Fig 1.
Overview of zebrafish lymphocyte cytosolic protein 1 (LCP1 or 'L-plastin').
A) Critical domains of the L-plastin protein. These include two EF-hand calcium-binding sites at the 5' end, and two actin-binding domains (ABD1 & 2) along the remainder of the peptide. Each actin-binding domain contains two serial calponin-homology domains (CH1 through 4). B) L-plastin in action. Each L-plastin monomer can bind two adjacent molecules of filamentous actin, stabilizing the parallel strands. Illustration by ZJC; L-plastin structure based on [8].
Fig 2.
Zebrafish orthologs of the plastins.
Maximum-likelihood phylogenetic analysis of plastin proteins from selected metazoans. All proteins shown were retrieved by mRNA-to-protein search (BLASTx) using the zebrafish L-plastin mRNA (ZDB-GENE-991213-5, NCBI RefSeq NM_131320.2) as a query. A calculated confidence level (percent of 100 bootstrap replicates) supports each node. For details of phylogenetic reconstruction, see Methods.
Fig 3.
Amino acid alignment of the zebrafish, mouse and human L-plastin (‘plastin 2’) proteins.
Zebrafish, mouse and human plastin 2 proteins were aligned using MUSCLE. D. rer. = NP_571395, M. mus. = NP_032905, and H. sap. = NP_002289. Colored bars indicate key protein residues or motifs. Amino acids are shaded according to alignment scores derived from the default blocks amino acids substitution matrix (BLOSUM). High BLOSUM scores (blue) reflect alignments that closely match the consensus and are highly improbable in a random model. ABD = actin-binding domain; CH = calponin-homology domain; Mst = mammalian Ste20-related protein kinase site.
Table 1.
The plastin gene family in zebrafish, mice and humans.
Fig 4.
CRISPR/Cas-9 targeting of zebrafish lcp1 exon 2.
A) Location of the L-plastin locus on zebrafish chromosome 9. B) Exon-intron structure of the locus (~42 kB). The most 5' and 3' exons are untranslated (UTR) and are separated from the intervening coding exons (here numbered 1 through 15) by large introns. C) Schematic of the CRISPR/Cas9 target site within zebrafish lcp1 exon 2. The exon 2 guide RNA (purple) binds the complementary genomic DNA (black) and docks with the Cas9 nuclease (gray). Upstream of the required protospacer-adjacent motif (PAM, in orange), the Cas9 cleavage site targets a Bsl1 restriction site (yellow), causing indels. D) PCR genotyping of gene-edited lcp1 exon 2. Top row: PCR primers flanking the PAM amplify a 175-bp product with three Bsl1 sites. Second row: An unedited PCR product digested with Bsl1 produces four fragments. Third row: An edited PCR product produces three fragments, with the large (156 bp) band being diagnostic. Bottom left: PCR genotyping of three uninjected embryos (24 hpf), showing a wild-type pattern. Right panel: PCR genotyping of three injected embryos, showing evidence of gene editing in the two leftmost lanes. E) Alignment of wild type and edited alleles of zebrafish lcp1. CH = ‘Charlie’, a 5 bp deletion; LM = ‘Lima”, a 7-bp deletion; FX = ‘Foxtrot’, a net 5 bp deletion. F) Predicted frame shifts, stop codons and truncated peptides derived from the mutant alleles. All predicted proteins are < 5% of the full-length version (image not to scale), and lack both actin-binding domains (ABDs).
Table 2.
Key oligos and primers.
Table 3.
Diagnostic restriction enzyme digests and fragments produced.
Fig 5.
Leukocyte staining is abolished in lcp1 null animals.
A) Flow chart of the experiment. lcp1 incrosses (+/- x +/-) were raised to 6 dpf and then processed for whole-mount immunostaining (head) and genomic DNA isolation (body). B) Representative genotyping results after genotyping PCR and Bsl1restriction enzyme digest. Homozygous wildtype, homozygous null, and heterozygous siblings are easily distinguished. C) Typical fluorescent immunostaining of a superficial leukocyte in wildtype and heterozygous animals. There is intense signal in the entire cytoplasm, including distant cellular projections; in contrast, the nucleoplasm is dark. D) Heterozygous and null siblings of the Charlie line (CH). In heterozygous fish, leukocytes are stained intensely, particularly in the gill area (arrow). In null fish, no leukocytes are visible. A faint, non-specific staining is present in all embryonic skeletal muscle. E) Heterozygous and null siblings of the Foxtrot line (FX). In the null animal, the LCP1-positive cells are undetectable. F) Null animals have neutrophils as seen in 2 dpf caudal fins stained with Sudan Black. G) Null animals have macrophages, as seen in 2 dpf caudal fins from embryos with green macrophages (Tg(mpeg1.1:Dendra2)). Nuclei are counterstained with DAPI.
Fig 6.
LCP1 protein is undetectable in null embryos and adults.
A) Chemiluminescent Western blot comparing 2 dpf total protein lysates from a wild type line (WT) and three independent null lines (CH, FX and LM). The top panel shows LCP1 signal (~65 kDa) and the middle panel shows actin signal (~ 43 kDa). The bottom panel reflects the total protein in each lane (Coomassie Blue). B) Chemiluminescent Western blot comparing 5 adult animals of one line (CH): two heterozygotes (+/-), one wild type (+/+), and two nulls (-/-).
Fig 7.
Survivorship analysis of lcp1 allele carriers.
A) Lima (LM) genotyping gel showing representative Mnl1 digest products against a low molecular-weight DNA ladder. The expected banding patterns for heterozygotes (+/-), wild types (+/+) and nulls (-/-) were all observed (see Table 3). B) Genotype distributions for the Charlie (CH), Foxtrot (FX), and Lima (LM) lines. All surviving fish were genotyped at either 7–8 weeks (CH and FX) or >1 year of age (LM). The black columns in each cluster represent the percentages expected for each genotype (25%, 50%, or 25%); the lighter columns represent the percentages observed. Counts and sample sizes are provided in the inset table.