MicroRNAs Establish Uniform Traits during the Architecture of Vertebrate Embryos

Abstract

Proper functioning of an organism requires cells and tissues to behave in uniform, well-organized ways. How this optimum of phenotypes is achieved during the development of vertebrates is unclear. Here, we carried out a multi-faceted and single-cell resolution screen of zebrafish embryonic blood vessels upon mutagenesis of single and multi-gene microRNA (miRNA) families. We found that embryos lacking particular miRNA-dependent signaling pathways develop a vascular trait similar to wild-type, but with a profound increase in phenotypic heterogeneity. Aberrant trait variance in miRNA mutant embryos uniquely sensitizes their vascular system to environmental perturbations. We discovered a previously unrecognized role for specific vertebrate miRNAs to protect tissue development against phenotypic variability. This discovery marks an important advance in our comprehension of how miRNAs function in the development of higher organisms.

Keywords: cardiovascular development; endothelial cells; environmental stress; miR-139; miR-223; miR-24; phenotypic variability; robustness; target gene networks; zebrafish mutants.

Figure 1. Generation of endothelial miRNA mutants in zebrafish

(A) Experimental procedure to identify miRNAs expressed in fluorescence activated cell (FAC)-sorted Kdrl:GFP⁺ endothelial cells and Kdrl:GFP⁻ non-endothelial cells during the four major stages of zebrafish vascular development. Dashed boxes outline the regions examined for cardiovascular phenotypes in endothelial miRNA mutant embryos (see Figure S2C). (B) Heat map depicts miRNA reads per million in Kdrl:GFP⁺ endothelial relative to non-endothelial cells for 2 biological replicates. Color scale ranges from the first quartile (Q1) to the third quartile (Q3) fold enrichment values for all 46 endothelial miRNAs identified (see Figure S1A). (C) Average mature miRNA levels relative to U6 snRNA expression as determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR) in FAC-sorted Kdrl:GFP⁺ endothelial cells at the indicated developmental stages. (D) Top, lateral trunk view of wild type embryos showing whole mount in situ hybridization (WISH) for mature miR-139, labeling cells within the ISV position. Confocal image shows the relative position of ISVs in the lateral trunk. Bottom, northern blot and respective quantification showing mature miR-139 expression relative to total RNA in three biological replicates of 32 hpf embryos treated as indicated. Consistent with miR-139 expression in ISVs, mature miR-139 levels were diminished in etv2 morphant embryos, which lack these trunk vessels (Pham et al., 2007). (E) Mature miR-24 localization in the ventral head of wild type embryos in relation to Kdrl:GFP⁺ vasculature at the indicated developmental stages. By 6 dpf, miR-24 remained in vascular cells, but was excluded from cartilaginous and bone structures. Arrows point to anatomic landmarks. Yellow arrows point to the region captured in zoomed-in images. (F) Mature miR-223 localization in the lateral Kdrl:GFP⁺ trunk vasculature of wild type embryos at the indicated stages. At 54 hpf, miR-223 is expressed in cells within the caudal hematopoietic tissue (CHT) located between the DA and CV. Arrows show examples of miR-223⁺ cells. Yellow arrows point to the region captured in magnified images. (G–I) Schematic representation of the genome editing strategies employed to mutagenize endothelial miRNAs. TALENs and a multiplexed CRISPR/Cas9 system were targeted to miRNA precursor genomic sequences to prevent mature miRNA formation. Grey boxes represent the wild type allele. Colored boxes reveal the nature of the mutant allele. See also Figure S2A. (J–L) qRT-PCR showing average mature miRNA expression normalized to U6 snRNA levels in miRNA mutant embryos (J), embryo heads (K) or adult fins (L) relative to wild type. For miR-24 mutants, genotypes were categorized as a single mutant when two (Δ2, miR-24-4Δ/Δ) and three (Δ3, ex. miR-24-1+/Δ 4Δ/Δ) miR-24 alleles were mutated, up to a quadruple mutant that lacked all eight miR-24 alleles (Δ8, miR-24-1Δ/Δ 2Δ/Δ 3Δ/Δ 4Δ/Δ). See also Figure S2B. Bar plots represent mean + stardard error of the mean (SEM) and significance calculations are relative to wild type embryos. n.s. (not significant, p > 0.05), *p ≤ 0.05, **p ≤ 0.01, and ****p ≤ 0.0001, two-tailed Student’s t test. qRT-PCR data represent 2–5 biological replicates. 10–20 embryos from at least two different clutches were examined by WISH. Abbreviations: AA, aortic arches; DA, dorsal aorta; CV caudal vein; E, eye; EC, endothelial cell; H, heart; HA, hypobranchial artery; ISV, intersegmental vessel; PA, pharyngeal arch; PCV, posterior cardinal vein.

