Skip to main content
. 2022 Aug 30;11:e75879. doi: 10.7554/eLife.75879

Figure 2. Transcription of slp1 and slp2 in medulla neuroblasts is regulated by two enhancers of lengths 220 bp and 850 bp, respectively.

(A) A schematic of the enhancer screening. slp1 and slp2 genes and transcriptional enhancers identified as regulating their expression are shown as black and grey boxes, respectively. Positions of slp1 coding locus (2 L:3,825,675.3,827,099 [+]), slp2 coding locus (2 L:3,836,840.3,839,185 [+]), GMR35H02 (2 L:3,817,265.3,821,014) and the REDfly enhancers u8772 (2 L:3,816,967.3,818,532) and d5778 (2 L:3,842,530.3,844,660) are shown relative to one another. A 220 bp enhancer located within the genomic segment covered by GMR35H02 and an 850 base pair enhancer element within the REDFly enhancer d5778 were identified as potential cis-regulatory elements of slp1 and slp2 genes. The distance between the start of GMR35H02 and the end of d5778 is around 27.394 kbp. Other fragments that were screened /cloned are shown as black bars with names on top. (B-B’’’) A 220 bp enhancer element located within the REDfly enhancer u8772 activates GFP expression in medulla neuroblasts in the same pattern as endogenous Slp1 and Slp2 in reporter assays. (C-C’’’) Reporter GFP expression driven by an 850 bp enhancer segment located within the REDfly enhancer d5778 also closely coincides with the expressions of endogenous Slp1 and Slp2, although it is initiated at a slightly later temporal stage than the 220 bp enhancer. (D-D’’’’) A CRISPR-Cas9 edited chromosome 2 with both enhancers deleted (indicated as SlpDED) when placed over the SlpS37A chromosome results in loss of Slp1 and Slp2 expression in medulla neuroblasts in affected flies (n=11). The effect is confined to neuroblasts, as Slp2 expression is retained in both lamina (la) and in surface glial cells (arrows), and Slp1 is also seen in laminar cells. This also confirms that coding sequences of slp1 and slp2 genes are unaffected by the CRISPR-Cas9 editing procedure. Control experiments where SlpDED was placed against a wild-type chromosome 2 (indicated by ‘+’) (E-E’’’’) (n=9) and where the SlpS37A chromosome was placed against a wild-type chromosome 2 (F-F’’’’) (n=11) show normal expression of Slp1 and Slp2 in medulla neuroblasts. Scale bars 20 µm.

Figure 2.

Figure 2—figure supplement 1. The identification of the 220 bp enhancer by promoter bashing.

Figure 2—figure supplement 1.

(A-A”’’) UAS-GFP driven by GMR35H02-Gal4 is initiated at the same time as endogenous Slp1 and Slp2 in neuroblasts marked by Dpn. (B-D’’) Expression of GFP reporters driven by SlpL1.5 (B-B’’) and its sub-segments Slpf1-220bp(C-C’’), Slpf2-592bp (D-D’’), and Slpf3-123bp (E-E’’). Of the three sub-segments of SlpL1.5, only the Slpf1 fragment (220 bp in size) shows enhancer activity. Scale bars 20 µm.
Figure 2—figure supplement 2. The two enhancers act partially redundantly.

Figure 2—figure supplement 2.

