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First published online 16 January 2008
doi: 10.1242/dev.016386

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Loss of seven-up from Drosophila R1/R6 photoreceptors reveals a stochastic fate choice that is normally biased by Notch

Institute of Molecular Biology, University of Oregon, 1370 Franklin Blvd, Eugene, OR 97403, USA.

View larger version (84K): [in this window] [in a new window]   Fig. 1. svp mutant R1 and R6 axons terminate in either the R8 or R7 target layer. Adult medullas in which the axon terminals of individual homozygous R1s, R6s and R7s created by GMR-FLP-mediated mitotic recombination were labeled with the synaptic vesicle marker synaptotagmin-GFP (green) using MARCM. All R axons were labeled with mAb24B10 (red). The approximate positions of the R8 recipient layer, M3, and the R7 recipient layer, M6, are indicated by broken lines. (A) Wild-type (FRT82) R7s terminate in the R7 recipient layer (arrows). (B) Some svp mutant R1/R6s terminate in the R8 recipient layer (arrowhead); a wild-type R7 is present in the same column (double arrow). Labeled axons in the R7 recipient layer (arrows) originate from R7s or from R1/R6s. (C,D) We used the sev mutation to remove R7s (residual mAb24B10 staining in the R7 recipient layer is derived from medulla neurons). (C) No wild-type R1/R6s terminate in the medulla. (D) svp mutant R1/R6s terminate in either the R8 (arrowheads) or the R7 recipient layer (arrows) with approximately equal frequency. (E) pros mutant R7s form synaptic boutons at both the R8 and R7 recipient layers (double arrows), but sometimes appear wild-type (not shown) or terminate in the R8 recipient layer (arrowhead). (F) Quantification of A,D,E. Red bars represent homozygous axons that form synaptic boutons in the R7 recipient layer only, blue bars represent those that terminate in the R8 recipient layer, and green bars represent those that form boutons in both the R8 and R7 recipient layers. Scale bar: 10 µm.

View larger version (42K): [in this window] [in a new window]   Fig. 2. svp mutant R1s and R6s express R8 or R7 rhodopsins with approximately equal likelihood. Adult retinas in which individual homozygous R1s and R6s created with GMR-FLP were labeled with mCD8-GFP (green) using MARCM. Rhabdomeres are visualized with phalloidin (red). Images are from the distal region of the retina, which contains the R1-R7 (identified in A) but not R8 rhabdomeres. (A-D) Retinas were labeled with antibodies against the two R8-specific rhodopsins Rh5 and Rh6 (both in blue). (A) Wild-type (FRT82) R1s and (B) R6s do not express R8 rhodopsins. Some svp mutant R1s (C) and R6s (D) express R8 rhodopsins. (E) Quantification of the experiment sampled in C and D. Blue bars represent mutant cells that adopted three R8 characteristics: a small, central rhabdomere, expression of Rh5 or Rh6 and a proximal nucleus. Green bars represent mutant cells that adopted three R7 characteristics: a small, central rhabdomere, a failure to express Rh5 or Rh6, and a distal nucleus. Gray bars represent mutant cells that retained R1/R6 characteristics. (F-I) Retinas were labeled with antibodies against the two R7-specific rhodopsins, Rh3 and Rh4 (both in blue). (F) Wild-type (FRT82) R1s and (G) R6s do not express R7 rhodopsins. Some svp mutant R1s (H) and R6s (I) express R7 rhodopsins. (J) Quantification of the experiment sample in H and I. Green bars represent mutant cells that adopted three R8 characteristics: a small central rhabdomere, a failure to express Rh3 or Rh4 and a proximal nucleus. Red bars represent mutant cells that adopted three R7 characteristics: a small central rhabdomere, expression of Rh3 or Rh4 and a distal nucleus. Gray bars represent mutant cells that retained R1/R6 characteristics. Scale bar: 5 µm.

