Figure 6. Identification of additional wit-responsive genomic fragments containing the BMP-LA motif.

We identified 10 genomic fragment reporters that exhibited expression in subsets of pMad-positive cells in the VNC and tested their wit-responsiveness. (A) EGFP reporter patterns of three genomic fragments that exhibit no wit-responsive loss of reporter expression in wit mutants (wit^A12/wit^B11) compared to controls (wit^A12/+) in late third Instar larval VNCs. (B) Nuclear EGFP expression patterns driven from seven genomic fragments containing conserved BMP-LAs that were down-regulated in wit mutants (wit^A12/wit^B11), compared to controls (wit^A12/+) in late third Instar larval VNCs. The observed down-regulation ranged from a near-total loss of all neuronal expression to loss of expression in a subset of neurons. Full z-projections though the whole VNC are shown. Genotypes: All control lines shown here were heterozygous (w;;CM#/+); wit mutants (w;;CM#,wit^A12/wit^B11).

Figure 6—figure supplement 1. BMP-LA containing reporter fragments with no reporter and pMad co-expression.

(A) No reporter expression was observed in the VNC of these reporter lines. (B) These nine reporter lines exhibited a range of reporter patterns in the VNC but lacked EGFP reporter and pMad stain overlap. Areas of high reporter expression were magnified and showed in insets as separate channels and overlay; reporter expressing cells were circled. No overlap in expression of EGFP reporter and pMad stain was observed. Full z-projections though the whole VNC are shown. Side panels indicate nuclei with GFP reporter (green) and/or pMad stain (magenta). Genotypes: All control lines shown here were heterozygous (w;;CM#/+); wit mutants (w;;CM#,wit^A12/wit^B11).

Figure 6—figure supplement 2. Quantification of reporter expressing cells reveals significant downregulation in wit mutants or pMad-binding site mutants compared to their respective controls.

(A) None of the three reporter fragments (CM8, CM9, CM10) showed a significant change in number of reporters expressing cells between controls and their respective mutants. (B) In the VNC, the CM1 (CM1,wit^A12/+ controls) reporter was expressed in 224 ± 43 nuclei, of which 56 ± 16 (25%) nuclei are pMad-positive (n = 7). In wit mutants (wit^A12/wit^B11), reporter activity was reduced by 61% to 88 ± 34 nuclei (n = 12). Once the pMad-binding site was mutated (CM1^Δmad), reporter activity was reduced by 27% to 159 ± 36 nuclei (n = 15) compared to controls that had reporter expression in 218 ± 46 nuclei (n = 6). CM2 (CM2,wit^A12/+ controls) reporter was expressed in 209 ± 53 nuclei, of which 111 ± 33 (53%) nuclei are pMad-positive (n = 10). In wit mutants (wit^A12/wit^B11), reporter activity was reduced by 88.5% to 24 ± 23 nuclei (n = 9). In the pMad-binding site mutant (CM2^Δmad), reporter activity was reduced by 41% to 131 ± 25 nuclei (n = 9) compared to controls that had reporter expression in 221 ± 36 nuclei (n = 6). CM3 (CM3,wit^A12/+ controls) reporter was expressed in 212 ± 50 nuclei, of which 68 ± 19 (32%) nuclei are pMad-positive (n = 9). In wit mutants, reporter activity was reduced by 64% to 77 ± 15 nuclei (n = 9). In the pMad-binding site mutant (CM3^Δmad), reporter activity was reduced by 67% to 76 ± 20 nuclei (n = 9) compared to controls that had reporter expression in 228 ± 52 nuclei (n = 9). CM4 reporter was expressed in 339 ± 62 nuclei, of which 134 ± 25 (34%) nuclei were pMad-positive (n = 16). In wit mutants, reporter activity was reduced by 47% to 181 ± 22 nuclei (n = 17). CM5 reporter was expressed in 805 ± 58 nuclei in the VNC, of which 217 ± 10 (27%) nuclei were pMad-positive (n = 7). In wit mutants, reporter activity was reduced by 30% to 592 ± 94 nuclei (n = 6). CM6 was expressed in 61 ± 13 nuclei (n = 5). In wit mutants, reporter activity was reduced by 50% to 30 ± 10 nuclei (n = 5). Finally, CM7 (CM7,wit^A12/+ controls) reporter was expressed in 49 ± 11 nuclei, of which 9 ± 2 (18%) nuclei are pMad-positive (n = 5). In wit mutants, reporter activity was reduced by 75% to 12 ± 3 nuclei (n = 5). In the pMad-binding site mutant (CM7^Δmad), reporter activity was reduced by 50% to 23 ± 9 nuclei (n = 7) compared to controls that had reporter expression in 46 ± 12 nuclei (n = 6). Significance was calculated with One-way ANOVA with a Tukey post hoc multiple comparisons test for the following genotypes: CM1 control versus wit mutants p<0.0001 and control versus pMad-binding site mutants p=0.0143; CM2 control versus wit mutant and control versus pMad-binding site mutants p<0.0001; CM7 control versus wit mutants p<0.0001 and control versus pMad-binding site mutants p=0.0021. Student`s t-test was used for: CM4 p<0.0001; CM5 p=0.0004; CM6 p=0.0028. For CM3, as the wit control samples were non-normally distributed (Shapiro Wilk test), significance was calculated with the Mann-Whitney U-test for control versus wit mutants p<0.0001; Student's t-test was used for control versus pMad-binding site mutants p<0.0001. Each point represents the total number of reporter-expressing cells in the VNC of a single animal, n indicates the number of VNCs analyzed and data is reported as mean ± SD. Genotypes: All control and pMad-binding site mutant lines examined here were heterozygous (w;;CM#/+); wit mutants (w;;CM#,wit^A12/wit^B11).