H15/mid null clones (–GFP) and H15/mid heterozygous mutant tissues expressing one copy of GFP (light green). (J–L) P3-staged pupal eye discs co-immunolabeled with (J) anti-Mid (green) and (K) anti-Sens (red) antisera. (L) WT discs exhibit an overlapping Mid and Sens expression pattern marking SOPs while (M–O) mid-RNAi discs show decreased expression of (M) Mid and (N) Sens where (O) represents the merged image. (A′–F′ and J′–O′) Higher magnification images of boxed regions in panels A–F and J–O from the boxed regions (dashed). Posterior is to the right.

Loss of H15/mid results in decreased Delta and Sca expression within the MF. A confocal image of a WT 3°L eye disc co-immunolabeled with anti-Mid and anti-Delta antisera shows that (A) Delta (red) predominantly labels proneural clusters within the furrow while (B) Mid (green) is expressed predominantly within photoreceptor neurons. (C) The merged image demonstrates that Delta and Mid exhibit a mutually exclusive expression pattern. (D–F) A mid-RNAi disc also co-immunolabeled with anti-Mid and anti-Delta antisera depicts (D) reduced levels of Mid (green) and (E) Delta (red) expression. (F) The merged image shows that Delta is expressed within a narrow band of cells in a vaguely defined furrow. (G) A 3°L eye disc harboring several large H15/mid LOF clones (–GFP) is immunolabeled with (H) anti-Delta antibody. (I) In the merged image, one clone spanning the MF and extending into zones anterior and posterior of the MF (bracket) exhibits reduced expression of Delta (red) while an additional clone generated within the MF directly above but flanked with Mid-expressing tissue (green) shows a WT Delta expression pattern. (J–L) A confocal image of a WT 3°L eye disc co-immunolabled with anti-Mid and anti-Sca antisera shows that (J) Mid (green) and (K) Sca (red) also exhibit a (L) non-overlapping expression pattern (merge). (M–O) A mid-RNAi disc lacks (M) Mid (green) and (N) exhibits a nearly complete loss of Sca (red) expression also detailed within the (O) merge. (P) A confocal image of H15/mid null clones (–GFP) generated in the 3°L eye disc. (Q) The disc is immunolabeled with anti-Sca antibody. Sca is expressed in a WT pattern within most areas of the MF and marks R8 cells posterior of the MF (). The only H15/mid null clone lacking Sca expression (bracket) spans the MF and extends anteriorly and posteriorly. The arrowhead shown in all panels marks the MF.

mid collaborates with senseless to regulate interommatidial bristle generation. (A–E) Light microscope images at 120× magnification depict (A) UAS-mid-RNAi/+;sens^E2/TM3, (B) +/CyO;sens^E2/GMR-Gal4 and (C) +/CyO;sens^E2/TM3 mutant eyes. While eyes depicted in panels (A–C) exhibit an ommatidial array and numbers of IOBs that are nearly equivalent to the WT OR eye (), placing a heterozygous mutant allele of (B) sens^E2 in the GMR-Gal4 background results in an ~18% loss of IOBs. (D) A UAS-mid-RNAi/+;GMR-Gal4/TM3 (mid-RNAi) eye exhibits an ~49% loss of IOBs and disorganized ommatidia. (E) Placing the heterozygous sens^E2 mutant allele in the mid-RNAi genetic background to generate a UAS-mid-RNAi/+;GMR-Gal4/sens^E2 eye partially suppresses the mutant IOB phenotype exhibited by a ~14% increase in IOBs. However, the ommatidial array and remaining IOBs are disorganized. (F–H) The scanning electron micrographs detail selected WT and mutant bristle phenotypes from panel A, D and E, respectively. (I) The bar graph represents mean bristle numbers ± SEM quantitated for 10 montaged light microscope images eyes per each genotype shown in panels A–E. Comparisons of data are indicated by brackets linking specific data sets. The full genotype of the mutant sens^E2 allele is sens^E2red¹e¹. (J) A table with columns labeled 1–5 in the left top corners represents the mean IOB numbers ± the SEM for each genotype represented in the bar graph. The inhibition of bristle numbers in mid-RNAi mutants was statistically significant comparing column 2 vs 4 where ***p < 0.0001. The suppression of IOB loss observed in mid-RNAi eyes in the absence of one functional copy of sens was also statistically significant comparing column 4 vs 5 where **p < 0.001. Statistical analyses were performed using Shaprio-Wilk's test and the Student's t-test.

