Figure 7. DIP-α–DIP-α interactions are required for proper MNISN-1s innervation of m4.

(a) DIP-α is expressed in MNISN-1s (green) neurons. The DIP-α-GAL4 allows for utilization of the UAS/GAL4 system and this gene trap is also a null allele (Ashley et al., 2019). In DIP-α-GAL4 heterozygous (het) larvae, both 1b (arrowhead) and 1 s (arrow) terminals are present on m4. (b) Removal of DIP-α results in loss of MNISN-1s innervation of m4. The MNISN-1s axon is still visible (green) and continues to innervate other dorsal muscles. These hemizygous male larvae retain GAL4 expression under the control of the endogenous DIP-α promoter. (c) Overexpression of UAS-DIP-α-Myc (shortened to UAS-DIP-α) does not affect innervation of m4 in a heterozygous DIP-α-GAL4 background. DIP-α localizes to the 1 s terminals (green in inset; Ashley et al., 2019). Note that DIP-α protein is labeled with anti-Myc. Green signal on muscles represents non-specific labeling of anti-Myc (see Figure 7—figure supplement 1e). (d) The DIP-α loss-of-function phenotype is rescued by reintroducing a UAS-DIP-α transgene in cells that normally express DIP-α. (e) UAS-DIP-α^I83A expression does not alter innervation of m4 and DIP-α^I83A localizes normally within the 1 s terminals (inset). (f) Expression of UAS-DIP-α^I83A fails to rescue the DIP-α loss-of-function phenotype (no 1 s innervation of m4). (g) Quantification of 1 s innervation of m4. Heterozygous background contains a single wild-type copy of DIP-α, while the hemizygous background only contains the loss-of-function allele. Expression of UAS-DIP-α completely rescues the loss-of-function phenotype, while expression of the UAS-DIP-α^I83A does not. Control UAS transgene background (no GAL4) does not affect m4 innervation. n: See Figure 7—source data 1. ***p<0.0001. Calibration bar, 10 μm.

Figure 7—figure supplement 1. Loss of DIP-α homophilic interactions does not affect expression or localization of DIP-α^I83A and innervation of m4 is not a sex-dependent variable.

(a) Normalized mean intensity of overexpressed UAS-DIP-α and UAS-DIP-α^I83A in motor neuron terminals that normally express DIP-α (see Materials and methods). (b) Quantification of 1 s innervation of m4 in male vs female larvae in each of the control transgene genotypes (UAS line crossed to a w¹¹¹⁸). Note that there is no statistical difference between males and females. (c) Rescue of the DIP-α loss-of-function phenotype by introducing a UAS-DIP-α (fused to Myc) in cells that normally express DIP-α. In c₁), DIP-α (green) properly localizes to the 1 s NMJ at m4. (d) Expression of UAS-DIP-α^I83A fails to rescue the DIP-α loss-of-function phenotype at m4 but DIP-α^I83A localizes normally at other 1 s synapses such as those on m12 (d₁). (e) Labeling with anti-Myc antibody in a control w¹¹¹⁸ preparation reveals background cross-reactivity of this antibody. (f) Quantification of 1 s innervation of m4 in flies either expressing UAS-DIP-α or UAS-DIP-α^I83A under the control of Eve^RN2-GAL4 (a GAL4 driver that expresses in the MNISN-1s neurons). Error bars represent S.E.M. n: See Figure 7—source data 1. Also see Figure 7.