Abstract
microRNAs are evolutionarily conserved non-coding RNAs that direct post-transcriptional regulation of target transcripts. We use the sea urchin embryo to achieve a comprehensive understanding of miR-1’s function in a developing embryo. Results indicate that miR-1 regulates gut contractions, specification, and positioning of serotonergic neurons, as well as mesodermally-derived muscles, pigment cells, and skeletogenic cells. Gain-of-function of miR-1 generally leads to more severe developmental defects than its loss-of-function. We identified that miR-1 directly suppresses Ets1/2, Tbr, and VegfR7 of the skeletogenic gene regulatory network, and Notch, Nodal, and Wnt1 signaling components. We found that miR-1’s direct suppression of Nodal may indirectly regulate FoxQ2 to impact serotonergic neurons. Excess miR-1 may lead to decreased Nodal and Notch that result in decreased circumpharnygeal muscle fibers and the number of pigment cells. The striking ectopic skeletal branching induced by miR-1 mimic injections may be explained by miR-1’s direct suppression of Nodal that leads to expression changes of Vegf3, and Fgfa that mediate skeletogenesis. This work demonstrates that miR-1 plays a diverse regulatory role that impacts tissues derived from all germ layers.
Summary statement This study identifies wide-ranging regulatory roles and regulatory mechanisms of miR-1 that impact structures derived from all three germ layers during embryonic development.
Introduction
microRNA-1 (miR-1) is classified as a myomiR because of its enriched expression in vertebrate muscle tissue and its function in myogenesis, angiogenesis, and vascularization (Mansfield et al., 2004; Sokol and Ambros, 2005; Wienholds et al., 2005; Zhao et al., 2005; McCarthy, 2011). miR-1 has also been implicated in regulating neuronal growth and survival by directly targeting Bdnf, Hes1, and potentially Hsp-70 (Varendi et al., 2014; Chang et al., 2016; Yi et al., 2016; Zheng et al., 2017). While there are extensive studies demonstrating miR-1’s role in angiogenesis and myogenesis, the exact molecular regulatory mechanisms of miR-1 are still not fully understood. Additionally, a systematic analysis of both loss and gain-of-function of miR-1 in a developing embryo is still lacking.
The miR-1 sequence is highly conserved amongst metazoans (Fig. S1). Most mammalian genomes contain two copies of miR-1 that perform similar functions (Heidersbach et al., 2013). Previously, we found that miR-1 is one of the most expressed miRNAs in the purple sea urchin embryo (Song et al., 2012). The sea urchin has ∼50 annotated miRNAs, which is relatively small in contrast to the 519 miRNAs in humans (Song et al., 2012; Fromm et al., 2015). The sea urchin embryo contains a single miR-1, making it feasible to use this embryo to provide a deeper understanding of miR-1’s function in development.
The developmental processes of the sea urchin and humans are remarkably similar at the cellular and molecular level (Adonin et al., 2020). Similar to vertebrates, they utilize highly conserved signaling pathways such as the canonical Wnt (cWnt)/β-catenin signaling for anterior-posterior (AP) axis formation (Wikramanayake et al., 1998; Angerer and Angerer, 2000; Komiya and Habas, 2008) and Nodal/Bmp signaling for specification of the dorsal-ventral (DV) secondary body axis (Duboc et al., 2004; Furtado et al., 2008; Chhabra et al., 2019). Immediately after fertilization, maternal inputs, zygotic transcription, and signaling mechanisms help define distinct gene regulatory networks (GRNs) (Fig. 1A) (Logan et al., 1999; Martik et al., 2016). By the mesenchyme blastula stage (24 hours post fertilization; hpf), germ layer specification has already occurred;during gastrulation, the three germ layers are differentiated by cross-regulation between signaling pathways, GRN interactions (Lyons et al., 2012), as well as post-transcriptional regulation mediated by miRNAs (Stepicheva and Song, 2015; Sampilo et al., 2018; Sampilo et al., 2021).
Although the body plans and structures of deuterostomes are diverse, the sea urchin embryo and vertebrates utilize conserved factors for analogous structures. For example, the sea urchin skeletogenesis is thought to be analogous to vertebrate angiogenesis and vascularization since these processes utilize a common set of transcription factors (TFs) (Ets1/2, Erg, Hex, Tel, and FoxO) and signaling pathways (Vegf, Nodal/Bmp, Notch, and Angiopoetin) (Duboc et al., 2005; Morgulis et al., 2019). In response to the Vegf ligand in the ectoderm, the sea urchin VegfR-expressing skeletogenic primary mesenchyme cells (PMCs) initiate the formation of the skeletal rudiment, differentiate, and pattern (Ettensohn and McClay, 1986; Duloquin et al., 2007; McIntyre et al., 2014). The migrating PMCs form a syncytium, connected by filopodial membranes between cell bodies where biomineralization enzymes form calcite granules (Ettensohn and McClay, 1986; Armstrong and McClay, 1994; Wilt, 2002). Similarly, the sea urchin larva utilizes similar TFs as vertebrates (FoxA, GataE, Xlox, Cdx) critical for gut differentiation (Annunziata et al., 2019). The tripartite gut is compartmentalized with the cardiac, pyloric, and anal sphincters (Fig. 2B) (Yaguchi and Yaguchi, 2019). For the nervous system, the sea urchin embryo has serotonergic neurons and ganglia located in the apical organ that is analogous to the vertebrate central nervous system and sensory and motor neurons within the ciliary band that is analogous to the peripheral nervous system that control swimming and feeding behaviors (Yaguchi and Katow, 2003; Wood et al., 2018; Yaguchi and Yaguchi, 2021). Additionally, the sea urchin embryo uses orthologous neuronal transcriptional factors as those expressed in the vertebrate forebrain (Six3, ZIC2, Achaete-scute, NKX2.1 and FEZ) (Wei et al., 2009). Thus, using the sea urchin as a simple and experimentally tractable organism, we can better understand complex molecular mechanisms.
We discovered that miR-1 regulates the endodermally-derived gut contractions, specification and positioning of ectodermally-derived serotonergic neurons, as well as mesodermally-derived circumpharyneal muscles, pigment cells (PCs), and skeletogenesis. Interestingly, gain-of-function of miR-1 leads to more severe developmental defects than its loss-of-function. We further identified miR-1 to directly suppress Ets1/2, Tbr, and VegfR7 of the PMC GRN, and signaling components Notch, Nodal, and Wnt1. We found that miR-1’s direct suppression of Nodal may indirectly regulate FoxQ2 to impact serotonergic neurons. miR-1 mimic-induced overexpression may lead to decreased Nodal and Notch that result in decreased circumpharnygeal muscle fibers and the number of pigment cells. Additionally, excess miR-1 also resulted in numerous ectopic skeletal branching that may be explained by miR-1’s direct suppression of Nodal that leads to expression changes of Vegf3 and Fgfa that mediate patterning and skeletogenesis, respectively. This study identifies wide-ranging regulatory roles and regulatory mechanisms of miR-1 that impact structures derived from all three germ layers during embryonic development.