Reducing expression of mid during third-instar larval and early pupal stages results in severe eye defects. Scanning electron micrograph (SEM) images of 1-day old adult eyes depict (A) a wild-type Oregon-R (OR) compound (WT) eye with a normal ommatidial array and (B) a mid-RNAi eye exhibiting a severe reduction of interommatidial bristles (IOBs) and a rough eye characterized by ommatidial fusion. (A′) Enlarged images of the WT pattern of ommatidia and (B′) a mid-RNAi eye detail the extent of ommatidial fusion and irregularly shaped bristles (B′). (C and D) The bar charts represent mean IOB numbers and the error bars denote the standard errors of the mean (SEM) quantitated from 10 scanning electron micrographs of WT and mid-RNAi eyes. (C) Comparisons of dorsal versus ventral and (D) anterior versus posterior fields of WT (white bars) and mid-RNAi eyes (black bars) show a significant reduction of IOBs under mid-RNAi conditions across all fields. The reduction of IOBs in the ventral field of mid-RNAi eyes compared to WT eyes is highly significant (p*** < 0.0001). Comparisons of data are indicated by brackets linking specific data sets (p* < 0.01, p** < 0.001) (E–G) Light microscope images taken at 120× magnification depict (E) a WT eye exhibiting a precisely patterned ommatidial array and (F) a mid-RNAi eye with a loss of IOBs, disorganized ommatidia and pigmentation defects. (G) An example of a mid-RNAi mutant eye exhibiting scarring due to tissue degeneration. (H) The bar chart represents mean IOB numbers ± SEM from 10 montaged images of adult fly eyes of WT and mid-RNAi eyes. The reduction of IOBs in mid-RNAi eyes is statistically significant (***p < 0.0001). Statistical analyses were performed using Shaprio-Wilk's test and the Student's t-test.

Reduced expression of Mid results in apoptosis within the pupal eye imaginal disc. (A–D) Compound eyes from representative genotypes of progeny generated from crossing UAS-p35/Y;+/+;+/+ males to w¹¹¹⁸/w¹¹¹⁸;UAS-mid-RNAi/CyO;GMR-Gal4/TM3 (mid-RNAi) females. (A) UAS-p35/w¹¹¹⁸;+/CyO;+/TM3 and (B) UAS-p35/w¹¹¹⁸;+/CyO;GMR-Gal4/+ flies exhibit normal numbers of bristles. (C) It was not possible to obtain female mid-RNAi flies from the cross because the UAS-p35 transgene is on the X-chromosome. For this reason we used parental mid-RNAi female flies for quantitating bristles for comparisons with other groups. (D) w¹¹¹⁸/UAS-p35;UAS-mid-RNAi/+;GMR-Gal4 eyes exhibit a partial suppression of the mid-RNAi mutant phenotype. (E) The bar graph depicts the mean number of bristles ± the SEM quantitated from 10 eyes from each represented genotype in panels A–D and indicates that the suppression of the mid-RNAi mutation by overexpressing p35 is statistically significant (*p < 0.0001). Comparisons of data represented by bar charts are indicated by brackets linking specific data sets. (F) Numeric values and statistical analyses of the bar graph are presented in table format. (G) A WT P3-staged pupal eye disc co-stained with anti-Caspase-3 antibody (red) and (G′) the nuclear marker DAPI (blue) exhibits a uniform array of photoreceptor neurons and SOP cells. (H) A stage-matched mid-RNAi pupal eye immunolabeled with anti-Caspase-3 (red) exhibits increased expression of Caspase-3 indicative of apoptosis. (H′) DAPI (blue) labeling of the same mid-RNAi disc shows tissue degeneration. (I–K) The comparison of (I) +/CyO;Df(3L)H99/TM3, (J) parental UAS-mid-RNAi/CyO;GMR-Gal4/TM3 and (K) UAS-mid-RNAi/+;Df(3L)H99/GMR-Gal4 compound eyes shows that placing mid-RNAi in a heterozygous Df(3L)H99 background removing one copy of the pro-apoptotic genes grim, reaper and hid partially recovers the mid-RNAi phenotype. (L) The bar graph depicts the mean number of bristles ± the SEM quantitated from montaged light microscope images of 10 eyes from each represented genotype in panels I–K and indicates that the suppression of the mid-RNAi mutation by overexpressing p35 is statistically significant (***p < 0.0001). Comparisons of data represented by the bar graph are indicated by brackets linking specific data sets. (M) A table showing the means ± the SEM values from the bar graph. Statistical analyses were performed using Shaprio-Wilk's test and the Student's t-test.

