Table 1.
qPCR primers.
Fig 1.
Snai2 endogenous expression in wild-type embryos.
Expression of snai2 was analyzed via whole mount in situ hybridization at 14 hpf (A), 18 hpf (B), and 24 hpf (C). Embryos were cryosectioned post-in situ at 18 hpf (D) and 24 hpf (F). Simplified schematics are provided (E and G). Double fluorescent in situ for snai2 with endothelial markers fli1a at 14 hpf (H) and etsrp at 26 hpf (I) was performed. Insets show a close-up view of the PLM. QPCR was used to compare snai2 enrichment within double positive HSPCs sorted from Tg(CD41:GFP/kdrl:mCherry) on 2 dpf to the rest of the embryo. Markers cmyb and kdrl, which should be enriched in this population, are displayed alongside for comparison. N: notochord; NT: neural tube; S: somite.
Fig 2.
Predicted genomic and protein effects of snai2 morpholinos.
Location of the splice-blocking and translation-blocking morpholinos are indicated on a schematic of the snai2 gene (A). The green arrow indicates the transcription start site, and the red line the endogenous stop codon. Predicted effects on the amino acid sequence (B) and protein structure (C) are shown for wild-type and SB morphant fish. 121 bp of exon 2 are lost due to the aberrant splicing event, leading to a truncation of the protein within the zinc-finger domain. Italicism of the amino acid sequence and the grey region of the structure indicate a region of missense amino acids prior to the early stop codon. E: exon; SB: splice-block; MO: morpholino.
Fig 3.
Snai2 morphants display a strong defect in HSC specification.
Expression of the HSC specification marker, runx1, was analyzed by in situ hybridization at ~26 hpf in embryos injected with SB MO, ATG MO, and their siblings (A). The effect of snai2 mRNA injection was also analyzed both alone and when coinjected with SB MO. Black arrowheads point to the middle of the aortic runx1 expression. Numbers in the lower right hand corner of each image depict the number of embryos with the phenotype pictured out of the total number of embryos assayed in each condition. Tg(gata2b:Gal4/UAS:LA-GFP/kdrl:mCherry) morphants and their siblings were imaged by confocal microscopy at 48 hpf, and Imaris imaging software was used to remove GFP signal outside of the vasculature (B). Pink coloration is indicative of double positive cells as filtered by the surfaces feature of Imaris. White arrowheads indicate separate putative HSPCs. Quantification for each fish was graphed and statistically analyzed by non-parametric t-test on Prism (C). Error bars are SEM.
Fig 4.
Snai2 SB morphants display depletion of sclerotome markers.
pax9, a marker of the sclerotome, was analyzed by WISH at 18 hpf (A). Black brackets highlight the difference in staining between morphants and siblings. qPCR on somitic, GFP+ cells sorted from morphant and control Tg(actc1b:GFP) embryos at ~17 hpf showed the sclerotome marker twist1b was decreased, while the pan-somitic marker remained normal in morphants (B). Wish for foxc1b showed clear diagonal expression in the anterior portion of the somites of uninjected embryos, while morphants lacked this distinctive stripe pattern (C). Error bars are calculated from technical replicates. Double fluorescent in situ hybridization for foxc1b and myoD (myogenic marker) followed by razor cutting for confocal analysis showed that while the muscle marker is consistent in both morphants and uninjected embryos, there is a notable decrease of positive staining for foxc1b, especially within the dorsal portion of the somites. A small schematic is provided to show greater detail of how embryos are oriented in Fig D. Numbers in the lower right-hand corner of each image depict the number of embryos with the phenotype pictured out of the total number of embryos assayed in each condition.
Fig 5.
Snai2 SB morphants have defective Notch signaling.
Aortic Notch activity was assessed by confocal microscopy of the Notch reporter Tg(TP1:GFP) (A). Median fluorescence intensity was calculated by the surfaces feature of Imaris and was graphed and statistically analyzed by a non-parametric t-test on Prism (B). Error bars are SEM. Using a combination of Tg(kdrl:miniGal4) and Tg(UAS:NICD-myc), we saw that ectopically activating Notch signaling within the endothelium was sufficient to rescue expression of the HSC marker runx1 in morphant embryos (C). Black arrowheads point to the middle of the aortic runx1 expression. Analysis by WISH displays that expression of the Notch ligands dlc and dld is decreased in morphants, especially within the more anterior somites (D). Black brackets are provided to highlight the differences in staining. This decrease was further confirmed by qPCR in somitic, GFP+ cells sorted from morphant Tg(actc1b:GFP) embryos as compared to uninjected siblings (E). Snai2 relative expression is included for comparison, since the misspliced transcript is consistently elevated in SB MO injected embryos. Error bars are calculated from technical replicates. Numbers in the lower right-hand corner of each image depict the number of embryos with the phenotype pictured out of the total number of embryos assayed in each condition.