Experimental Procedures
Animals
Adult purple sea urchin, Strongylocentrotus purpuratus (Sp), were obtained from Point Loma Marine Invertebrate Lab, (Lakeside, CA) and Marinus Scientific, LLC (Long Beach, CA). Adult males and females were intracoelomically injected with 0.5 M KCl to obtain sperm and eggs. Filtered natural seawater (FSW) (collected from Indian River Inlet; University of Delaware) or artificial seawater (ASW) made from Instant Ocean© was used for embryo cultures incubated at 15°C.
Cloning
For generating 3’UTR luciferase reporter constructs of predicted miR-1 targets, PCR primers, or FragmentGENE DNA fragments (Genewiz, South Plainfield, NJ) were designed based on sequence information available from the sea urchin genome (echinobase.org) (Table S1). Amplified PCR products of Bmp2/4, Ets1/2, Notch, Nodal, and Tbr 3’UTRs were first cloned into ZeroBlunt vector (Thermo Fisher Scientific, Waltham, MA), and then subcloned into the Renilla luciferase (Rluc) reporter construct. Wildtype (WT) constructs of IgTM and Nodal were commercially synthesized prior to cloning. VegfR7 and Wnt1 3’UTRs were previously cloned (Stepicheva and Song, 2015; Sampilo et al., 2021). Mutations were generated within the miR-1 seed sequences using the QuikChange Lightning or QuikChange Multi Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, California) to disrupt miR-1’s binding and regulatory function (Gregory et al., 2008; Stepicheva et al., 2015). The predicted miR-1 seed sites (canonical site: 5’ ACATTCC 3’) within the 3’UTRs of Ets1/2, Dri, Tbr, VegfR7, Notch, Nodal, Bmp2/4, Wnt1, and IgTM were modified at the third and fifth base pair (Table S1). We have previously demonstrated that truncated miRNA seed sequence differing in one nucleotide at the 5’ end is sufficient in miRNA-mRNA target recognition and function (Sampilo et al., 2021). Nodal and Wnt1 contain truncated miR-1 sites (6 out of 7 bps), and Bmp2/4 and IgTM contain mismatched miR-1 seed sites which differ in one nucleotide within the seed sequence (Table. S1). Only two of the three potential miR-1 binding sites within Nodal 3’UTR were mutated due to sequence complexity. Firefly luciferase was used as a normalization control as previously described (Stepicheva et al., 2015). Each of the construct sequences were verified by DNA sequencing (Genewiz, South Plainfield, NJ). Luciferase constructs containing Ets1/2, VegfR7, Nodal, and IgTM 3’UTRs were linearized with EcoRI, while Tbr, Bmp2/4, and Wnt1 3’UTRs were linearized with NotI. The luciferase constructs and Firefly luciferase mRNA were in vitro transcribed using the Sp6 mMessage machine kit (Ambion Inc, Austin, Texas). In vitro transcribed mRNAs were purified using Macherey-Nagel Nucleospin® RNA Clean-up kit (Macherey-Nagel, Bethlehem, PA) prior to injections.
Microinjections
Microinjections were performed as previously described (Cheers and Ettensohn, 2004; Stepicheva and Song, 2014) with modifications. All injection solutions were prepared in a 2.5 µl solution consisting of 20% glycerol and 0.4 μg/µl of 10,000 MW neutral non-fixable Texas Red dextran (Thermo Fisher Scientific, Waltham, MA). Approximately 1-2 picoliter was injected into each newly fertilized egg based on the size of the injection bolus at about one-fifth of the egg diameter. miR-1 miRCURY LNA miRNA Power Inhibitor and miRCURY LNA miRNA mimic were obtained from Qiagen (Germantown, MD). miR-1 inhibitor (Hsa-miR-1-3p, ID# YI04100840; 5’-UGGAAUGUAAAGAAGUAUGUAU-3’) and miR-1 miRCURY LNA miRNA mimic (ID#YM00472818; 5’-UGGAAUGUAAAGAAGUAUGUAU-3’) were used at 10 µM, 30 µM and 40 µM concentrations. Cel-miR-39-3p LNA mimic (ID#YM00479902; 5’-UCACCGGGUGUAAAUCAGCUUG-3’) was used as an injection control (not present in the sea urchin or humans). miR-1 inhibitor and miR-1 mimic were co-injected at a 1:1 molar ratio (40 µM miR-1 inhibitor + 40 µM miR-1 mimic) to test the specificity of miR-1 inhibitor.
Dual-luciferase quantification
The injection solutions for the dual-luciferase assay contained 20% sterile glycerol, 0.4 μg/µl 10,000 MW Texas Red lysine-charged dextran, 100-200 ng/μl Firefly mRNA, and 100 ng/μl Rluc mRNA (Ets1/2, Dri, Tbr, VegfR7, Notch, Nodal, Bmp2/4, Wnt1, and IgTM). 20-50 embryos were collected at the mesenchyme blastula stage (24 hpf). Dual-luciferase assays were performed using the Promega Dual-Luciferase Reporter (DLR™) Assay Systems with the Promega GloMax 20/20 Luminometry System (Promega, Madision, WI) (Stepicheva et al., 2015; Stepicheva and Song, 2015; Sampilo et al., 2018; Sampilo et al., 2021). The Rluc values were normalized to the Firefly signal to account for microinjection volume differences. Rluc data with mutated miR-1 seed sites were normalized to the Rluc with wildtype (WT) 3’UTR constructs. P-value was analyzed using Student’s t-test. All error bars represent Standard Error (SEM).
Immunofluorescence
Gastrulae and larvae were fixed in 4% paraformaldehyde (PFA) (20% stock; EMS, Hatfield, PA) in FSW overnight at 4°C (1D5, Serotonin, and Sp1), or fixed with 3.7% PFA for 20 min at RT followed by 1 min post-fix with ice-cold methanol (for SynB). 10X Phosphate Buffered Saline (PBS) (Bio-Rad, Hercules, CA) was diluted at 1:10. Three PBS-Tween (0.05% Tween-20 in 1X PBS for 1D5) or PBS-Triton (0.1% Triton in 1X PBS for Serotonin, Sp1, and SynB) washes were performed, followed by 1 h block with 4% sheep serum (MilliporeSigma, St. Louis, MO). 1D5 antibody was used at 1:50 to visualize PMCs (McClay et al., 1983); serotonin at 1:500 (MilliporeSigma, Cat#S5545) to visualize serotonergic neurons; Sp1 at 1:200 to visualize pigment cells (Developmental Studies Hybridoma Bank, Iowa City, Iowa, Cat#531884); and SynB at 1:200 to visualize mature and functional neurons (Burke et al., 2006; Leguia et al., 2006) (gift from Dr. Gary Wessel, Brown University). Antibodies were diluted in PBS-Tween/Triton with 4% sheep serum, and embryos were incubated overnight to 3 days at 4°C and washed 3 times with PBS-Tween/Triton, followed by goat-anti mouse (1D5 and Sp1) or anti-rabbit (Serotonin and SynB) secondary antibody (Thermo Fisher Scientific, Waltham, MA) at 1:300 for 1 h at RT. Embryos were then washed 3 times with PBS-Tween/Triton and mounted on slides for confocal imaging. For visualization of DNA, embryos were counterstained with Hoechst dye (Lonza, Walkersville, MD), DAPI (Thermo Fisher Scientific, Waltham, MA), or VECTASHIELD® Antifade Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA).
