CFTR is required for the migration of primordial germ cells during zebrafish early embryogenesis

Abstract

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene affect fertility in both sexes. However, the involvement of CFTR in regulating germ cell development remains largely unknown. Here, we used zebrafish model to investigate the role of CFTR in primordial germ cells (PGCs) development. We generated a cftr frameshift mutant zebrafish line using CRISPR/Cas9 technique and investigated the migration of PGCs during early embryo development. Our results showed that loss of Cftr impairs the migration of PGCs from dome stages onward. The migration of PGCs was also perturbed by treatment of CFTRinh-172, a gating-specific CFTR channel inhibitor. Moreover, defected PGCs migration in cftr mutant embryos can be partially rescued by injection of WT but not other channel-defective mutant cftr mRNAs. Finally, we observed the elevation of cxcr4b, cxcl12a, rgs14a and ca15b, key factors involved in zebrafish PGCs migration, in cftr-mutant zebrafish embryos. Taken together, the present study revealed an important role of CFTR acting as an ion channel in regulating PGCs migration during early embryogenesis. Defect of which may impair germ cell development through elevation of key factors involved in cell motility and response to chemotactic gradient in PGCs.

Figure 1

Targeted indel mutation induced by engineered Cas9/gRNA at the cftr gene in zebrafish. (A) The target sites highlighted by yellow and the PAM sequence marked by red underlined text. Deletions of cftr mutant are shown as dashes. Boxes show the start codon of WT and destroyed start codon of cftr mutant. (B) Gel shows T7E1 digestion of PCR products amplified from adult tail genomic DNA of F1 heterozygous generation. The uncleaved and cleaved PCR products are indicated. After digestion with T7E1, the cleaved PCR product of the adult tail represents the fragments containing the mutation. (C) Sequencing results show that F1 heterozygous generation fish carrying cftr mutant produces overlapping peaks marked by dashed box. (D) Western blot assay indicates the significant reduced Cftr protein level in offspring embryos from mutant line. (E) The got genotyped homozygous cftr mutant embryo also demonstrated the absent of Kupffer’s vesicle (KV) lumen (pointed by arrow) at 8-somite stage.

Figure 2

cftr mutant induces nanos1/vasa-marked PGCs disorder in early zebrafish embryo. Analysis of localization of nanos1/vasa positive cells in offspring embryos from WT and mutant line by WISH at 4-cell stage (A), Dome stage (B), 50% Epiboly stage (C), 8-somite stage (D) and Prim-5 stage (E). Embryo orientations: 4-cell, Dome stage and 50% Epiboly stage, top view; 8-somite, dorsal view with anterior oriented at the top; Prim-5 stage, lateral views with anterior oriented toward the left. Arrows show the normal location of PGCs, arrowheads demonstrate the aberrant position of PGCs in cftr mutant. Region between two dotted circles on embryo shows normal location of PGCs. The numbers indicated in each picture are the number (left) of affected embryos with phenotype similar to what is shown in the picture and the total number (right) of observed embryos. The same number labeling was used thereafter.

Figure 3

CFTR inhibitor CFTR_inh172 leads to PGCs disorder in early zebrafish embryo. Analysis of localization of nanos1/vasa positive cells in embryos by WISH at 50% Epiboly stage with top view.

Figure 4

CFTR mutants with loss of ion channel function fail to recover PGCs development. Analysis of localization of nanos1/vasa positive cells in embryos by WISH at 50% Epiboly stage with top view.

Figure 5

Aberrant expression of key factors (A: cxcr4b; B: cxcl12a; C: rgs14a; D: ca15b) is detected in offspring embryos from mutant line. 50%-epiboly stage embryos were used in these assays. All of the factors were detected by qPCR; ca15b was also detected by WISH.