microRNA-124 directly suppresses Nodal and Notch to regulate mesodermal development

Abstract

MicroRNAs regulate gene expression post-transcriptionally by destabilizing and/or inhibiting translation of target mRNAs in animal cells. MicroRNA-124 (miR-124) has been examined mostly in the context of neurogenesis. This study discovers a novel role of miR-124 in regulating mesodermal cell differentiation in the sea urchin embryo. The expression of miR-124 is first detectable at 12hours post fertilization at the early blastula stage, during endomesodermal specification. Mesodermally-derived immune cells come from the same progenitor cells that give rise to blastocoelar cells (BCs) and pigment cells (PCs) that must make a binary fate decision. We determined that miR-124 directly represses Nodal and Notch to regulate BC and PC differentiation. miR-124 inhibition does not impact the dorsal-ventral axis formation, but result in a significant increase in number of cells expressing BC-specific transcription factors (TFs) and a concurrent reduction of differentiated PCs. In general, removing miR-124's suppression of Nodal phenocopies miR124 inhibition. Interestingly, removing miR-124's suppression of Notch leads to an increased number of both BCs and PCs, with a subset of hybrid cells that express both BC- and PC-specific TFs in the larvae. Removal of miR-124's suppression of Notch not only affects differentiation of both BCs and PCs, but also induces cell proliferation of these cells during the first wave of Notch signaling. This study demonstrates that post-transcriptional regulation by miR-124 impacts differentiation of BCs and PCs by regulating the Nodal and Notch signaling pathways.

Keywords: Blastocoelar cells; Pigment cells; Post-transcriptional regulation; Sea urchin.

Figure 1.. Blastocoelar cells are specified before pigment cells.

(A) Schematic of combined work from our research and others to demonstrate how blastocoelar and pigment cells specify and differentiate in the purple sea urchin. Mesodermal development is initiated by the activation of Pmar in the large micromeres that will become the primary mesenchyme cells (PMCs). Pmar inactivates HesC to allow the expression of Delta in the presumptive PMCs to initiate the first wave of Notch signaling that will initiate the specification of the presumptive NSM. Nodal signaling will first set up dorsal-ventral axis prior to 10hpf (morula) and then activates Not to allow specification of BCs. Lastly, before ingression of PMCs into the blastocoel, Delta will signal to Notch in the BCs and PCs to further activate genes important for their differentiation prior to their ingression into the blastocoel to perform their immune functions (Duboc et al., 2010; Range et al., 2008; Revilla-i-Domingo et al., 2007). (B) FISH was performed at several time points to reveal the timing of BC and PC specification in the purple sea urchin. Gcm is expressed in BC and PC progenitor cells starting at 8hpf. Ese is used to follow specified BCs, and Pks1 is used to follow specified PCs. Gcm becomes restricted in PCs to the aboral side at 14–16hpf (mid- to late blastula). Initiation of specification of BCs begins with Ese expression at 10hpf (morula) (indicated by the dotted box), and specification of PCs begins when Pks1 is expressed at detectable levels at 14hpf (mid-blastula) (indicated by the dotted box). A range of 3–8 confocal images slices were compiled into a single maximum projection image. Scale bar=50μm. 3 biological replicates.

Figure 2.. miR-124 is expressed during Nodal signaling and towards the end of the first wave of Notch signaling.

(A) miR-124 qPCR was performed at different developmental timepoints. Individual blue circles represent the three replicates, with the bar graph indicating the averages. SEM is graphed. miR-124 is expressed at 18hpf. (B) Embryos were collected at different developmental timepoints and hybridized with either miR-124 or the scrambled control LNA RNA probe. The expression of miR-124 is first detectable at 12hpf, peaks at 16hpf, and subsides after 18hpf. V=ventral ectoderm; D=dorsal ectoderm (C) Double FISH with miR-124 (green) and ventral marker Not (magenta) experiment indicates that miR-124 is more enriched on the dorsal/aboral side of the embryo. This asymmetric expression of miR-124 persists to the 24hpf (swimming blastula stage). Not seems to be highly expressed in cells that are dividing. (D) Double FISH with miR-124 (green) and Notch (magenta) was performed. miR-124 co-expresses with Notch in NSM cells in late blastula stage. A=anterior; P=posterior. For each developmental stage, we examined approximately 40 embryos. At least 3 biological replicates were performed. Single confocal images are presented.

Figure 3.. Inhibition of miR-124 results in increased BCs at the expense of differentiated PCs.