Whole mount in situ hybridization (WMISH)
Partial coding sequences of Bmp2/4, FoxA, FoxQ2, Krl, Nodal, Not1, Vegf3, and Wnt1 were cloned into ZeroBlunt vector to generate RNA probes (Thermo Fisher Scientific, Waltham, MA) Constructs were linearized using FastDigest™ (Thermo Fisher Scientific, Waltham, MA) and in vitro transcribed with DIG RNA Labeling Kit (Millipore Sigma, St. Louis, MO) (Table S2). FoxA, Krl, Vegf3 and Wnt1 RNA probes were previously cloned (Stepicheva and Song, 2015; Sampilo et al., 2021). WMISH was conducted according to previous publications (Arenas-Mena et al., 2000; Minokawa et al., 2004; Stepicheva et al., 2015; Stepicheva and Song, 2015). Probes were used at 1 ng/µl and incubated at 50°C for 5-7 days.
To test endodermal differentiation, we examined the activity of alkaline phosphatase (Kumano and Nishida, 1998; Drawbridge, 2003). Larvae 5 days post fertilization (5 dpf) were fixed in MOPS-paraformaldehyde based fixative (4% paraformaldehyde, 100 mM MOPS pH 7, 2 mM MgSO4, 1 mM EGTA, and 0.8 M NaCl) for 10 min at RT as previously described (Stepicheva et al., 2015). Embryos were then washed with alkaline phosphatase buffer 3 times (100 mM Tris pH 9.5, 100 mM NaCl, 50 mM MgCl2, 0.1% Tween-20), followed by staining until the desired color was developed with the staining solution (0.1 M Tris pH 9.5, 50 mM MgCl2, 0.1 M NaCl, 1 mM Levamisole, 10% Dimethylformamide, 50 mg/ml NBT in 70% dimethyl formamide and 50 mg/ml BCIP in 100% dimethyl formamide). Staining was stopped with multiple washes of MOPS buffer (0.1 M MOPS pH 7, 0.5M NaCl, and 0.1% Tween-20).
Imaging and Phenotyping
Representative images were taken with Zeiss LSM 880 scanning confocal microscope using Zen software or ZEISS Observer Z1 using AxioVision software (Carl Zeiss Microscopy, LLC, White Plains, NY). For videos of pigment cells and gut contractions, live embryos were collected 5 dpf and mounted in FSW onto protamine sulfate (PS)-coated coverslips, creating a positively-charged surface (Stepicheva and Song, 2014). For live behavior examination, control injected, miR-1 inhibitor, and miR-1 mimic-injected embryos were mounted on the same multichambered PS-coated coverslip to avoid variability of environmental conditions and response. To measure dorsoventral connecting rod (DVC) length or PMC migration, ZEISS Observer Z1 microscope was used to take Z-stacks of differential interference contrast (DIC) and 1D5-immunolabeled images. ZEISS AxioCam105 color camera was used to take in situ images. AxioVision or Zen 3.1 software (Carl Zeiss Microscopy, White Plains, NY) was used to measure the length of DVCs, PMC migration distance, in situ expression domains and to determine the center of gastrulae in vegetal views to measure angles of Vegf3, Wnt1, Nodal, Not1, and Bmp2/4 expression domains, ventral ectodermal (VE), and dorsal ectodermal (DE) domains. N is the total number of embryos examined unless otherwise stated. NS = not significant, *p < 0.05, **p < 0.001, ***p < 0.0001. All error bars represent SEM.
To measure the average intensity levels of arbitrary fluorescent units (intensity) in larvae immunolabeled with serotonin and SynB, maximum intensity projection images were analyzed with ImageJ (Schneider, 2012; Remsburg et al., 2021). Serotonin and SynB expression within the ciliary band was measured and the background was subtracted.
Real-Time, quantitative PCR (qPCR)
To examine the levels of endogenous miR-1 expression within a developing embryo, 200-500 embryos were collected at various developmental stages (Fig. 1C). Purification of total RNA was done using miRNAeasy Micro Kit (QIAGEN, Germantown, MD). cDNA synthesis of 100 ng total RNA was performed with miRCURY LNA RT Kit (10 µl volume reaction) which adds a 5’ universal tag of a poly(A) tail to mature miRNA templates (QIAGEN, Germantown, MD). cDNA template was diluted 1:10, and miRNA qPCR was performed using miRCURY LNA miRNA PCR Assays (QIAGEN, Germantown, MD) in QuantStudio 6 Real-Time PCR cycler system (Thermo Fisher Scientific, Waltham, MA). Sea urchin miR-200 were used as normalization controls due to its similar expression from the cleavage to the larval stages (Song et al., 2012). Results are shown as fold changes compared to the egg stage using the Ct-2ΔΔ method as previously described (Stepicheva et al., 2015). miRCURY LNA miRNA PCR Primer Mix is against human miR-1 (Hsa-miR-1-3p).
To measure the transcriptional changes of key TFs for ectodermal, endodermal, and mesodermal specification, transcripts that encode TFs of the skeletogenic GRN, and biomineralization enzymes, we injected zygotes with control, miR-1 inhibitor, and miR-1 mimic. 100 of these blastulae were collected at 24 hpf. Total RNA was extracted by using the Macherey-Nagel Nucleospin® RNA Clean-up XS kit (Macherey-Nagel, Bethlehem, PA). cDNA was synthesized using iScript cDNA synthesis kit (Bio-Rad, Hercules, CA). qPCR was performed using 2.5 embryo equivalents for each reaction with the Fast SYBR or PowerUp Green PCR Master Mix (Thermo Fisher Scientific, Waltham, MA) in the QuantStudio 6 Real-Time PCR cycler system (Thermo Fisher Scientific, Waltham, MA). Results were normalized to the mRNA expression of ubiquitin and depicted as fold changes compared to control embryos using the Ct-2ΔΔ method as previously described to analyze the relative changes in gene expression (Stepicheva et al., 2015). Primer sequences were designed using the Primer 3 Program (Rozen and Skaletsky, 2000) and are listed in Table S3. 3-6 biological replicates were conducted. Statistical significance was calculated using two-tailed unpaired Student’s t-tests.
