Fig. 3

TcRobo2/3 expression reproduces Robo3’s expression pattern in the robo3 ^TcRobo2/3 allele. a–d Stage 16 Drosophila embryos stained with anti-HRP (magenta; labels all axons) and anti-Robo3 (green) antibodies. Lower images show anti-Robo3 channel alone from the same embryos. In wild-type embryos, endogenous Robo3 protein is detectable on longitudinal axons within the outer two-thirds of the neuropile (a, arrowhead). Robo3 protein is undetectable in embryos homozygous for the loss of function robo3 ¹ allele (b, arrowhead with asterisk) [7, 8]. There are no large-scale defects detectable with anti-HRP in the axon scaffold of robo3 ¹ mutants. In embryos in which the robo3 gene has been replaced with an HA-tagged robo3 cDNA, Robo3 protein expressed from the modified locus reproduces its normal expression pattern (c, arrowhead) [8]. In our CRISPR-modified embryos in which robo3 has been replaced by TcRobo2/3, Robo3 protein is undetectable, consistent with the removal of robo3 coding sequences (d, arrowhead with asterisk). e, f Stage 16 embryos stained with anti-HRP (magenta) and anti-HA (green) antibodies. Lower images show anti-HA channel alone from the same embryos. Anti-HA staining in robo3 ^robo3 embryos detects the Robo3 protein expressed from the modified locus and reproduces the staining pattern seen with anti-Robo3 (e, arrowhead). In robo3 ^TcRobo2/3 embryos, the HA-tagged TcRobo2/3 protein reproduces Robo3’s expression pattern and is detectable on longitudinal axons within the lateral two-thirds of the neuropile (f, arrowhead). Schematics of the two modified robo3 alleles are shown at lower left. The robo3 ^robo3 allele was generated by Spitzweck et al. [8]