Lrrk2 modulation of Wnt signaling during zebrafish development

Abstract

Mutations in leucine-rich repeat kinase 2 (lrrk2) are the most common genetic cause of Parkinson's disease. Difficulty in elucidating the pathogenic mechanisms resulting from disease-associated Lrrk2 variants stems from the complexity of Lrrk2 function and activities. Lrrk2 contains multiple protein-protein interacting domains, a GTPase domain, and a kinase domain. Lrrk2 is implicated in many cellular processes including vesicular trafficking, autophagy, cytoskeleton dynamics, and Wnt signaling. Here, we generated a zebrafish lrrk2 allelic series to study the requirements for Lrrk2 during development and to dissect the importance of its various domains. The alleles are predicted to encode proteins that either lack all functional domains (lrrk2^sbu304 ), the GTPase, and kinase domains (lrrk2^sbu71 ) or the kinase domain (lrrk2^sbu96 ). All three lrrk2 mutants are viable, morphologically normal, and display wild-type-like locomotion. Because Lrrk2 modulates Wnt signaling in some contexts, we assessed Wnt signaling in all three mutant lines. Analysis of Wnt signaling by studying the expression of target genes using whole mount RNA in situ hybridization and a transgenic Wnt reporter revealed wild-type domains of Wnt activity in each of the mutants. However, we found that Wnt pathway activation is attenuated in lrrk2^{sbu304/sbu304} , which lacks both scaffolding and catalytic domains, but not in the other alleles during late embryogenesis. This supports a model in which Lrrk2 scaffolding functions are key to a context-dependent role in promoting canonical Wnt signaling.

Keywords: CRISPR-Cas9; Lrrk2; Parkinson's disease; Wnt; zebrafish.

FIGURE 1

Zebrafish lrrk2 Loss-of-Function Allelic Series. (a) Schematics of lrrk2 loss-of-function mutants created with CRISPR-Cas9. (b) lrrk2^sbu96 encodes a 5bp insertion and 1bp deletion in the kinase domain, lrrk2^sbu71 encodes a 4bp deletion in the ROC domain, and lrrk2^sbu304 encodes an 8bp deletion in the armadillo repeat domain. All deletions in the lrrk2 zebrafish lines create a premature stop codon indicated by a red asterisk. b (Left): Nucleotide sequences of the loss-of-function mutants and b (Right): Predicted amino acid translations of the loss-of-function mutants. (c) Ratio of adults recovered for each lrrk2 allele from heterozygous intercross. (d) lrrk2 mRNA expression in each of the lrrk2 mutants Student’s t test, (lrrk2^sbu96, n = 6, p = 0.23), (lrrk2^sbu71, n = 6, p = 0.17), and (lrrk2^sbu304, n = 3, p = 0.97)

FIGURE 2

Assessment of Locomotor Behavior of lrrk2 Mutants. (a) Visual-motor paradigm for assessing spontaneous locomotor behavior in 6 dpf zebrafish. (b,e,h) Spontaneous movement count per minute in the light and dark (illumination is removed at minute 15 of the trial) for (b) lrrk2^+/+ (n = 28) and lrrk2^sbu96/sbu96 (n = 21), (e) lrrk2^+/+ (n = 34) and lrrk2^sbu71/sbu71 (n = 35), (h) lrrk2^+/+ (n = 24) and lrrk2^{sbu304/sbu304} (n = 24). (c,f,i) The average total number of movements for each genotype recorded during the 15 min in the light (p = 0.41, p = 0.65, p = 0.82, Student’s t test, for lrrk2^sbu96, lrrk2^sbu71, lrrk2^sbu304 respectively). (d,g,j) Distance traveled per second upon light change in response to the removal of illumination (photic response) at second 11 of the trial

FIGURE 3

th1 mRNA Expression and cell count for lrrk2^sbu304 Mutant. (a,b) RNA in situ hybridization of th1 on 72 hpf wild type and lrrk2^{sbu304/sbu304} mutant embryos from heterozygous intercrosses. Images taken on a dorsal view, focused on the midbrain. (c) Cell counts of th1 positive cells in wild types and lrrk2^{sbu304/sbu304} mutants. Student’s t test, lrrk2^+/+ (n = 8) and lrrk2^{sbu304/sbu304} (n = 7), p = 0.32

FIGURE 4

Wnt Target Gene Expression in lrrk2 Mutants. (a-f) RNA in situ hybridization of sp5l in 10-somite embryos from lrrk2^sbu96, lrrk2^sbu71, lrrk2^sbu304 heterozygous intercrosses, dorsal view of the tailbud region. (g-l) RNA in situ hybridization of dkk1 in 27 hpf embryos from lrrk2^sbu96, lrrk2^sbu71, lrrk2^sbu304 heterozygous intercrosses; dorsal view of the hindbrain expression. (m-r) RNA in situ hybridization of lef1 in 48 hpf embryos from lrrk2^sbu96, lrrk2^sbu71, lrrk2^sbu304 heterozygous intercrosses; lateral view of the head. (s-x) RNA in situ hybridization of sp5 in 72 hpf embryos from lrrk2^sbu96, lrrk2^sbu71, lrrk2^sbu304 heterozygous crosses; dorsal view of the brain