Figure 2. Genome-wide identification of vascular target gene networks regulated by endothelial miRNAs

(A–D) Strategy to identify vascular transcripts regulated by endothelial miRNAs. Kdrl:GFP⁺ endothelial cells were FACS-sorted from dissected 27 hpf wild type, miR-139Δ/Δ, and miR-223Δ/Δ trunk tissue (A, C) or 51 hpf wild type and miR-24Δ/Δ head tissue (B) and used to generate Quantseq 3′ mRNA libraries to identify differentially expressed genes (DEG) between wild type and mutant samples. miR-24Δ/Δ samples represent a mixed clutch of double, triple, and quadruple mutant genotypes. Standard mRNA-seq libraries were generated to assess endothelial cell gene expression levels in whole embryos across the indicated developmental times (D). (E) Circos plot depicting the dynamic regulation of vascular genes upregulated (log₂-fold change (FC) ≥0.26, p-value ≤0.05) in miR-139Δ/Δ trunk (blue sector), miR-24Δ/Δ head (red sector), and miR-223Δ/Δ trunk (green sector) endothelial cells compared to wild type controls. Colored and gray links indicate upregulated vascular genes that contain or lack a computationally-predicted target site for the indicated miRNA, respectively. Links connect to stage specific heat maps in the gray sector, which show overall expression levels of genes differentially expressed between wild type and mutant Kdrl:GFP⁺ endothelial cells in terms of FPKM (fragments per kilobase of exon per million mapped reads). (F–H) Bar plots of the most significantly enriched Gene ontology terms amongst upregulated (log₂-fold change (FC) ≥0.26) vascular target genes for each miRNA. GO terms have −log₁₀ (Enrichment P value < 0.05).

Figure 3. miR-139 homozygous mutants exhibit heterogeneity in endothelial cell filopodia number

(A) Lateral view of Kdrl:GFP⁺ trunk vasculature at the indicated developmental stages. Arrows point to ISVs. (B) Average ISV length (μm) was determined from confocal projections (n = 9–18). Bar plots show mean + SEM (C) Representative images of single endothelial cells expressing tol2-Fli1a-H2B-BFP-p2A-EGFP-Farnesyl vector in wild type and miR-139Δ/Δ ISVs. miR-139Δ/Δ images depict endothelial cells with excessive (top) or few (bottom) filopodia. Endothelial cell nuclei are in blue and membranes are in green. (D) Bar plots show the average of replicate means + SEM of endothelial cell filopodia number (n = 9 replicates). (E) Violin plots show filopodia number probability density distributions. Solid lines in box plots depict median values. Phenotypic variability was significantly different from wild type ***p = 9.46 × 10⁻⁴ by Levene’s test (n = 36 cells). (F) Bar plots show average replicate standard deviation (SD) + SEM of endothelial cell filopodia number (n = 9 replicates). Significance calculations were relative to wild type embryos. n.s. (not significant, p > 0.05), *p ≤ 0.05, ***p ≤ 0.001, two-tailed Mann-Whitney U-test unless otherwise indicated. Abbreviations as defined in Figure 1. See also Figure S3.

Figure 4. Enhanced phenotypic variability in hypobranchial artery sprouting with diminishing miR-24 activity

(A) sox9a WISH labeling chondrocyte progenitors (left) and alcian blue stained cartilage (right) in the ventral head of wild type and miR-24 mutant embryos (n ≥ 6 for all genotypes except n = 2 for quadruple mutants). Numbers indicate the position of pharyngeal arches 1–7. miR-24 genotypes are categorized as in Figure S2B. (B) Ventral view of 54 hpf head vasculature. Magnified images depict the spectrum of HA phenotypes (dotted outlines) for each genotype. The HA sprout most representative of the genotype is indicated with a yellow arrow and is depicted in the zoomed-in picture with a yellow HA label. White arrows point to aortic arches (AA). (C) Bar plots show the average of replicate means + SEM of HA length (μm) at 54 hpf (n = 4 replicates). (D) Violin plots show 54 hpf HA length (μm) probability density distributions. Solid lines in box plots depict median values. Phenotypic variability was statistically different from wild type as determined by the Levene’s test (n = 40 embryos). single: *p = 0.04; double: ****p = 5.77 × 10⁻⁶; triple: ***p = 3.46 × 10⁻⁴; quadruple: ***p = 7.57 × 10⁻⁴ (F) Bar plots show average replicate standard deviation (SD) + SEM of HA length (μm) at 54 hpf (n = 4 replicates). For all bar blots, S = single, D = double, T = triple, Q = quadruple mutant. Significance calculations were relative to wild type embryos. n.s. (not significant, p > 0.05), *p ≤ 0.05, ***p ≤ 0.001, ****p ≤ 0.0001, two-tailed Mann-Whitney U-test unless otherwise indicated.