View larger version (57K): [in this window] [in a new window]   Fig. 3. Rhodopsin expression and target selection are correlated in svp mutant R1/R6s. Adult medullas in which the axon terminals of individual homozygous R1s, R6s and R7s created with GMR-FLP were labeled with synaptotagmin-GFP (green) using MARCM. All R axons were labeled with mAb24B10 (red). The approximate positions of the R8 recipient layer, M3, and the R7 recipient layer, M6, are indicated by broken lines. (A-D) Animals contained a transgene expressing lacZ under the control of the Rh6 promoter (Rh6-lacZ; blue). (A) Wild-type (FRT82) R7s never express Rh6-lacZ (arrows), whereas 70% of wild-type R8s do (asterisks). (B) Approximately 70% of svp mutant R1/R6 axons that terminate in the R8 recipient layer express Rh6-lacZ (arrowhead), whereas most svp mutant R1/R6 and R7 axons that terminate in the R7 recipient layer do not (arrows). (C,D) We used the sev mutation to remove R7s. (C) Wild-type R8s all express Rh6-lacZ when R7s are absent. (D) svp mutant R1/R6 axons that terminate in the R7 recipient layer do not express Rh6-lacZ (arrow), while those that terminate in the R8 recipient layer do (arrowheads). (E-H) Animals contained a transgene expressing lacZ under the control of the Rh4 promoter (Rh4-lacZ; blue). (E) Approximately 70% of wild-type (FRT82) R7s express Rh4-lacZ (arrow; asterisks indicate heterozygous R7s expressing Rh4-lacZ); those that do not (double arrow) presumably express Rh3. (F) Approximately 70% of svp mutant R1/R6 and R7 axons that terminate in the R7 recipient layer express Rh4-lacZ (arrows), whereas most svp mutant R1/R6 axons that terminate in the R8 recipient layer do not (arrowhead). (G,H) We used the sev mutation to remove R7s. (G) When R7s are removed in wild type, there is no Rh4-lacZ expression. (H) Approximately 70% of svp mutant R1/R6s that terminate in the R7 recipient layer express Rh4-lacZ (arrow), while those that terminate in the R8 recipient layer do not (arrowheads). (I) Quantification of the experiments sampled in B and F, as well as of analogous experiments using Rh5-lacZ and Rh3-lacZ. Rh-lacZ expression was quantified in axons terminating in the R7 or R8 target layers of the medulla; each Rh-lacZ was examined in a separate experiment. Scale bar: 10 µm.

View larger version (39K): [in this window] [in a new window]   Fig. 4. svp mutant R3s and R4s can become either R7s or R8s. Adult retinas in which individual homozygous cells created with ey3.5-FLP were labeled with mCD8-GFP (green) using MARCM. The R1-R6-specific rhodopsin Rh1 was visualized with antibodies (red). Images are all from the distal region of the retina, which contains the R1-R7 (labeled in A and E) but not R8 rhabdomeres. (A-C) Retinas were labeled with antibodies against the two R8-specific rhodopsins, Rh5 and Rh6 (both in blue). (A) Wild-type (FRT82) R3s (arrow) and R4s (arrowhead) have large outer rhabdomeres that express Rh1 but not R8 Rhs. (B) Most svp mutant R4s (arrowhead) have small central rhabdomeres that no longer express Rh1, of which approximately half gain expression of R8 rhodopsins. (C) Most svp mutant R3s (arrow) retain large outer rhabdomeres that express Rh1 but occupy R4-like positions in ommatidia with reversed chirality. (D) Quantification of the experiment sampled in B and C. Blue and green bars represent mutant cells that adopted R8 or R7 characteristics, respectively (see Fig. 2E; in addition these lost Rh1 expression). Gray and black bars represent mutant cells that retained large outer rhabdomeres expressing Rh1 in ommatidia with normal or reversed chirality, respectively. (E-G) Retinas were labeled with antibodies against the two R7-specific rhodopsins, Rh3 and Rh4 (both in blue). (E) R7s but not wild-type R3s (arrow) or R4s (arrowhead) express R7 rhodopsins. (F) Approximately half of svp mutant R4s with small central rhabdomeres (arrowhead) express R7 rhodopsins (quantified in H). svp mutant R1/R6s generated by ey3.5-FLP resemble those generated by GMR-FLP (double arrow indicates an R1). (G) As in C, most svp mutant R3s (arrow) retain large, outer rhabdomeres that express Rh1 but occupy R4-like positions in ommatidia with reversed chirality (quantified in H). (H) Quantification of the experiment sampled in F and G. Green and red bars represent mutant cells that adopted R8 or R7 characteristics, respectively (see Fig. 2J; in addition these lost Rh1 expression). Gray and black bars are as in D. Scale bar: 5 µm.