mid collaborates with extramacrochaetae to regulate interommatidial bristle generation. (A–E) Light microscope images at 120× magnification depict (A) UAS-mid-RNAi/+;emc¹/TM3, (B) +/CyO;GMR-Gal4/TM6 and (C) +/CyO;GMR-Gal4/emc¹ eyes exhibiting a well-organized ommatidial array and numbers of IOBs that are nearly equivalent to the WT OR eye phenotype. (D) A UAS-mid-RNAi/+;GMR-Gal4/TM6 (mid-RNAi) eye exhibits a ~50% loss of IOBs and disorganized ommatidia. (E) Placing the heterozygous emc¹ mutant allele in the mid-RNAi background to generate a UAS-mid-RNAi/+;GMR-Gal4/emc¹ eye partially suppresses the mutant IOB phenotype by a ~25% increase in IOBs, partially recovers the integrity of the ommatidial array and restores a large area of pigmentation in the tissue. (F–H) Scanning electron micrograph images show greater details of IOBs and ommatidial arrangements. (F) The +/CyO;GMR-Gal4/emc¹ eye depicts a few duplicated bristles within the center. (G) The mid-RNAi eye exhibits large regions of IOB loss, ommatidial fusion and surviving IOBs are disorganized. (H) Although a few ommatidia are fused and there are size differences among them, the UAS-mid-RNAi/+;GMR-Gal4/emc¹ eye exhibits a significant recovery of IOBs throughout the eye field. The ommatidia of the UAS-mid-RNAi/+;GMR-Gal4/emc¹ represented in (H) were counted and nearly equal in number (701 counted) to ommatidia of WT OR eyes (750 counted). (I) The bar graph represents mean bristle numbers ± SEM quantitated for 10 eyes for each genotype shown in panels A–E as well as additional genotypes generated from the cross including +/CyO;emc¹/TM3, UAS-mid-RNAi/+;TM3/TM6 and +/CyO;TM3/TM6. Comparisons of data are indicated by brackets linking specific data sets. The full genotype of the mutant emc¹ allele is emc¹P{neoFRT}80B. (J) A table with columns labeled 1–8 in the left top corners represents the mean IOB numbers ± the SEM for each genotype represented in the bar graph. The inhibition of bristle numbers in mid-RNAi mutants was statistically significant comparing columns 1 versus (vs) 7 where ***p < 0.0001. The suppression of IOB loss observed in mid-RNAi eyes in the absence of one functional copy of emc was also statistically significant comparing column 7 vs 8 where ***p < 0.0001. Statistical analyses were performed using Shaprio-Wilk's test and the Student's t-test.

mid collaborates with atonal to regulate interommatidial bristle generation. (A–E) Light microscope images at 120× magnification depict (A) UAS-mid-RNAi/+;ato¹/TM3, (B) +/CyO;ato¹/GMR-Gal4 and (C) +/CyO;ato¹/TM3 eyes exhibiting normal ommatidial arrays and numbers of IOBs that are nearly equivalent to those counted in the WT OR eye (). (D) A UAS-mid-RNAi/+;GMR-Gal4/TM3 (mid-RNAi) eye exhibits a significant loss of IOBs by ~59% and disorganized ommatidia as previously shown (). (E) Placing the heterozygous ato¹ mutant allele in the mid-RNAi genetic background to generate a UAS-mid-RNAi/+;GMR-Gal4/ato¹ eye partially suppresses the mutant IOB phenotype with a ~16% gain of IOBs. The integrity of the ommatidial array is not restored and remaining IOBs are disorganized. (F–H) The scanning electron micrographs detail selected WT and mutant bristle phenotypes from panel A, D and E, respectively. (I) The bar graph represents mean bristle numbers ± SEM quantitated from montaged light microscope images taken of 10 eyes representing each genotype shown in panels A–E. Comparisons of data are indicated by brackets linking specific data sets. (J) A table with columns labeled 1–5 in the left top corners represents the mean IOB numbers ± the SEM for each genotype represented in the bar graph. The inhibition of bristle numbers in mid-RNAi mutants was statistically significant comparing column 2 vs 4 where ***p = 0.0002. The suppression of IOB loss observed in mid-RNAi eyes in the absence of one functional copy of ato was also statistically significant comparing column 4 vs 5 where ***p = 0.0003. Statistical analyses were performed using Shaprio-Wilk's test and the Student's t-test (K–L) Confocal images of 3°L eye discs immunolabeled with anti-Ato antibody. (K) A WT Oregon-R disc details several Ato-expressing (red) intermediate groups (IGs) (bracket) and R8-equivalence groups (R8 EGs) (arrowhead) within the MF. R8 PNs (arrow) emerge posterior of the furrow. (L) The ato¹/TM6 and (M) mid-RNAi mutant discs exhibit a WT expression pattern of Ato while the (N) UAS-mid-RNAi/+;GMR-Gal4/ato¹ mutant disc lacks the distinct labeling of Ato-expressing cell populations. Right is posterior.