Figure 5. miR-223 does not regulate phenotypic variability of HSPC number

(A) WISH of HSPC marker cmyb at transient sites of hematopoiesis, namely the DA ventral wall at 32 hpf, the caudal hematopoietic tissue between the DA and CV at 54 hpf, and the thymus, a definitive site of hematopoiesis, at 6 dpf (n = 10–20 embryos). Arrows show examples of cmyb⁺ cells. Yellow arrows indicate the region of the lateral trunk that is depicted in magnified images. (B) Mean cmyb expression in miR-223Δ/Δ embryos normalized to actb1 levels and compared to wild type as determined by qRT–PCR for 3 biological replicates. Bar plots show mean + S.E.M., two-tailed Student’s t test (C) Representative micrographs of the lateral trunk in embryos expressing Tg(kdrl:ras-mCherry)^s896 and Tg(cmyb:GFP)^zf169. Arrows point to examples of kdrl⁺ cmyb⁺ cells budding from the DA ventral wall at the peak of hematopoiesis. Yellow arrows show region of the lateral trunk that is captured in zoomed-in images. (D) Bar plots show the average of replicate means + SEM of kdrl⁺ cmyb⁺ cell number at 36 hpf (n = 4 replicates). (E) Violin plots show kdrl⁺ cmyb⁺ cell number probability density distributions. Solid lines in box plots depict median values. Phenotypic variability was not significantly different from wild type p = 0.24 by Levene’s test (n = 36 embryos). (F) Bar plots show average replicate standard deviation (SD) + SEM of kdrl⁺ cmyb⁺ cell number (n = 4 replicates). Significance calculations were relative to wild type embryos. n.s. (not significant, p > 0.05), *p ≤ 0.05, **p ≤ 0.01, two-tailed Mann-Whitney U-test unless otherwise indicated. Abbreviations as defined in Figure 1.

Figure 6. miR-139 and miR-24 mutants are broadly affected by stress

(A–B) Schematic depicts duration of chemical exposure or environmental condition used to test stress sensitivity of miR-139-regulated (A) and miR-24-regulated (B) vascular traits in absence of miRNA activity. Stressors were administered at doses that minimally affect wild type as indicated (see Table S1). (C–D) Arrows point to ectopic ISV branches in 54 hpf miR-139Δ/Δ embryos treated with pro-angiogenic drugs: BIO (n = 24) and GSI (n = 50–55 embryos). Controls were treated with DMSO vehicle (n = 17–24 embryos). (E) Arrows highlight stunted ISV branches in 27 hpf miR-139Δ/Δ embryos treated with anti-angiogenic VEGF inhibitor SU5416 (n = 25–27 embryos). Controls were treated with DMSO vehicle (n = 17–25 embryos). (F) Arrows show ectopic ISV branches in 54 hpf miR-139Δ/Δ embryos exposed to high temperature stress. (n = 31–44 embryos/genotype) (G) Arrows point to hypersprouting of DLAV endothelial cells in 52 hpf miR-139Δ/Δ embryos exposed to 4 hours of hypoxia (n = 40–45 embryos/genotype). (H) Arrows indicate the HA of BIO-treated embryos. (I–M) Quantification of 56 hpf HA length (μm) upon treatment of BIO or DMSO (n = 12–24 for all genotypes except n = 6 for quadruple mutant embryos, I); GSI or DMSO (n = 15, J); SU5416 or DMSO (n = 11–16, K); high or control temperature (n = 38 – 50, L), and hypoxia or normoxia (n = 24, M). S = single, D = double, T = triple, Q = quadruple mutant embryos All bar plots represent mean + SEM and significance comparisons between samples are as indicated. n.s. (not significant, p > 0.05), *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 and ****p ≤ 0.0001, two-tailed Mann-Whitney U-test.

Figure 7. Phenotypic heterogeneity determines trait stress sensitivity

(A) Schematic depicts duration of NO donor SNAP exposure to test stress sensitivity of kdrl⁺ cmyb⁺ cell number in the absence of miR-223 activity. SNAP was given at a low dose that minimally affects wild type as indicated (See Table S1). (B) Arrows show kdrl⁺ cmyb⁺ cells budding from the DA ventral wall in untreated or SNAP-treated 36 hpf and miR-223 Δ/Δ (n = 11–12 embryos). (C) Bar plots represent mean kdrl⁺ cmyb⁺ cell number + SEM. n.s. (not significant, p > 0.05), two-tailed Mann-Whitney U-test. (D) Proposed model for miRNA-mediated regulation of developing traits in vertebrates. miRNA targeting of single or multiple mRNAs in complex genetic networks can control the construction and/or robustness of a phenotypic trait. Upon loss of miRNA activity, a change only in phenotypic distribution (Gaussian or otherwise) predetermines a trait’s sensitivity to changing environments.