Results
Expression of miR-1 peaks at gastrula stage and is enriched in the larval gut and ciliary band
miR-1 is expressed maternally. At the 32-cell stage, the expression of miR-1 is enriched in the perinuclear region (Fig. 1B). miR-1 expression peaks at the gastrula stage with ubiquitous expression. At the larval stage, miR-1 is enriched in the gut and ciliary band (Fig. 1B). These results are consistent with relative quantitative levels of miR-1 using real-time, qPCR (Fig. 1C).
miR-1 modulates endodermal differentiation and gut function
To examine the function of miR-1 in early development, we injected miR-1 inhibitor for loss-of-function studies or miR-1 mimic for gain-of-function studies. To test the efficacy of the miR-1 inhibitor, zygotes were injected with control or miR-1 inhibitor, followed by miR-1 FISH (Fig. 2A). We observed a reduction of miR-1 in 100% of miR-1 inhibitor-injected gastrulae, indicating the effectiveness of the miR-1 inhibitor.
Results indicate that miR-1 perturbed embryos have aberrant gut morphology compared to the control (Fig. 2B). In miR-1 inhibitor-injected larvae, width of midgut (MG) is smaller compared to control (Fig. 2Bi). Conversely, in miR-1 mimic-injected larvae, width of MG and hindgut (HG), in addition to length of MG is much wider compared to control (Fig. 2Bi-iii). We further used alkaline phosphatase (ALP) staining as a marker for endodermal differentiation (Drawbridge, 2003; Stepicheva et al., 2015). Control-injected embryos have ALP activity within the MG and part of the HG, whereas miR-1 perturbed larvae exhibited a loss of ALP within the HG and/or in the MG (Fig. 2Biv). Interestingly, both miR-1 inhibitor and miR-1 mimic-injected larvae have significantly less foregut and hindgut contractions compared to the control, indicating a potential defect in gut functions (Fig. 2C, Movies 1-3).
We examined the spatial expression of Krl and FoxA that are important for specifying the foregut and midgut (Oliveri et al., 2006; Yamazaki et al., 2008). In miR-1 perturbed embryos, the spatial expression of Krl and FoxA expanded, and Krl’s expression also shifted anteriorly compared to control (Fig. 2D). The spatial expression changes of key TFs involved in endodermal specification could in part contribute to the morphological changes in the larval gut. Results indicate that miR-1 may regulate gut morphology, differentiation, and function.
Perturbation of miR-1 results in circumpharyngeal muscle defects
Since defects in gut contraction may attribute to gut muscle defects, we examined the structure of the circumpharyngeal muscles (Fig. 3). The number of filamentous actin-rich fibers (F-actin) within the muscle fiber ring was significantly decreased and less structured in both miR-1 perturbed larvae compared to the control. Almost 20% of miR-1 mimic-injected larvae had a complete loss of detectable F-actin (Fig. 3). The miR-1 inhibitor-induced defects were rescued by co-injection with the miR-1 mimic, indicating that the muscle fiber defects are due to miR-1 perturbation.
Perturbation of miR-1 impacts neuronal development
Since gut contractions are in part mediated by serotonin (Yaguchi and Yaguchi, 2021), we examined the level of serotonin, number, and positioning of serotonergic neurons (Fig. 4A). Compared to control, miR-1 inhibitor-injected embryos had decreased levels of serotonin and fewer serotonergic neurons, while miR-1 mimic-injected embryos had a higher serotonin levels and more serotonergic neurons (Fig. 4Ai-ii). Additionally, we observed that serotonergic neurons in control-injected larvae were positioned linearly in the animal plate, whereas serotonergic neurons in miR-1 mimic-injected larvae are dispersed throughout the apical organ or within the ciliary band (Fig. 4Aiii). We also observed miR-1 perturbed embryos exhibited a significant decrease in mature and functional neurons compared to the control, as indicated by the immunolabeling of SynB-expressing neurons (Fig. 4B).
Since FoxQ2 is required for serotonergic neural specification and must be downregulated for their differentiation (Yaguchi et al., 2012), we examined its expression. We observed that miR-1 inhibitor-injected embryos have expanded FoxQ2 expression domain, while miR-1 mimic-injected embryos have decreased FoxQ2 expression domain compared to control (Fig. 4C).
miR-1 regulates pigment cell number and morphology
PCs are derived from the mesoderm and has an essential role in the innate immune defense system (Fig. 1A) (Tokuoka et al., 2002; Smith et al., 2006). In control embryos, PCs have long filopodial extensions (Fig. 5). In miR-1 inhibitor-injected larvae, PCs are clumped or rounded, whereas PCs in miR-1 mimic-injected larvae have multiple long filopodial extensions (Fig. 5Ai).
In addition, while inhibition of miR-1 did not affect the number of PCs, miR-1 mimic-injections resulted in a significant decrease of PCs compared to control (Fig. 5Aii).
We further examined the behavior of PCs and observed that PCs of control larvae travel in a linear fashion with occasional bifurcated filopodial extensions (Fig. 5Ai, Movie 4). In miR-1 inhibitor-injected larvae, their rounded PCs move with membraneous blebs, whereas miR-1 mimic-injected larvae form multifurcated filopodial extensions that travel in multiple directions (Fig. 5Ai, Movies 5,6).
We examined the spatial expression of blastocoelar cell (BC) TF Ese (oral/ventral mesoderm) and pigment cell marker Pks1 (aboral/dorsal mesoderm) in the mesenchyme blastula stage, when PCs and BCs become specified (Fig. 5B) (Materna et al., 2013). We observed that both inhibitor and miR-1 mimic-injections resulted in increased Ese and decreased Pks1 expression domains, with miR-1 mimic-injected embryos having a more dramatic decrease in the expression domain of Pks1 (Fig. 5).
miR-1 perturbations result in less PMCs and a delay in PMC ingression
To assess how miR-1 perturbation impacts PMC specification and/or ingression, we used the VegfR10 RNA probe to mark and follow PMCs during early gastrulation in a time-course experiment. While undergoing epithelial to mesenchymal transition (EMT), PMCs appear as ‘bottle-shaped’, whereas cells that have completed EMT have a ‘round cell shape’ (Lyons et al., 2012; Sampilo et al., 2021). Using the cell shape as a criterion for EMT, we found miR-1 perturbation resulted in a significant delay of PMC ingression during gastrulation (Fig. 6Ai,Bi). miR-1 perturbed embryos also had an average of three PMCs less than control embryo (Fig. 6Aii,Bii).