Signaling pathways essential for R8 and SOP cell specification (A) Notch (N) signaling is inhibited in the SOP cell (right) by the co-repressors Su(H) and H that inhibit E(spl) expression. This leads to the potential reduction of emc expression or change in Emc activity. With Emc inactive, a subsequent increase in da as well as increased ac, ato, and sens gene expression occurs. High levels of sens further activate ac expression (). Conversely, in the Delta-receiving cell (left) Notch signaling is active. Su(H) and the N_ICD activate E(spl) expression. E(Spl) then positively modulates either emc expression or Emc activity. Emc can now physically sequester Da and Ato resulting in reduced expression of the proneural genes ac and sens. Low levels of sens are predicted to inhibit ac expression (). mid is placed downstream of Notch and upstream of both E(Spl) and emc. The dashed lines indicate a loss of mid that affects gene expression within the pathway. (B) A series of Notch–Delta signaling events illustrated in schematics compares WT and mid mutant conditions in the generation of SOP cells and SOP daughter cells. (B1) Under WT conditions, lateral inhibition by Notch–Delta signaling results in the generation of a Ganglion Mother Cell (GMC) from an SOP while neighboring cells assume the default epidermal fate. The GMC divides giving rise to the PIIa and PIIb cells. The PIIa cell divides to generate the shaft and socket cells. The black crescent shown in the SOP cell, GMC and PIIa precursor cells depicts the asymmetric inheritance of the Numb protein. (B2) H15/mid LOF mutant conditions appear to result in a Notch GOF phenotype that results in an abnormal expansion of epithelial cells at the expense of SOP cells. (B3) mid overexpression results in the generation of small shaft cells within the dorsal region of the eye from an apparent Notch LOF phenotype. SOP cells are duplicated at the expense of default epithelial cells (black circles) (not shown) and shaft cells are duplicated at the expense of socket cells. (C) A Dpp morphogenic gradient is established from the posterior (high concentration) to anterior regions of the eye imaginal disc (low concentration) (black graded shading). Only one gradient is shown but fills the spaces in a reiterative pattern outlined below. High-affinity Dpp receptor binding sites expressed by undifferentiated cells in the pre-proneural zone (white background) may initiate the selection of Grandmother Precursor-SOP cells (GPS-cells; open circles). Low affinity Dpp receptor binding sites expressed by Ato-positive cells (pink circles) in the proneural zone (yellow background) regulate Delta expression (genetic schematic). Column 0 represents a row of R8 equivalence groups where the pre-R8 cell (dark blue) is selected from the group and emerges posterior of the MF to reside in column 1 (light blue background). The remaining columns are not indicated. Fully assembled R1–R8 PNs are depicted as Mid-expressing clusters (dark green) in the posterior (P) region (light green background). Although not shown, the R7 is located directly above the R8 PN in the center of clusters. We predict that the selected pre-SOP cells are situated close to the R7/R8 PN pair. The interleaved and scalloped configuration of a Dpp gradient filling the areas demarcated by solid and dashed black lines is shown to depict a pattern that may establish the coordinates where GPS-cells are selected before they transition to pre-SOP cells within the proneural zone. The illustrated genetic pathways below the eye disc are partly adopted from a review by .