Select microRNAs are essential for early development in the sea urchin

Abstract

microRNAs (miRNAs) are small noncoding RNAs that mediate post-transcriptional gene regulation and have emerged as essential regulators of many developmental events. The transcriptional network during early embryogenesis of the purple sea urchin, Strongylocentrotus purpuratus, is well described and can serve as an excellent model to test functional contributions of miRNAs in embryogenesis. We examined the loss of function phenotypes of major components of the miRNA biogenesis pathway. Inhibition of de novo synthesis of Drosha and Dicer in the embryo led to consistent developmental defects, a failure to gastrulate, and embryonic lethality, including changes in the steady state levels of transcription factors and signaling molecules involved in germ layer specification. We annotated and profiled small RNA expression from the ovary and several early embryonic stages by deep sequencing followed by computational analysis. miRNAs as well as a large population of putative piRNAs (piwi-interacting RNAs) had dynamic accumulation profiles through early development. Defects in morphogenesis caused by loss of Drosha could be rescued with four miRNAs. Taken together our results indicate that post-transcriptional gene regulation directed by miRNAs is functionally important for early embryogenesis and is an integral part of the early embryonic gene regulatory network in S. purpuratus.

Fig. 1. Dynamic expressions of the sea urchin miRNAs

Hierarchical clustering of miRNAs based on gene expression patterns is plotted as a heat map. Most miRNAs are expressed maternally and are upregulated by 24 and 48 hpf. The “*” corresponds to the minor miRNA precursor product found at lower concentration. Three novel miRNAs are spu-miRDeep2-35240, spu-miRDeep2-5317, and spu-miRDeep2-30364.

Fig. 2. Sea urchin small RNA composition and expression profile

Small RNAs from the ovary, egg, 32-cell stage, blastula (24 hpf), gastrula (48 hpf), and pluteus (72 hpf) were sequenced and annotated. miRNAs are 22 nts and putative piRNAs are between 27 and 29 nts. Putative piRNAs that overlap with each other in consistency with conserved ‘ping-pong’ mode of biogenesis (see Methods) are labeled ‘piRNA’ while the remaining are labeled ‘Unknown’. The figure is scaled to yield identical vertical extend of the 22 nucleotide miRNA fraction. Compare to figure S3 for an illustration with static scaling.

Fig. 3. Dicer is required for early sea urchin development

(A,B) Dicer morpholino antisense oligonucleotide (MASO)-injected embryos have dose-dependent developmental defects. (B) Barplots depicting the percentages of normal embryos in each experimental treatment relative to the percentage of normal embryos in the mock injected control at 48 hpf. n is the number of embryos analyzed. Unpaired Student T-test was used to determine the significance of the knockdown compared to the mock control. p-value was 0.025 for the Dicer MASO 24nM sample. (C) Dicer knockdowns resulted in a decrease of miRNAs. A miRNA unique to the sea urchin spu-miR-2009 and miR-31, a conserved miRNA, were decreased in Dicer knockdown embryos compared to the control mock-injected embryos at 24 hpf. Standard deviation error bars are from 4 replicates.

Fig. 4. Drosha knockdown display similar developmental defects as the dicer knockdown

Morpholino antisense oligonucleotides of variable concentrations were injected into newly fertilized eggs. (A,C) DIC images of embryos at 24 hpf, 48 hpf, and 72 hpf. Scale bar is 50 μm. (B,D,E) Barplots depicting the percentages of normal embryos in each experimental treatment relative to the percentage of normal embryos in the mock injected control at 48 hpf. n is the total number of injected embryos. Unpaired Student T-test was used to determine the significance level between the knockdown and the mock control. The p-values are 0.015 and 0.009 for Drosha MASO 12nM and Drosha MASO 24nM, respectively (A,B) Drosha MASO-injected embryos have dose-dependent developmental defects, whereas only very few (C,D) DGCR8 MASO injected embryos show the same phenotype and most do not. (E) Injecting morpholinos against DGCR8 + Drosha or DGCR8 + Dicer leads to a decreased percentage of normal embryos as compared to each MASO-injection alone. A lower concentration of Drosha and Dicer MASO at 16 nM instead of 24 nM (lanes 2 and 3) was used so that we can observe the further decrease in the percentage of normal embryos in MASO co-injections. An average of two biological replicated is presented.

Fig. 5. miRNAs regulate broad gene sets

Embryos at 48 hpf were immunolabeled with Endo1 (A) and Meso1 (B) monoclonal antibodies to identify cell-type specific differentiation markers of endoderm and mesoderm, respectively. Scale bar is 20μm. (C) RT-QPCR measurement of mRNAs levels of molecules that mediate transcriptional regulation, cell signaling, cell adhesion, cell movement, and cell proliferation. QPCR results from 2–3 and 3–6 independent biological experiments are measured in 17h and 24h embryos, respectively.

Fig. 6. Four miRNAs rescue developmental defects from Drosha loss of function

Embryos were injected with Drosha MASO with or without exogenous dsRNAs. Barplots depicting the percentages of normal embryos in each experimental treatment relative to the percentage of normal embryos in the mock injected control at 48 hpf. (A) Drosha knockdown embryos (24nM) were complemented with 160nM or 32nM of dsRNA duplexes, miR-1, -31, -2012, -71, -153, and -375, or a dsRNA negative control (NCI). Injections of dsRNA-1, -31, -2012, or -71 alone did not cause developmental defects (data not shown). n is the total number of injected embryos. (B) Embryos with Drosha knockdown (24nM) complemented with dsRNA duplexes (160nM) developed into normal larvae.

Fig. 7. miR-71 rescues significant Drosha knockdown phenotype

Single and combinations of dsRNAs corresponding to miR-1, -31, -71, and -2012 Dicer substrates were coinjected with the Drosha MASO into newly fertilized eggs. The percentages of normal and abnormal embryos (excluding dead embryos) are tabulated at 24 hpf. Two independent biological experiments were conducted.