Perturbation of miR-1 results in skeletal defects
During gastrula stage, we observed that miR-1 inhibitor and miR-1 mimic-injections resulted in decreased length of DVCs in a dose-dependent manner (Fig. 7A,B). miR-1 inhibitor-induced skeletal defects were rescued by co-injection of a miR-1 mimic, indicating that this defect is specifically induced by miR-1 inhibition (Fig. 7A). Interestingly, both miR-1 perturbations resulted in supernumerary tri-radiate rudiments and a similar ectopic skeletal branching off one of the tri-radiate arms (Fig. 7C).
miR-1 inhibitor-injected larvae were underdeveloped, lacked body rod (BR) convergence, and/or occasionally exhibited ectopic BR branching (Fig. 7D). miR-1 mimic-injected larvae had a dose-dependent severity of abnormal and supernumerary skeletal branching off the postoral rods (POR), anterolateral rods (ALR), and BRs (Fig. 7D). We also observed independent skeletal elements developed perpendicular to the larval BRs. Based on the dose-response data, 40 µM of miR-1 inhibitor and 30 µM of miR-1 mimic were used throughout this study. Overall, these results indicate that miR-1 plays a critical role in the initial formation and elongation of the skeletal spicules and that miR-1 mimic-injections induced a more severe skeletal defect than miR-1 inhibitor-injections.
Perturbation of miR-1 results in PMC patterning defects
Since we observed skeletal branching defects (Fig. 7), we examined the patterning of PMCs, which are the only cells that make the skeleton (Ettensohn and McClay, 1986) (Fig. 8). In miR-1 inhibitor-injected gastrulae, we found that while the patterning of PMCs was not greatly affected, PMCs were clustered posteriorly and had less anterior migration compared to control (Fig. 8Ai-ii). This decreased migration in miR-1 inhibitor-injected embryos was rescued by co-injection of miR-1 mimic, indicating that this defect is due to inhibition of miR-1. In miR-1 mimic-injected gastrulae, the PMCs were dispersed and migrated to the animal pole (Fig. 8Bi-ii). miR-1-inhibitor-injected larvae had occasional PMCs positioned off a skeletal branch (Fig. 8Ci), whereas miR-1 mimic-injected larvae had many PMCs positioned off all branches (Fig. 8Cii).
miR-1 directly targets components of skeletogenic GRN and signaling pathways
To reveal the regulatory molecular mechanism of miR-1, we bioinformatically searched for potential miR-1 binding sites within transcripts that encode regulators of the PMC GRN and key components of signaling pathways. Using Rluc reporter construct and site-directed mutagenesis, we identified miR-1 to directly repress Ets1/2, Tbr, VegfR7, Notch, Nodal, and Wnt1. miR-1 may have weak miRNA-mRNA binding affinity with Dri (Fig. 9A,B).
miR-1 mimic-injections result in ectopic expression domains of several factors
To identify the molecular mechanism of how miR-1 regulates skeletogenesis, embryos were collected at gastrula stage and hybridized with Vegf3, Wnt1, Nodal, Not1, and Bmp2/4 RNA probes (Fig. 10). In miR-1 inhibitor-injected gastrulae, the lateral and vegetal expression domains of Vegf3 were significantly decreased compared to control (Fig. 10Ai). Concurrently, the vegetal expression domains of Nodal and Not1 were expanded, without expression domain change of Bmp2/4 (Fig. 10Bi). In miR-1 mimic-injected gastrulae, the lateral expression of Vegf3 had no expression domain change; however, its vegetal expression domain was significantly expanded into both the VE and DE domains (Fig. 10Aii,Bii). In contrast to miR-1 inhibitor-injected embryos, the miR-1 mimic-injected embryos had Nodal, Not1, and Bmp2/4 decreased in the vegetal expression domains, while Wnt1 was increased compared to control. Results indicate that Vegf3 expression domains in miR-1 perturbed embryos correlated to changes in expression domains of Nodal, Bmp2/4, and Wnt1.
miR-1 regulates expression of key endodermal, ectodermal, and mesodermal transcripts
To identify the underlying molecular mechanism of how miR-1 impacts structures derived from the three germ layers, we tested expression levels of key TFs involved in endodermal, ectodermal, and mesodermal specification (Fig. 11A). Results indicate that miR-1 inhibition resulted in a 2-fold increase in Krl and FoxA, while Onecut decreased 2-fold. miR-1 mimic-injections resulted in almost a 2-fold increase in Onecut, and Ese, while Tropo1, MHC, and Pks had a >2-fold decrease.
To identify the underlying molecular mechanism that led to PMC patterning and skeletal branching defects, we examined the relative expression levels of transcripts that encode PMC specification and patterning (Ets1/2, Dri, Tbr, Snail, Nodal, Bmp2/4, Not1, Vegf3, Wnt1, FgfA and CDC42) (Röttinger et al., 2004; Duloquin et al., 2007; Röttinger et al., 2008; Duboc et al., 2010; Adomako-Ankomah and Ettensohn, 2013; Sepúlveda-Ramírez et al., 2018), biomineralization enzymes (P19, p58A, SM30, SM50) (Cheers and Ettensohn, 2005; Livingston et al., 2006; Adomako-Ankomah and Ettensohn, 2011), PMC-specific cell surface protein Msp130 (Leaf et al., 1987), and PMC adhesion protein KirrelL (Ettensohn and Dey, 2017), and markers of dorsal PMCs (Tbx2/3 and GataC) (Duboc et al., 2010) (Fig. 11B). Results indicate that inhibition of miR-1 results in a 2-fold increase in transcript levels of Ets1/2, Notch, and CDC42, while miR-1 mimic-injections resulted in decreased transcript levels of these same genes. Inhibition of miR-1 resulted in an almost 2-fold increase in Dri, Tbr, and Snail. Both miR-1 inhibitor and miR-1 mimic-injections resulted in ∼2-fold decrease in GataC, Tbx2/3, SM30, and SM50.
Discussion
We identified miR-1 to have a diverse role in embryogenesis, in addition to its conserved function in muscle-specific tissues. In the sea urchin, miR-1 is one of the most abundantly expressed miRNAs, and was able to rescue severe embryonic lethality induced by knockdown of Drosha and/or Dicer when co-injected with the three other highly sequenced miRNAs in the sea urchin (miR-31, miR-71, and miR-2012) (Song et al., 2012), suggesting that miR-1 is required for proper early development. We identified miR-1 to not only regulate mesodermally-derived structures, which include muscles, PCs, and skeletogenesis, but also gut morphology and function, and specification and positioning of serotonergic neurons.
We observed that the gut morphology and contractions in miR-1 perturbed larvae were altered compared to control (Fig. 2Bi-iii,2C, Movies 1-3). Both miR-1 inhibitor and mimic-injected embryos have expanded expression domains of Krl and FoxA (Fig. 2B-D). Previously, Krl knockdown resulted in missing foregut and delayed gastrulation (Howard et al., 2001; Yamazaki et al., 2008), while FoxA knockdown reduces gut size (Oliveri et al., 2006). Consistent with our in situ data, miR-1 inhibition resulted in a 2-fold increase of Krl and FoxA (Fig. 11A). Since Krl and FoxA do not contain canonical miR-1 binding sites, miR-1 may indirectly modulate them to impact gut development. In addition, we found that miR-1 directly suppresses Notch and Wnt1 (Fig. 9). Consistent with prior work, Notch2 is a known miR-1 target (Liu et al., 2018; Hua et al., 2019). Notch knockdown embryos resulted in decreased Krl levels (Yamazaki et al., 2008), and Wnt1 knockdown resulted in decreased FoxA levels (Cui et al., 2014). Thus, miR-1 inhibitor-injected embryos may have increased Notch and Wnt1, which may result in increased Krl and FoxA. While Krl and FoxA expression domain expanded in miR-1 mimic-injected embryos, their levels did not increase (Figs. 2D, 11A). How miR-1 mimic-injection resulted in expanded Krl and FoxA is unclear. miR-1 may also regulate transcripts that encode other undefined factors important for endodermal differentiation, gut morphology, and function.