Fig 6.
Predicted genomic and protein effects of the mutant snai2 alleles.
A schematic of the snai2 gene displays where the three guide RNAs (gRNAs) were designed for CRISPR/Cas9 directed mutagenesis (red arrowheads) as well as the single nucleotide polymorphism (SNP) location in the mutagenesis derived snai2sa24539 allele (red *) (A). The endogenous stop codon is indicated by the red line in exon 3. Predicted effects on the amino acid sequence (B) and protein structure (C) of wild-type, snai2112Δ, and snai2sa24539 are depicted. For the snai2112Δ allele, a 112 bp deletion within the beginning of exon 2 leads to a truncation prior to the zinc-finger domain. Italicism of the amino acid sequence and the grey region of the structure indicate a region of missense amino acids prior to the early stop codon. The snai2sa24539 allele SNP is a stop codon.
Fig 7.
Snai2112Δ and snai2sa24539 mutants have no embryonic defects in HSC or sclerotome formation.
WISH analysis was performed on embryos derived from heterozygote in-crosses including probing for the hematopoietic markers runx1 (A) and cmyb (B) and the sclerotome marker foxc1b (C). Wild-type, heterozygotes, and mutants are all included for the Snai2112Δ allele since genotyping without sequencing was possible. For the snai2sa24539 allele, genotyping by PCR was sufficient to determine which embryos lacked the wild-type SNP, but not to distinguish wild-type from heterozygote. Thus, images are included for mutants versus “siblings”. For all markers, no obvious defect is detected. Black arrowheads point to the middle of the aortic runx1 or cmyb expression. Numbers in the lower right-hand corner of each image depict the number of embryos with the phenotype pictured out of the total number of embryos assayed in each condition. HSC specification was also analyzed in Tg(CD41:GFP/kdrl:mCherry) fish on the snai2112Δ background by confocal microscopy at 48 hpf, and Imaris imaging software was used to remove GFP signal outside of the vasculature (D). Pink coloration is indicative of double positive cells as filtered by the surfaces feature of Imaris. White arrowheads indicate separate putative HSPCs, and quantification for each fish was graphed and statistically analyzed by a non-parametric t-test on Prism (E). Error bars are SEM.
Fig 8.
Snai2112Δ mutant adult hematopoiesis appears normal.
We analyzed the adult whole-kidney marrow (WKM) of 6-month-old Snai2112Δ mutants and their siblings by flow cytometry. Analyzing the cells by forward and side-scatter (A) displayed no significant difference between the various hematopoietic populations via two-way ANOVA (B). Error bars are SEM.
Fig 9.
Preliminary investigation of morphant vs. mutant phenotype.
WISH within 17 hpf embryos from an in-cross of snai2+/112Δ displayed that the Snail family members snai1a and snai1b are not differentially expressed in mutant embryos as compared to heterozygote and wild-type siblings (A). However, qPCR in pooled embryonic trunks at 26 hpf showed a different trend: all 3 additional members of the Snail family show increased expression (B). This graph presents the average of two independent experiments in which embryonic heads were removed and genotyped, followed by pooling of trunks of the same genotype. Error bars are SD. Snai2 reverse primer is designed within the mutant deletion, so transcript decrease reflects present of mutant transcript. We also observed the effect on emerging HSCs when the SB MO was injected into mutant embryos and their siblings by observing both WISH for runx1 at 26 hpf (C), and the double positive population in Tg(CD41:GFP/kdrl:mCherry) embryos at 48 hpf. Double positive cells were filtered by the surfaces function on Imaris, quantified, and submitted to statistical testing by a non-parametric t-test on Prism (D). Error bars are SEM. By both analyses, HSC specification was affected by SB MO in all genetic backgrounds. Black arrowheads point to the middle of the aortic runx1 expression. **** represents p<0.0001. WT: Wild-type.