CRISPR-Cas9 deletions of the u8772 enhancer (A-A’’’) and the d5778 enhancer (B-B’’’) individually do not abolish Slp1/2 expression suggesting possible redundance. Scale bars: 20 µm. (C) Middle panel: comparison of widths of expression domains normalized to brain size for Slp1 in wild type and enhancer deleted brains. p Value of ordinary one way ANOVA is <0.0001 and is statistically significant because p<0.05. Adjusted p values from Dunnett’s multiple comparison test between wild type and the u8772 220 bp enhancer deletion is 0.0041, between wild type and d5778 850 bp enhancer deletion is 0.0187. Both are statistically significant since p<0.05. Data from seven brains of each genotype are quantified (n=7). Right panel: expression domain width normalized to brain size for Slp2 compared between wild type and enhancer deleted brains shows no statistically significant differences relative to wild type (Adjusted p values from Dunnett’s multiple comparison test relative to wild type are as follows: for u8772 220 bp enhancer deletion p=0.0730, for d5778 850 bp enhancer deletion p=0.0965. Neither are statistically significant since p>0.05). The difference between u8772 deletion and d5778 deletion is significant (p value from ordinary one way ANOVA = 0.0019, statistically significant because p<0.05). Data from seven brains of each genotype are quantified (n=7). Left panel: expression domain width normalized to brain size for Dpn also shows no significant differences between wild type and enhancer deletion mutants (p value from ordinary one-way ANOVA is 0.5277 and is >0.05, hence not statistically significant. Adjusted p values from Dunnett’s multiple comparisons test between enhancer deletion mutants and the wild type are as follows: for the u8772 220 bp enhancer deletion p=0.6310, for the d5778 850 bp enhancer deletion p=0.9414. Neither are statistically significant being >0.05). Data from seven brains of each genotype are quantified (n=7). This shows that deletion of the enhancers doesn’t affect Dpn expression.
Figure 2—figure supplement 2—source data 1. Quantification and comparison of Dpn expression in wild type control, u8772 enhancer deletion, and d5778 enhancer deletion brains.
Figure 2—figure supplement 2—source data 2. Quantification and comparison of Slp1 expression in wild type control, u8772 enhancer deletion, and d5778 enhancer deletion brains.
Figure 2—figure supplement 2—source data 3. Quantification and comparison of Slp2 expression in wild type control, u8772 enhancer deletion, and d5778 enhancer deletion brains.
Figure 2—figure supplement 2—source data 4. Expression level quantification of Dpn, Slp1 and Slp2 in wild type control, u8772 enhancer deletion, and d5778 enhancer deletion brains.
Figure 2—figure supplement 3. Screen of REDfly enhancers identifies d5778 as an enhancer of slp1/2 transcription in medulla neuroblasts.

Figure 2—figure supplement 3.

(A-R’’) Results of the screen showing lacZ reporter expressions driven by REDfly enhancers. Of the reporters tested, u8772 (A-A’’) and u8781 (B-B’’) recapitulated the expression patterns driven by the previously identified 220 bp slp enhancer, thereby supporting our findings. Both u8772 and u8781 contain within them the 220 bp enhancer element. Additionally, patterns of lacZ expression driven by u8166 (D-D’’) and u5547 (F-F’’) are also interesting in that they somewhat overlap with the expression of endogenous Slp2. However, the u5547 enhancer was contained within the GMR35H02 sequence, and enhancer bashing experiments encoding this part of GMR35H02 did not show reporter expression. The u8166 line drove reporter expression primarily in neurons than in neuroblasts. For these reasons, the u5547 and u8166 enhancers were not considered for further analysis. However, the screen of REDfly enhancers identified d5778 as a potential enhancer of slp1 and slp2 transcription since it drove reporter expression in neuroblasts in a pattern overlapping endogenous Slp2 (Q-Q’’). Scale bars 20 µm.
Figure 2—figure supplement 4. The d5778 850 bp enhancer drives reporter expression predominantly in neuroblasts.

Figure 2—figure supplement 4.

(A-A’’’) The d5778 850 bp enhancer is activated slightly after the initiation of Slp2 expression. The wild type d5778 850 bp reporter drives GFP expression (A’) predominantly in neuroblasts identifiable by their expression of the neuroblast marker Dpn (A’’’’). Slp2 is expressed in some surface glial cells expressing the marker Repo (A, A’’, A’’’). (B-B’’’’) Neuroblast-specific suppression of 850 bp reporter GFP expression by GFP-RNAi results in no visible GFP signals from neuroblasts. (C-C’’’’) Glial specific suppression of 850 bp reporter GFP by GFP-RNAi preserves GFP expression in neuroblasts and establishes that the d5778 enhancer is indeed prominently active in medulla neuroblasts. Scale bars: 20 µm.