The defective gut contractions in miR-1 perturbed larvae may be due to problems with the muscle fibers and/or neural coordination. We observed that miR-1 perturbed larvae have decreased number of F-actin muscle fiber rings (Fig. 3). Previous work has shown Delta/Notch signaling to play critical roles in vertebrate myogenesis (Kopan et al., 1994; Conboy and Rando, 2002; Schuster-Gossler et al., 2007), and knockdown of either Delta or Notch in the sea urchin embryos resulted in decreased muscle fibers (Sweet et al., 2002; Range et al., 2008). We showed that miR-1 directly suppresses Notch, indicating that the defective muscle fibers in miR-1 perturbed larvae may be in part explained by miR-1’s direct suppression of Notch (Fig. 9A). Additionally, we bioinformatically identified miR-1 to have predicted truncated and/or mismatched miR-1 binding sites within MHC and Tropo1, which are conserved muscle differentiation genes (Mu et al., 2007; Andrikou et al., 2013; Annunziata et al., 2014; Jin et al., 2016). In the sea urchin, both are expressed in circumesophageal muscle fibers, and MHC is also expressed in all the sphincters (Andrikou et al., 2013; Annunziata et al., 2014). In vertebrates, various MHC isoforms are expressed in the upper esophageal sphincter and have distinct contractile properties (Mu et al., 2007). The exact function of Tropo1 in the sea urchin is unknown; however, in vertebrates, Tropo1 is critical in cardiac development and contractile function of skeletal muscles (Jin et al., 2016; England et al., 2017). While the levels of MHC and Tropo1 transcripts in miR-1 inhibitor-injected embryos did not change compared to the control, their levels were decreased 2-fold in miR-1 mimic-injected embryos (Fig. 11A). Interestingly, miR-1 is enriched in the larval gut (Fig. 2B), suggesting that it may post-transcriptionally regulate MHC and Tropo1 that may impact the organization of the F-actin of the muscle fibers and/or gut contractions. Additionally, since serotonin can regulate the pyloric opening of the larval gut (Yaguchi and Yaguchi, 2021), aberrant serotonin expression in miR-1 perturbed embryos may also contribute to decreased gut contractions (Figs. 2C, 4A).
We found that in comparison to control larvae, miR-1 inhibitor-injected larvae had lower serotonin levels and fewer serotonergic neurons, while miR-1 mimic-injected larvae had increased serotonin levels and a higher number of serotonergic neurons (Fig. 4A). We propose that miR-1 regulates the specification of serotonergic neurons, potentially through FoxQ2. FoxQ2 is required early for the specification of serotonergic neurons and must be removed from the apical plate by late gastrula stage to allow differentiation of serotonergic neurons (Yaguchi et al., 2008; Yaguchi et al., 2012). In miR-1 inhibitor-injected blastulae, the expression domain of FoxQ2 is expanded (Fig. 4C), consistent with prior findings where expanded FoxQ2 in LRP6-morphants correlated with a complete loss of serotonergic neurons (Yaguchi et al., 2016). In miR-1 mimic-injected blastulae, FoxQ2 expression domain became more restricted which may result in increased serotonergic neurons (Figs. 4A,C). It appears that while the spatial expression of FoxQ2 was altered in miR-1 perturbed embryos, FoxQ2 levels did not change (Fig. 11A). Since FoxQ2 does not contain potential miR-1 binding sites, we speculate that miR-1 may indirectly regulate expression of FoxQ2 in early development.
miR-1’s regulation of FoxQ2 may be through its direct suppression of Nodal. (Fig. 9). Nodal has been implicated in promoting neuronal specification in humans and in sea urchins (Smith et al., 2008; Patani et al., 2009; Yaguchi et al., 2016; McClay et al., 2018). In Nodal morphants, FoxQ2 expression domain in blastulae became more restricted (Yaguchi et al., 2016), and this restriction of FoxQ2 expression domain is similar to what was observed in miR-1 mimic-injected blastulae (Fig. 3C). Thus, miR-1 may suppress Nodal to indirectly restrict FoxQ2. Interestingly, serotonergic neurons of Nodal morphants were dispersed throughout the apical domain (Yaguchi et al., 2016), similar to what we observed in miR-1 mimic-injected larvae (Fig. 3A). Nodal/Bmp signaling along the DV axis is critical for regulating the specification of the neuroectoderm and patterning within the apical domain (Duboc et al., 2004; Yaguchi et al., 2016). In the sea urchin, Nodal signaling suppresses serotonergic neuronal differentiation from the oral/ventral side of the animal plate (Yaguchi et al., 2007; Yaguchi et al., 2016). Thus, potentially, aberrant Nodal expression induced by miR-1 overexpression may lead to mispositioning of these serotonergic neurons.
Additionally, we found that miR-1 perturbation resulted in a significant decrease in SynB-positive neurons compared to the control (Fig. 4B). SynB mediates neurotransmitter release of synaptic vesicles in mature and functional neurons (Burke et al., 2006; Yang et al., 2015). Since neuronal TFs SoxB1 and SoxC, which regulate neuronal specification (Angerer et al., 2005; Garner et al., 2016; Niwa et al., 2016), were not significantly altered under miR-1 perturbation (Fig. 11A), this indicates that miR-1 may regulate neuronal development by suppressing Nodal, and indirectly FoxQ2, as well as other TFs during neuronal differentiation.
We then examined mesodermally-derived structures in miR-1 perturbed embryos. In the sea urchin, PCs migrate throughout the larval ectoderm to carry out immune defense (Takata and Kominami, 2003; Smith et al., 2006). miR-1 mimic-injected larvae have fewer PCs, correlating to decreased expression of Pks1 (Figs. 5, 11A). During development, PCs and BCs share the same progenitor cells and make a binary fate decision during the blastula stage (Sweet et al., 1999). Their specification is regulated by both Nodal and Notch signaling pathways, where Nodal initiates specification by activating TFs that specify BCs and inhibiting TFs that specify PCs; later Notch signaling promotes differentiation of both BCs and PCs (Duboc et al., 2010). Nodal overexpression resulted in a lack of Pks1-expressing PCs and expanded expression of BC markers GataC and Ese (Duboc et al., 2010). Knockdown of Notch resulted in fewer PCs and BCs (Sweet et al., 1999; Peterson and McClay, 2005). Taken together, miR-1’s regulation of both Nodal and Notch is likely to contribute to BC and PC specification. PCs in miR-1 perturbed larvae exhibited different morphology and behavior when compared to control (Fig. 5, Movies 4-6). How miR-1 regulates the morphology and movement of PCs remains unclear; however, miR-1 perturbation affects the expression of CDC42, which may be important for mediating general cell movement (Fig. 11) (Yamao et al., 2015; Sepúlveda-Ramírez et al., 2018).
