Distinct roles for the cell adhesion molecule Contactin2 in the development and function of neural circuits in zebrafish

Abstract

Contactin2 (Cntn2)/Transient Axonal Glycoprotein 1 (Tag1), a neural cell adhesion molecule, has established roles in neuronal migration and axon fasciculation in chick and mouse. In zebrafish, antisense morpholino-based studies have indicated roles for cntn2 in the migration of facial branchiomotor (FBM) neurons, the guidance of the axons of the nucleus of the medial longitudinal fascicle (nucMLF), and the outgrowth of Rohon-Beard (RB) central axons. To study functions of Cntn2 in later stages of neuronal development, we generated cntn2 mutant zebrafish using CRISPR-Cas9. Using a null mutant allele, we detected genetic interactions between cntn2 and the planar cell polarity gene vangl2, as shown previously with cntn2 morphants, demonstrating a function for cntn2 during FBM neuron migration in a sensitized background of reduced planar cell polarity signaling. In addition, maternal-zygotic (MZ) cntn2 mutant larvae exhibited aberrant touch responses and swimming, suggestive of defects in sensorimotor circuits, consistent with studies in mice. However, the nucMLF axon convergence, FBM neuron migration, and RB outgrowth defects seen in morphants were not seen in the mutants, and we show here that they are likely off-target effects of morpholinos. However, MLF axons exhibited local defasciculation in MZcntn2 mutants, consistent with a role for Cntn2 in axon fasciculation. These data demonstrate distinct roles for zebrafish cntn2 in neuronal migration and axon fasciculation, and in the function of sensorimotor circuits.

Keywords: Axon guidance; CRISPR/Cas9; Cell adhesion molecule; Cntn2; Facial branchiomotor neuron; Morpholino; Neuronal migration; Vangl2; Zebrafish; nucMLF.

Figure 1. Generation and validation of CRISPR-generated cntn2 mutant

(A) Genomic structure of cntn2 containing 19 exons, with the CRISPR target site (sgRNA) in the 4^th exon. The target site is highlighted in blue while the 7bp insertion and the 11bp deletion in cntn2^zou20 and cntn2^zou22 alleles are highlighted in green and indicated by dash marks, respectively. (B) Domain structure of Cntn2 containing six immunoglobulin (Ig) domains, four fibronectin (FN) domains, and a glycosylphosphatidylinositol (GPI)-anchor linked to the plasma membrane. Predicted amino acid sequences of wildtype (WT) cntn2, and cntn2^zou20 and cntn^zou22 alleles containing multiple stop codons (*). The highlighted AAs (blue) correspond to the CRISPR target site in the gene. (C) A PCR product (528bp, zou20; 510bp, zou22) spanning the target site digested with BanI differentiate between three genotypes: Wildtype (2 cut bands), heterozygote (1 uncut and 2 cut bands) and homozygote (1 uncut band). (D) Dorsal views of wildtype (WT) and mutant (zou20) hindbrains processed for cntn2 in situ hybridization (ISH) (upper panels) and anti-Cntn2 immunohistochemistry (IHC) (lower panels). Arrowheads indicate migrated FBM neurons and asterisks mark sensory ganglia. In cntn2 (zou20) mutants, cntn2 expression is greatly reduced, and Cntn2 protein is not detectable. Scale bar = 50 μm. (E) Western blot analysis of Cntn2 in cntn2^zou20 and cntn^zou22 embryos at 48 hpf. Cntn2 protein is not detectable in cntn2 mutants. Loading control is α-tubulin.

Figure 2. FBM neuron migration is affected in cntn2 morphants but not in cntn2 mutants

Panels A-D show dorsal views of the hindbrain with anterior to the left. Tg(isl1:gfp) embryos were fixed at 48 hpf, and processed for immunohistochemistry with zn5 antibody (red) to label hindbrain commissural neurons and axons at rhombomere boundaries, and anti-GFP antibody (green) to label FBM neurons (arrowheads). (A) FBM neurons (arrowheads) migrate normally into r6 and r7 in an uninjected embryo. (B) FBM neurons largely fail to migrate out of r4 in a cntn2 MO-injected embryo. (C, D) FBM neurons migrate normally in zygotic mutant (Zcntn2−/−) (C), and maternal-zygotic mutant (MZcntn2−/−) (D) embryos. Scale bar in D, 50 μm for A-D. (E) Quantification of FBM neuron migration defects. Number in parenthesis denotes number of embryos. Data are from 3 to 4 experiments.

Figure 3. cntn2 interacts genetically with vangl2 but not with lamc1

Panels A-D and F-H show dorsal views of the hindbrain with anterior to the left. Tg(isl1:gfp) embryos were fixed at 48 hpf, and processed for immunohistochemistry with zn5 antibody (red) to label hindbrain commissural neurons and axons at rhombomere boundaries, and anti-GFP antibody (green) to label FBM neurons (arrowheads). (A) FBM neurons migrate normally in a control embryo. (B) FBM neurons migrate normally in a cntn2 heterozygous (cntn2+/−) embryo. (C) FBM neurons migrate poorly in a vangl2 heterozygous (vangl2+/−) embryo, with neurons located along the entire migratory pathway from r4 to r6. (D) FBM neurons fail to migrate out of r4 in a cntn2; vangl2 double heterozygote (cntn2+/−; vangl2+/−). Scale bar in D, 50 μm for A-D. (E) Quantification of genetic interaction data. Number in parenthesis denotes number of embryos. **Chi-square test at p<0.001; NS: not significant. Data are from 2 to 4 experiments. (F-H) Offsprings of vangl2+/− heterozygous and cntn2−/− homozygous mutants exhibit normal, partial block, and severe block phenotypes for FBM neuron migration. Embryos exhibiting partial block (10/10) and severe block (10/10) were all identified as cntn2; vangl2 double heterozygote (cntn2+/−; vangl2+/−) and a majority of embryos (8/10) exhibiting normal migration were identified as cntn2+/−; vangl2+/+ by genotyping. (I) Quantification of non-migrated FBM neurons in r4, partially migrated FBM neurons in r5 and fully migrated FBM neurons in r6, and r7. Number in parenthesis denotes number of cells. Scale bar in F, 50 μm for F-H.

