The crlf3 gene is expressed in hematopoietic and other tissues during zebrafish embryogenesis. (A–R). WISH with sense (s) or antisense (as) crlf3 probes at the indicated time points on wild-type embryos (wt/wt, A–G, J–P) or those injected with 1 mM standard control morpholino (sc mo, H) or lycat morpholino (lycat mo, I) or bathed from 56 hpf in DMSO vehicle control (DMSO, Q) or JAK3 inhibitor (JAK3 inh, R). Embryos are dorsal view with anterior to the left (A, B, D, F, L, N, Q, R); anterior view with dorsal to the left (C); lateral view with anterior to the left (E, J, K, M), except panel O that is ventral view with anterior to the left and P is anterior view with dorsal to the top. The indicated structures are: AC (adaxial cells), ALM (anterior lateral plate mesoderm), DMMB (dorsal midline of midbrain), EP (exocrine pancreas), ICM (intermediate cell mass), PLM (posterior lateral plate mesoderm), R (retina), and T (thymus).

Generation of zebrafish crlf3 mutants using genome editing. (A) Schematic representation of the CRLF3 protein, consisting mostly of a cytokine receptor homology domain (rounded rectangle) containing two conserved cysteines (C, thin lines) and a WSXWS motif (thick line). (B) The intron/exon structure of the zebrafish crlf3 gene, with exons represented as numbered boxes, showing untranslated (green) and translated (gray) regions, and introns represented with intervening lines. (C) Sequence trace of homozygous wild-type crlf3^wt/wt (wt/wt) and its corresponding nucleotide sequence and encoded amino acids shown below. Nucleotides deleted in mdu14 and mdu15 alleles are boxed in tan and blue, respectively. (D) Targeting of exon 2 with TALENs, with the PstI site used in RFLP analysis underlined, to generate the mdu14 allele, with the sequence of a homozygous crlf3^mdu14/mdu14 (mdu14/mdu14) mutant shown. This represents a 1 bp deletion that causes a frameshift resulting in translation from an alternative reading frame (red) followed by a stop codon (*) that prematurely truncates the protein. (E) Targeting of exon 2 with CRISPR, with target site italicized and PAM site indicated, to generate the mdu15 allele, with the sequence of a homozygous crlf3^mdu15/mdu15 (mdu15/mdu15) mutant shown. This 14 bp deletion also causes a frameshift and premature stop.

Mutation of crlf3 impacts primitive hematopoiesis. (A–AD). Homozygous crlf3^wt/wt (wt/wt), and crlf3^mdu14/mdu14 (mdu14/mdu14) embryos were subjected to WISH at 14 hpf with scl(A, B), fli1(E, F), spi1(I, J) and gata1(M, N), at 20 hpf with ikzf1(P, Q), and at 22 hpf with fli1(S, T), lcp1(V, W), mpo(Y, Z) and hbbe(AB, AC) (scale bar = 200 μm; red and blue dotted areas depict caudal and rostral expression domains, respectively). Individual embryos were assessed for area of staining or cell number at the indicated locations for scl(C, D), fli1(G, H, U), spi1(K, L), gata1(O), ikzf1(R), lcp1(X), mpo(AA) and hbbe(AD), with the mean and SEM shown in red and level of statistical-significance indicated (***p < 0.001, **p < 0.01, *p < 0.05, ns, not significant). Welch’s correction was used for panel (R).

Mutation of crlf3 impacts early definitive hematopoiesis. (A–X). Homozygous wild-type crlf3^wt/wt (wt/wt) and crlf3^mdu14/mdu14 (mdu14/mdu14) embryos were subjected to WISH with cmyb(A, B) at 4 dpf, and lcp1(D, E), mpo(G, H), hbbe(J, K), ikzf1(M–N), rag1(P, Q) and tcra(S, T) at 5 dpf (scale bar = 200 μm), or underwent blood analysis (V–W) at 5 dpf (scale bar = 10 μm). Individual embryos were assessed for the area of staining of cmyb CHD region (dotted boxes in panels A and B) (C), hbbe(L), ikzf1(O), rag1(R) and tcra(U), as well as the number of lcp1⁺(F) and mpo⁺(L) cells or for blood differential counts (X), with mean and SEM in red and statistical significance indicated (***p < 0.001, **p < 0.01, *p < 0.05, ns, not significant). Welch’s correction was used for panels (C, L).

Mutation of crlf3 fails to affect other relevant embryonic tissues. (A–H) Homozygous wild-type crlf3^wt/wt (wt/wt), and crlf3^mdu14/mdu14 (mdu14/mdu14) embryos at the indicated ages were subjected to WISH with myod(A–D), pax6(E, F) and trypsin(G, H), presented as dorsal view (A, B), lateral view (C–F) or ventral view (G, H) with anterior to the left in each case.