Another mesodermally-derived tissue that is regulated by miR-1 is the larval skeleton. To understand how miR-1 may be regulating PMC development and skeletogenesis, we identified miR-1 to directly suppress Ets1/2, Tbr, and VegfR7 of the PMC GRN, and Notch, Nodal, Bmp2/4, and Wnt1 of signaling pathways (Fig. 9). Ets1/2 and Tbr are critical for PMC specification and function. Ets1/2 is an important modulator of MAPK pathway that is required for the specification of PMCs and their ability to undergo EMT (Kurokawa et al., 1999; Röttinger et al., 2004). Additionally, Tbr plays an essential role in specification of the skeletogenic mesoderm and formation of the larval skeleton where Tbr knockdown resulted in a complete loss of skeleton (Oliveri et al., 2002). The percentage of PMCs undergoing EMT is significantly and consistently less than the control (Fig. 6Ai,6Bi). Additionally, we observed both miR-1 perturbed embryos have a decrease of 3-4 PMCs compared to control which would likely not have detrimental effects (Fig. 6Aii,6Bii). Thus, miR-1’s regulation of Ets1/2 and Tbr may potentially contribute to the decreased PMCs and EMT defect in miR-1 perturbed embryos. Moreover, the delay in PMC ingression could be due to miR-1’s potential regulation of G-cadherin and Snail, both of which have predicted miR-1 binding sites. Snail represses the transcription of cadherin in order for PMCs to detach from basal lamina and ingress into the blastocoel (Wu and McClay, 2007). The delay in PMC ingression could potentially affect PMC pattering by disrupting the time and distance-sensitive interaction between VegfR10-expressing PMCs and the Vegf3 ligand expressed in the ectoderm, as Vegf3 expression becomes restricted to the Veg1 ectoderm by 30 hpf (Li et al., 2014). Additionally, the function of another Vegf receptor, VegfR7 is not well understood; however we have previously found that blocking miR-31’s direct suppression of VegfR7 resulted in mislocalized PMCs, indicating VegfR7’s potential role in PMC patterning (Stepicheva and Song, 2015). Thus, miR-1’s direct suppression of Ets1/2, Tbr, and VegfR7 may affect not only PMC ingression but also affect PMC patterning.
A possible mechanism for miR-1 perturbation-induced ectopic branching can be explained by our observation that the PMCs of miR-1 perturbed larvae migrate off the main skeletal elements and continue to form syncytial cables (filopodia) (Fig. 8C). Since PMCs are solely responsible for the formation of the larval skeleton (Ettensohn and McClay, 1986), these PMCs in miR-1 perturbed larvae appear to be mispatterned to areas where we observed ectopic skeletal elements (Figs. 7D, 8C). Additionally, ectopic branching can also be explained by miR-1’s direct suppression of Nodal (Fig. 9B). Of note is that miR-1 mimic-injected embryos have the characteristic radialized morphology in the vegetal view, similar to Nodal morphant-induced morphology (Figs. 10Aii,10Bii) (Duboc et al., 2010). Also, although the level of Nodal transcripts was not greatly altered in miR-1 mimic-injected embryos, we observed a >3-fold decrease of Nodal target, Not1 (Li et al., 2012). This result indicates that miR-1 mimic-injected embryos have decreased Nodal protein, resulting in decreased Not1 transcripts (Fig. 11B). miR-1’s direct suppression of Nodal could potentially explain the skeletal defects in both contexts of loss-of-function and gain-of-function of miR-1 (Figs. 7,9), since knockdown or overexpression of Nodal results in ectopic skeletal branching (Duboc et al., 2004). Additionally, Nodal and Bmp2/4 restrict Fgfa expression to the PMC VLC (Guss and Ettensohn, 1997; Röttinger et al., 2008). Our results indicate that miR-1 inhibition led to increased Nodal, which may lead to a >2-fold decrease of Fgfa; while miR-1 mimic-injections may lead to decreased Nodal which may lead to a >2-fold increase of Fgfa (Fig. 11B). During the gastrula stage, Fgfa is expressed in ectoderm near the regions of the tri-radiate spicule rudiment branches which form the BRs and the PORs (Horstadius, 1973; Röttinger et al., 2008). Inhibition of Fgfa prevents skeletogenesis, whereas overexpression of Fgfa resulted in supernumerary tri-radiates leading to ectopic skeletal branching. Increased Fgfa in miR-1 mimic-injected embryos could in part explain the multiple spicules in gastrulae and ectopic BR and POR branches observed in larvae (Fig. 7C,D). Interestingly, Bmp2/4 knockdown also result in larvae with multiple skeletal rods and ectopic branching (Duboc et al., 2004). Since results indicate that miR-1 does not directly regulate Bmp2/4, any changes in Bmp2/4 spatial expression and/or transcript levels are likely due to miR-1’s suppression of Nodal which activates Bmp2/4 (Duboc et al., 2004). Thus, we propose that mispatterned PMCs and aberrant skeletal branching in miR-1 mimic-injected embryos may attribute to miR-1’s repression of Nodal to increase Fgfa (Duboc et al., 2010) and miR-1’s indirect regulation of Bmp2/4.
