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CRISPR-based gene replacement reveals evolutionarily conserved axon guidance functions of Drosophila Robo3 and Tribolium Robo2/3 - PubMed

  • ️Sun Jan 01 2017

CRISPR-based gene replacement reveals evolutionarily conserved axon guidance functions of Drosophila Robo3 and Tribolium Robo2/3

Timothy A Evans. Evodevo. 2017.

Abstract

Background: Axon guidance receptors of the Roundabout (Robo) family regulate a number of axon guidance outcomes in bilaterian animals in addition to their canonical role in Slit-dependent midline repulsion. In the fruit fly Drosophila melanogaster, three Robo paralogs (Robo1, Robo2, and Robo3) each have specialized roles in regulating midline crossing and the formation of longitudinal axon pathways in the embryonic ventral nerve cord. The number of robo genes differs in other insects, and it is unknown whether the roles and/or signaling mechanisms of Drosophila Robos are shared in other insect species. To directly compare the axon guidance activities of Robo receptors in Drosophila and the flour beetle Tribolium castaneum, I have used a CRISPR/Cas9-based approach to replace Drosophila robo3 with Tribolium robo2/3.

Results: I show that when expressed from the robo3 locus in Drosophila embryos, Tribolium Robo2/3 (TcRobo2/3) protein is properly translated and localized to axons, where it reproduces the normal expression pattern of Drosophila Robo3. In embryos expressing TcRobo2/3 in place of robo3, two distinct subsets of longitudinal axons are guided properly to their normal positions in the intermediate neuropile, indicating that TcRobo2/3 can promote Robo3-dependent axon guidance decisions in developing Drosophila neurons.

Conclusions: These observations suggest that the mechanism by which Drosophila Robo3 promotes longitudinal pathway formation is evolutionarily conserved in Tribolium, where it is performed by TcRobo2/3. The CRISPR/Cas9-based gene replacement approach described here can be applied to comparative evolutionary developmental studies of other Drosophila genes and their orthologs in other species.

Keywords: Axon guidance; CRISPR; Drosophila; Longitudinal pathways; Roundabout; Slit; Tribolium.

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Figures

Fig. 1
Fig. 1

Sequence comparison of Drosophila Robo3 and Tribolium Robo2/3. a Schematic comparison of the two receptors showing conserved domain structure and percent identity between individual ectodomain elements. The highest degree of sequence conservation occurs within the Slit-binding Ig1 domain (70% identity). While both proteins share the evolutionarily conserved CC0 and CC1 motifs, the TcRobo2/3 cytodomain (206 aa) is less than half the length of the Robo3 cytodomain (452 aa). b Protein sequence alignment. Structural features are indicated below the sequence. Fn domains have been re-annotated relative to Evans and Bashaw [11] based on revised predictions of beta strand locations. Identical residues are shaded black; similar residues are shaded gray. Ig immunoglobulin-like domain, Fn fibronectin type III repeat, Tm transmembrane helix, CC conserved cytoplasmic motif

Fig. 2
Fig. 2

CRISPR-based gene replacement of robo3. a Schematic of the robo3 gene showing intron/exon structure and location of gRNA target sites, robo3 TcRobo2/3 homologous donor plasmid, and the final modified robo3 TcRobo2/3 allele. Endogenous robo3 coding exons are shown as purple boxes; 5′ and 3′ untranslated regions are shown as light gray boxes. The start of transcription is indicated by the bent arrow. Introns and exons are shown to scale, with the exception of the first intron, from which approximately 13 kb has been omitted. Red arrows indicate the location of upstream (gRNA 1) and downstream (gRNA 2) gRNA target sites. Gray brackets demarcate the region to be replaced by sequences from the donor plasmid. Arrows indicate the position and orientation of PCR primers. b Partial DNA sequences of the unmodified robo3 gene and the modified robo3 TcRobo2/3 allele. Black letters indicated endogenous DNA sequence; red letters indicate exogenous sequence. Both DNA strands are illustrated. The gRNA protospacer and PAM sequences are indicated for both gRNAs. The first five base pairs of robo3 exon 2 are unaltered in the robo3 TcRobo2/3 allele, and the robo3 coding sequence beginning with codon H21 is replaced by the HA-tagged TcRobo2/3 cDNA. The endogenous robo3 transcription start site, ATG start codon, and signal peptide are retained in exon 1. The PAM sequences and portions of both protospacers are deleted in the modified allele, ensuring that the robo3 TcRobo2/3 donor plasmid and modified robo3 TcRobo2/3 allele are not targeted by Cas9. UTR untranslated regions, 5′H 5′ homology region, 3′H 3′ homology region, HA hemagglutinin epitope tag, gRNA guide RNA, HDR homology-directed repair, PAM protospacer adjacent motif

Fig. 3
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]

Fig. 4
Fig. 4

Tribolium Robo2/3 can substitute for Drosophila Robo3 to promote axon pathway formation in Drosophila embryos. a–d Stage 16 Drosophila embryos stained with anti-HRP (magenta) and anti-FasII (green) antibodies. Lower images show anti-FasII channel alone from the same embryos. In wild-type embryos, FasII-positive axons form three distinct longitudinal pathways on either side of the midline, one each in the medial, intermediate, and lateral zones of the neuropile. The intermediate FasII pathway is distinct from the medial and lateral pathways in every hemisegment in wild-type embryos (a, arrow). In robo3 1 embryos, FasII-positive axons that normally form the intermediate pathway are displaced medially, and the intermediate pathway fails to form (b, arrow with asterisk). Intermediate pathways form correctly in embryos in which the robo3 gene is replaced with a robo3 cDNA (c, arrow). When robo3 is replaced with a TcRobo2/3 cDNA, intermediate pathways form correctly in over 88% of hemisegments (d, arrow), indicating that TcRobo2/3 can substitute for robo3 to promote axon pathway formation in the intermediate region of the neuropile. Bar graph shows quantification of intermediate FasII pathway defects in the genotypes shown in a–d. Error bars indicate standard error of the mean. Number of embryos scored for each genotype is indicated in parentheses. e–h Embryos carrying the sema2b-TauMyc transgene and stained with anti-HRP (blue), anti-FasII (red), and anti-Myc (green) antibodies. The sema2b-TauMyc transgene labels the cell bodies and axons of 2–3 neurons per hemisegment in abdominal segments A4–A8. These axons normally project across the midline and then extend anteriorly in the intermediate region of the neuropile (e, arrowhead). In robo3 1 embryos, these axons are displaced medially (f, arrowhead with asterisk), but their normal intermediate position is restored in both robo3 robo3 (g, arrowhead) and robo3 TcRobo2/3 embryos (h, arrowhead)

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