View larger version (110K): [in this window] [in a new window]   Fig. 5. svp mutant R neurons co-express R7 and R8 markers during larval but not pupal development. Eye discs from wandering third instar larvae (L3; A-D'') or pupae 25 hours after puparium formation (P25; E-F''). ey-FLP3.5 and the EGUF/hid method (Stowers and Schwarz, 1999) were used to create eyes homozygous for a wild-type (FRT82; A,C,E) or svp mutant (B-B'',D-D'',F-F'') chromosome. R cell nuclei were visualized with anti-Elav antibodies (red). Images are all from the apical region of the disc, which contains the R7 nuclei; R8 nuclei are not visible, except as noted in B. A proportion of R1/R6 and R3/R4 nuclei are visible. (A-B'') L3 eye discs were labeled with antibodies against the R7 and R8 marker Sal (green) and the R8 marker Sens (blue). Images are from row 20. (A) In wild-type L3 eye discs, R7s express Sal but not Sens, and R1/R6s and R3/R4s express neither. R8s (not visible) express both. (B-B'') In svp mutant L3 eye discs, most R1/R6s (solid circles) express Sal; some co-express Sens. R7s (asterisks) express Sal but not Sens, and R8s (the single visible R8 is marked with a carat) express Sal and Sens, as in the wild type. R3/R4/MCs (dashed circles) can also express either both Sal and Sens or Sal alone. (C-D'') L3 eye discs were labeled with antibodies against the R7 marker Pros (green) and the R8 marker Sens (blue). Images are from row 20. (C) In wild-type L3 eye discs, R7s express Pros but not Sens, R1/R6s and R3/R4s express neither, and R8s (not visible) express Sens but not Pros. (D-D'') In svp mutant L3 eye discs, most R1/R6s (solid circles) express Pros or Sens, and some express both (arrowhead). R7s (asterisks) express Pros only, and R8s express Sens only (not shown). R3/R4/MCs (dashed circles) can also co-express Pros and Sens (arrows). (E-F'') P25 eye discs were labeled with antibodies against Pros (green) and Sens (blue). (E) In wild-type P25 eye discs, R7s express Pros, and R8s (not shown) express Sens. (F-F'') In svp mutant P25 eye discs, svp mutant R neurons express either Pros or Sens but only rarely both (arrow). The disorganization of the svp disc at this stage prevents unambiguous identification of R1/R6 versus R3/R4 versus R7 neurons, but each ommatidium contains extra Pros or Sens-expressing R cells (see text). Scale bar: 5 µm.

View larger version (80K): [in this window] [in a new window]   Fig. 6. Notch activation in R1/R6s represses both Svp and Sens expression. L3 eye discs. Images are all from the apical region of the disc, containing the R3/R4 and R1/R6 nuclei (labeled in A), and the R7 nuclei (asterisks) but not the R8 nuclei; all panels are presented in the same orientation. Images are from rows 15-17. (A-B') Wild-type or sev-Nact L3 eye discs were stained with antibodies against Sal (green), Svp (red) and Sens (blue). (A,A') In wild-type L3 eye discs, R7s express Sal, R1/R6s and R3/R4s express Svp, and R8s (not shown) express Sens. (B,B') The sev-Nact transgene expresses the intracellular domain of N in R1/R6s, R3/R4s and R7s. R1/R6s expressing activated N lose Svp expression and, like svp mutant R1/R6s, gain Sal expression. Unlike svp mutant R1/R6s, they never express Sens. (Activated N does not affect Svp expression in R3/R4s.) (C-D'') sev-Nact or sev-Nact; svp mutant L3 eye discs were stained with antibodies against Pros (green), Elav (red) and Sens (blue). (C-C'') All R1/R6s expressing activated N gain Pros expression but never Sens, consistent with adoption of the R7 fate (compare with wild-type R1/R6s in Fig. 5C). A small number of R1/R6s gain Pros but also lose the neuronal marker Elav, consistent with adoption instead of the cone cell fate (arrow). R3/R4s gain neither Pros nor Sens expression, consistent with their continued expression of Svp and failure to express Sal (see B,B'). (D-D'') In svp mutant L3 eye discs, R1/R6s (solid circles) expressing activated N always express Pros and not Sens. Activated N does not affect the ability of svp mutant R3/R4s (dashed circles) to express Sens. Scale bar: 5 µm.

View larger version (57K): [in this window] [in a new window]   Fig. 7. Proposed model for the specification of fates in the R1/R6/R7 equivalence group. Because N is not activated in the R1 and R6 precursors, they express Svp, which prevents expression of Sal (and likely other determinants of R7 and R8 fate), resulting in adoption of the default R1/R6 fate. By contrast, Dl in R1 and R6 activates N in the R7 precursor. N represses svp, allowing expression of Sal (and other determinants of R7 and R8 fate), which promotes both R7 and R8 fates; mutual negative feedback between the two programs would result in stochastic adoption of either the R7 or the R8 fate, but, in parallel, N represses sens, resulting in exclusive adoption of the R7 fate. See text for details.

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