FIGURE 5

Wnt Reporter Tg(7xTCF-Xla.Siam:GFP)^ia4 in lrrk2^sbu304 Mutants with Wnt Activator BIO Treatment. (a-d) Fluorescent images of lrrk2^sbu304 mutants in a Siam:GFP background at 56 hpf. Response to Wnt activator: (a,b) DMSO control 56 hpf lrrk2^+/+ and lrrk2^{sbu304 sbu304}. (c,d) Treated with BIO (2.5 μM) 56 hpf lrrk2^+/+ and irrk2^{sbu304/sbu304}. Images taken in lateral view

FIGURE 6

mRNA Expression of Wnt Target Genes in lrrk2^sbu304 Mutants. (a-d) Real-time qPCR showing the expression level of (a) dkk1 (n = 6), (b) lef1 (n = 6), (c) sp5 (n = 6) and (d) sp5l (n = 6) for lrrk2^+/+ and lrrk2^{sbu304/sbu304} at 6 hpf for vehicle (DMSO)-and BIO (2.5uM)-treated embryos. (a) Expression levels of dkk1 comparing lrrk2^+/+ control and treated (p = 0.00022) and lrrk2^{sbu304/sbu304} control and treated (p = 0.000034). (b) Expression levels of lef1 comparing lrrk2^+/+ control and treated (p = 0.0019). (c) Expression levels of sp5 comparing lrrk2^+/+ control and treated (p = 6.7e-8) and lrrk2^{sbu304/sbu304} control and treated (p = 1.2e-6). (d) Expression levels of sp5l comparing lrrk2^+/+ control and treated (p = 0.000082) and lrrk2^{sbu304/sbu304} control and treated (p = 0.028). (e-h) Real-time qPCR showing the expression of (e) dkk1(n = 3), (f) lef1(n = 3), (g) sp5 (n = 3), and (h) sp5l (n = 3) for lrrk2^+/+ and lrrk2^{sbu304/sbu304} at 56 hpf for vehicle (DMSO)- and BIO (2.5uM)-treated embryos. (e) Expression levels of dkk1 comparing lrrk2^+/+ control and treated (p = 3.1e-7), lrrk2^{sbu304/sbu304} control and treated (p = 0.0082), and lrrk2^+/+ treated and lrrk2^{sbu304/sbu304} treated (p = 2.8e-6). (f) Expression levels of lef1 comparing lrrk2^+/+ control and treated (p = 0.000025) and lrrk2^{sbu304/sbu304} control and treated (p = 0.00032). (g) Expression levels of sp5 comparing lrrk2^+/+ control and treated (p = 0.00001) and lrrk2^{sbu304/sbu304} control and treated (p = 0.023), and lrrk2^+/+ treated and lrrk2^{sbu304/sbu304} treated (p = 0.00012). (h) Expression levels of sp5l comparing lrrk2^+/+ control and treated (p = 0.0012) and lrrk2^{sbu304/sbu304} control and treated (p = 0.00076). Statistical analysis for pairwise comparisons was done with a post hoc Tukey’s test. A two-way analysis of variance was conducted to test if there was a significant interaction between genotype and treatment. We found a significant interaction between genotype and treatment for (e) dkk1(F_(1,8) = 105.7, n = 3, p = 6.9e-6)) and (g) sp5 (F₍₁₈₎ = 33.89, n = 3, p = 0.00039) at 56 hpf. p < 0.05 (*), p < 0.01 (**), p < 0.001 (***)

FIGURE 7

mRNA Expression of Wnt Target Genes in lrrk2^sbu71 and lrrk2^sbu96 Mutants. (a-d) Real-time qPCR showing the expression level of (a) dkk1 (n = 11), (b) lef1 (n = 11), (c) sp5 (n = 11), and (d) sp5l (n = 111) for lrrk2^+/+ and lrrk2^sbu71/sbu71 56 hpf for vehicle (DMSO)- and BIO (2.5uM)-treated embryos. (a) Expression levels of dkk1 comparing lrrk2^+/+ treated and lrrk2^sbu71/sbu71 treated (p = 0.0073) and lrrk2^sbu71/sbu71 control and treated (p = 0.000067). (c) Expression levels of sp5 comparing lrrk2^+/+ control and treated (p = 0.000025). (e-h) Real-time qPCR showing the expression of (e) dkk1 (n = 6), (f) lef1(n = 6), (g) sp5 (n = 6) and (h) sp5l (n = 6) for lrrk2^+/+ and lrrk2^sbu96/sbu96 at 56 hpf for vehicle (DMSO)- and BIO (2.5 μM)-treated embryos. (e) Expression levels of dkk1 comparing lrrk2^+/+ control and treated (p = 0.000041), lrrk2^{sbu96/ sbu96} control and treated (p = 0.0023). (g) Expression levels of sp5 comparing lrrk2^+/+ control and treated (p = 0.0016). Statistical analysis for pairwise comparisons was done with a post hoc Tukey’s test. p < 0.05 (*), p < 0.01 (**), p < 0.001 (***)