(A) The number of PCs were counted based on their natural purple pigmentation. miR-124 inhibitor-injected larvae had decreased differentiated PCs compared to the control embryos. 3 biological replicates. Black arrows point to some PCs. (B) PCs were immunolabeled with PC-specific Sp1 antibody (green) and counterstained with DAPI to label DNA (blue). We observed a similar decrease in PCs in the miR-124 inhibitor-injected larvae in comparison to the controls. C=control; Inh=miR-124 inhibitor; R=miR-124 inhibitor co-injected with miR-124 mimic. 3 biological replicates. Student T-test. Maximum intensity projection of Z-stack confocal images is presented. (C) miR-124 inhibitor-injected larvae had more 185/333 and MacpfA2-positive BCs (green) than the control. Larvae were counterstained with DAPI (blue). C=control; Inh=miR-124 inhibitor. 3 biological replicates. Student T-test. Maximum intensity projection of Z-stack confocal images is presented.

Figure 4.. Nodal is a direct target of miR-124 and miR-124 inhibition leads to expanded Not expression domain.

(A) Dual luciferase assay results indicated that miR-124 directly suppresses Nodal. Each biological replicate contained 50 embryos. 3 biological replicates. SEM is graphed. Student’s t-test was used. WT= wild type miR124 site; D=deleted miR-124 site. (B) miR-124 inhibitor-injected blastulae had increased and expanded Not expression compared to the control. The expression domain of Not is measured from anterior to posterior region and presented as a ratio to the height of the embryo. 3 biological replicates. Scale bar = 50μm.

Figure 5.. Removing miR-124’s direct suppression of Nodal results in increased BCs and a concurrent decrease in differentiated PCs.

(A) Nodal TP-injected blastulae at 24hpf were subjected to RNA in situ hybridization with BC and PC-specific probes. A significant increase in BC-specific Ese and Scl expression domains was observed in the Nodal TP-injected embryos compared to the control TP-injected embryos. Schematic indicates the measurement of the expression domains. CTP=Control TP; NTP=Nodal TP. Student T-test. 3 biological replicates. (B) Control and Nodal TP-injected larvae were hybridized with BC markers 185/333 and MacpfA2. BCs are increased in Nodal TP-injected larvae. (C) Nodal TP-injected embryos have increased expression domains of PC-specific Gcm and Pks1 at the blastula stage. (D) Nodal TP-injected embryos were cultured to 72hpf and exhibited 2-fold decrease in PCs. (E) PCs were immunolabeled with PC-specific Sp1 antibody (green) and counterstained with DAPI (blue). We observed a similar decrease in PCs in the Nodal TP-injected embryos in comparison to the controls. 3 biological replicates. Maximum intensity projection of Z-stack confocal images is presented. CTP=Control TP; NTP=Nodal TP. Student T-test is used for statistical analyses.

Figure 6.. Removal of miR-124’s suppression of Notch results in expanded expression domains of BC and PC-specific TFs as well as the number of BCs and PCs.

(A) Double FISH was conducted on Notch TP and Control TP-injected embryos at the blastula stage, using BC-specific TF Ese and PC-specific TF Pks1 RNA probes. Notch TP-injected embryos exhibited an expanded expression domain of both Ese and Pks1 during the blastula stage compared to the Control TP-injected embryos. (B) Notch TP-injected larvae have increased numbers of both Ese-positive cells and Pks1-positive cells, with a few hybrid cells expressing both markers (arrows). (C) Number of BCs, PCs, and hybrid cells (arrows) in Control and Notch TP-injected embryos are presented in a graph. Images indicate enlarged views of hybrid cells in Notch TP-injected embryos. CTP=Control TP (N=38); NTP=Notch TP (N=36). Student T-test. 3 biological replicates.

Figure 7.. Removal of miR-124’s suppression of Notch results in an increased number of both BCs and PCs.

(A) Embryos were incubated with Click-It EdU to detect cell proliferation for 30 min at 16hpf. At 24hpf, blastulae were fixed and incubated with BC and PC-specific markers and counterstained with DAPI to visualize DNA. Notch TP-injected embryos have more BCs and PCs that are undergoing cell division compared to control. Dotted white lines indicate the inset. (B) Inset images show enlarged areas of posterior region of the embryos. Graph indicates the number of proliferating BCs and PCs in Control and Notch TP-injected embryos. CTP=control TP (N=39) and NTP=Notch TP (N=31). Student T-test. 3 biological replicates. Maximum intensity projection of Z-stack confocal images is presented. *p<0.05; **p<0.001.

Figure 8.. Model of regulatory mechanism of miR-124 in BC and PC development.

(A) Summary of results. (B) Early in sea urchin development, BCs and PCs share the same progenitor cells. Initiation of specification of BCs begins with Nodal expression (blue) in the oral ectoderm that activates Not (orange), which activates BC-specific TFs and inhibits PC-specific TFs on the ventral/oral side of the embryo. miR-124 directly suppresses Nodal and Notch. miR-124 inhibition results in increased Not, leading to increased BCs at 14–16hpf. miR-124 continues its expression to 18hpf, when it regulates Notch signaling to impact BC and PC differentiation and proliferation. (C) Expression of miR-124 in various organisms throughout phylogeny is summarized. miR-124 is enriched in the neural system of all organisms.