Figure 4. cntn2 mutants show MLF defasciculation but lacks nucMLF defects seen in morphants

Panels A-D show ventral views of the midbrain with anterior to the left. (A-D) Confocal projections of Tg(pitx2c:gfp) embryos labeled with anti-GFP antibody at 24 hpf. Panels F-I show ventral views of the midbrain and anterior hindbrain region with anterior to the left. (A) In a control MO-injected embryo, the nucMLF is found as bilateral groups of tightly clustered cells (delineated by dashed outline). Their axons form tight fascicles (arrow) immediately posterior to the neuron clusters. (B) In a cntn2 MO-injected embryo, the nucMLF neurons are loosely packed, and their axons are defasciculated. (C, D) The nucMLF neurons and axons converge normally in zygotic (Zcntn2−/−) and maternal-zygotic (MZcntn2−/−) mutants. (E) Quantification of nucMLF defects. Number in parenthesis denotes number of embryos. Data are from 2 to 4 experiments. (F-I) Zn-12 antibody labeling of the MLF axons in 24 hpf embryos. MLF axons in a cntn2+/+ form a tight fascicle (F); however, MLF axons are defasciculated (arrowheads) in MZcntn2−/− embryos (G-I). Black dotted line in F shows the cut-off point (for scoring) where the trigeminal sensory axons enter the hindbrain in r2. (J) Quantification of MLF defasciculation defects. Number in parenthesis denotes number of embryos. Data are from 2 experiments. Scale bar in D, 50 μm for A-D; Scale bar in F, 50 μm for F-I.

Figure 5. Some neuronal defects in cntn2 morphants are likely to be off-target effects

(A) Experiments to distinguish between genetic compensation in MZcntn2−/− mutants, and off-target effects of the cntn2 MO. Normal development of FBM and nucMLF neuron in MZcntn2−/− mutants injected with cntn2 MO would suggest compensation. However, defective development of both cell types in these embryos would suggest off-target effects. (B, D, F, H) Dorsal views of the hindbrain with anterior to the left. Tg(isl1:gfp) embryos were fixed at 48 hpf, and processed for immunohistochemistry with zn5 antibody (red) to label hindbrain commissural neurons and axons at rhombomere boundaries, and anti-GFP antibody (green) to label FBM neurons (arrowheads). (B, D) FBM neurons migrate normally in control MO-injected cntn2+/+ (B) and in MZcntn2−/− (D) embryos. (F, H) Migration of FBM neurons is greatly reduced in cntn2 MO-injected cntn2+/+ (F) and MZcntn2−/− (H) embryos. (C, E, G, I) Ventral views of the midbrain, with anterior to the left, of Tg(pitx2c:gfp) embryos labeled with anti-GFP antibody. (C, E) Normal nucMLF development in control MO-injected cntn2+/+ (C) and MZcntn2−/− (E) embryos. (G, I) Defective nucMLF development in cntn2 MO-injected cntn2+/+ (G) and MZcntn2−/− (I) embryos. (J) Quantification of data presented in B, D, F and H. Number in parenthesis denotes number of embryos. (K) Quantification of data presented in C, E, G and I. Number in parenthesis denotes number of embryos. Scale bar in B, 50 μm for B, D, F, and H; Scale bar in F, 50 μm for C, E, G, and I.

Figure 6. cntn2 mutants exhibit defective touch responses

(A, B) Distribution of touch-evoked escape responses of 2 dpf cntn2+/+ and MZcntn2−/− embryos following a head touch (A) or a trunk touch (B). The larval responses were binned into three categories: No response (no movement after touch), Weak Response (muted movement with larva remaining in the field of view), and Strong Response (rapid and vigorous movement with larva swimming out of the field of view). MZcntn2−/− mutants responded similarly to control cntn2+/+ larvae when touched on the head. However, they exhibited much weaker escape responses compared to cntn2+/+ larvae when touched in the trunk. Data pooled from 2 experiments (number of embryos in parenthesis). *Chi-square test at p<0.05; NS: not significant.

Figure 7. cntn2 mutants exhibit swimming deficits

(A) Swimming assay and analysis. (B, C) Distance moved and moving duration are compared between cntn2+/− heterozygote and MZcntn2−/− mutant larvae during Lights off (B) and Lights on (C) phases. There were significant differences between cntn2+/− (n=43) and MZcntn2−/− (n=27). MZcntn2−/− larvae moved less than cntn2+/− siblings during both lights off (unpaired t-test, *p<0.05) and lights on (unpaired t-test with Welch’s correction, **p<0.001) phases (B, C). MZcntn2−/− larvae also moved for shorter duration compared to cntn2+/− siblings during both lights off (unpaired t-test, *p<0.05) and lights on (unpaired t-test, **p<0.001) phases (B, C). Error bars show Mean ± SD.