To further elucidate the molecular mechanism of how miR-1 regulates PMC patterning, we examined the spatial expression domains and transcript levels of Vegf3, Wnt1, Nodal, Not1, and Bmp2/4 (Figs. 10,11). Both Nodal and Bmp2/4 signaling pattern the PMCs by restricting Vegf3 expression to the two lateral ectodermal domains and posterior ventral corners where the PMC clusters reside to form the skeletal rudiments within the border ectoderm-dorsal ventral margin (BE-DVM) intersection (Fig. 12A) (Duboc et al., 2010; McIntyre et al., 2013; Layous, 2020). In the ventral ectoderm, Nodal activates the expression of itself, Not1, Bmp2/4 and Vegf3 at 8-9 hpf in the ventral ectoderm (Lapraz et al., 2009; Li et al., 2012). Subsequently, Not1 restricts Vegf3 expression from the ventral domain by 27 hpf (Li et al., 2012), while Bmp2/4 protein translocates to the dorsal side by late blastula to eliminate Wnt1 from the dorsal posterior cells to induce dorsal fates (Lapraz et al., 2009; Wei et al., 2012). Wnt1 then restricts Nodal from the posterior ventral region (Wei et al., 2012), and indirectly activates Vegf3 (Sampilo et al., 2021). In miR-1 inhibitor-injected gastrulae, both the lateral and vegetal expression of Vegf3 was significantly decreased (Fig. 10A). In contrast, miR-1 mimic-injected gastrulae had no change in lateral Vegf3 expression (Fig. 10Ai), but vegetal Vegf3 expression had significantly expanded into both the VE and DE domains (Fig. 10Aii). The expanded Vegf3 expression domain may contribute to ectopic supernumerary skeletal branching (Duloquin et al., 2007; Morgulis et al., 2019). Previous studies in vertebrates have shown miR-1 to directly target VegfA (Stahlhut et al., 2012; Zhu et al., 2018). In the sea urchin, a partial Vegf3 transcript contains one predicted miR-1 binding site; however, the sequence data on SpVegf3 3’UTR is incomplete and difficult to clone. Thus, although the change in Vegf3 expression domain may be due to miR-1’s direct suppression, our results indicate that the expression of Vegf3 in the ventral-dorsal axis may be a result of miR-1’s direct regulation of Nodal. Additionally, a previous study has shown that a loss of Bmp2/4 resulted in the position of the PMC ring to shift towards the animal pole and a radialized the embryo (Angerer et al., 2000; Layous, 2020). Interestingly, in miR-1 mimic-injected gastrulae, we observed mispatterned PMCs that migrated near the animal pole (Fig. 8B), decreased Bmp2/4 expression domain, and radialized embryos (Fig. 10Bii), indicating that these defects are likely due to miR-1’s suppression of Nodal. Animal pole PMCs that ectopically migrated from the DVCs may become PMCs that give rise to ectopic branches off the ALRs (Sun and Ettensohn, 2014).
The expanded expression domain of Wnt1 in miR-1 mimic-injected embryos may restrict the expression domain of Nodal, leading to expansion of Vegf3 expression domain. Although Wnt1 is directly suppressed by miR-1, Wnt1 can activate its own transcription and is part of highly cross-regulated positive feedback circuitry (Cui et al., 2014; Sampilo et al., 2021). The expanded expression domain of Wnt1 in miR-1 mimic-injected gastrulae is likely due to decreased Bmp2/4 expression domain via miR-1’s direct suppression of Nodal (Figs. 9, 10Bii, 11B). The changes we observed in the spatial expressions of Vegf3, Nodal, Not1, Bmp2/4, and Wnt1 in miR-1 mimic-injected embryos may all contribute to PMC mispatterning (Figs. 8B, 8Cii, 10Aii, 10Bii). Overall, miR-1 mimic-injected embryos have reminiscent larval branching defects as Nodal and Bmp2/4 knockdown embryos (Duboc et al., 2004), and Vegf3 and Fgfa overexpressing embryos (Fig. 7D) (Duloquin et al., 2007; Röttinger et al., 2008).
Previously, we identified that miR-31 in the sea urchin regulates skeletogenesis by directly suppressing Eve and Wnt1 (Sampilo et al., 2021). Depletion of miR-31 resulted in expanded vegetal spatial expression of Vegf3 (Stepicheva and Song, 2015; Sampilo et al., 2021), similar to miR-1 mimic-injections. miR-1 directly suppresses genes Ets1/2, Tbr, VegfR7 and Wnt1, of which Ets1/2 and Tbr are downstream of miR-31 targets Pmar1 and Eve (Stepicheva and Song, 2015; Sampilo et al., 2021). Thus, both miR-31 and miR-1 target critical components within the PMC GRN and co-regulate skeletogenesis (Fig. 12B).
Overall, we identified miR-1 to regulate not only mesodermally-derived structures, but also mediate the development of the endodermal gut and ectodermally-derived nervous system. This study identifies novel functions of miR-1, by identifying its direct targets and revealing miR-1 as a multifunctional miRNA that regulates structures derived from all three germ layers in a developing embryo.
Supplementary Figures
Fig. S1 miR-1 is evolutionarily conserved. Alignment of mature miR-1 sequences in several metazoan species is shown. Red nucleotides indicate the highly conserved miR-1 seed sequence. miR-1 in protostomes is shown in blue and deuterostomes in purple. Yellow indicates variable nucleotides. miR-1 sequences were obtained from miRBase (Kozomara and Griffiths-Jones, 2014) and aligned with Clustal Omega Multiple Sequence Alignment program (Sievers et al., 2011).
Movie 1. Gut contractions in control embryos. Live embryos were collected 5 dpf and recorded. Average of contractions was analyzed within 4 min. Scale bar = 20 µm.
Movie 2. Gut contractions in miR-1 inhibitor-injected embryos. Live embryos were collected 5 dpf and recorded. Average of contractions was analyzed within 4 min. Scale bar = 20 µm.
Movie 3. Gut contractions in miR-1 mimic-injected embryos. Live embryos were collected 5 dpf and recorded. Average of contractions was analyzed within 4 min. Scale bar = 20 µm.
Movie 4. Pigment granules of control-injected larvae travel linearly. Live embryos were collected 5 dpf and recorded. Pigment granules within the PCs of control larvae travel in a linear fashion that occasionally bifurcate into two filopodial extensions. Scale bar = 20 µm.
Movie 5. Pigment cells in miR-1 inhibited larvae move with blebbing. Live embryos were collected 5 dpf and recorded. In miR-1 inhibitor-injected larvae, pigment granules within immunocytes migrate in clumped or rounded manner. Scale bar = 20 µm.
Movie 6. Pigment granules of miR-1 mimic-injected larvae exhibit scattered and multifurcated extensions. Live embryos were collected 5 dpf and recorded. In miR-1 mimic-injected larvae, PCs exhibit scattered pigment granules that form multifurcated filopodial extensions that travel in multiple directions. Scale bar = 20 µm.
Acknowledgements
The authors would like to thank David McClay (Duke University) for his kind gift of 1D5 antibody and Gary Wessel (Brown University) for the SynB antibody. This work is funded by University of Delaware Graduate Fellowship to NFS. NSF CAREER (IOS 1553338) and MCB 2103453 to JLS and NIH NIGMS P20GM103446.
Abbreviations
- ALP
- alkaline phosphatase
- ALR
- anterolateral rod
- BC
- blastocoelar cells
- BR
- body rod
- BE-DVM
- border ectoderm-dorsal ventral margin
- cWnt
- canonical Wnt
- DE
- dorsal ectoderm
- Dpf
- days post fertilization
- DVC
- dorsoventral connecting rod
- EMT
- epithelial to mesenchymal transition
- GRN
- gene regulatory network
- PC
- pigment cells
- Hpf
- hours post fertilization
- PC
- pigment cell
- PMCs
- primary mesenchyme cells
- POR
- postoral rod
- TF
- transcription factor
- VE
- ventral ectoderm
- Vegf
- vascular endothelial growth factor