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In early embryonic development, the cross-regulation of transcription factors and signaling pathways are critical in mediating developmental and physiological processes. Additionally, many studies have shown the importance of post-transcriptional regulation of signaling and network components mediated by microRNAs (miRNAs); however, how miRNAs are transcriptionally regulated is poorly understood. miRNAs are critical fine-tuners of many biological processes and their dysregulation leads to a variety of diseases and developmental defects. Previously, we have shown that miRNAs are dynamically expressed throughout sea urchin development, suggesting that miRNAs are likely to be under transcriptional regulation. Here, we used pharmacological inhibitors, genetic constructs, and loss-of-function reagents to assess the impact of key signaling pathways (Wnt, Nodal, MAPK, Sonic Hedgehog, Delta/Notch, VEGF, and BMP) and transcription factors (Alx1, Ets1/2, and Tbr) on the transcript levels of the evolutionarily conserved miR-1, miR-31, miR-92 and miR-124; the invertebrate-specific miR-71; and the echinoderm-specific miR-2002, miR-2007, and miR-2012. We also used computational methods to identify potential transcription factor binding sites of these miRNAs. Lists of binding motifs for transcription factors (TFs) were acquired from the MEME-Suite Motif Database and used as inputs for the algorithm FIMO (Find Individual Motif Occurrences), which detects short nucleotide motifs within larger sequences. Based on experimental data on miRNA expression in conjunction with bioinformatic predictions, we propose that the transcription factors Tbr, Alx1, and Ets1 regulate Sp miR-1, Sp miR-31, and Sp miR-71, respectively. We additionally observed significant effects on miRNA levels as a result of perturbations to Wnt, Nodal, MAPK, and Sonic Hedgehog signaling pathways, while no significant change on miRNA levels were observed with perturbations to Delta/Notch, VEGF, or BMP signaling pathways. Overall, this study provides insights into the transcriptional regulation of miRNAs by signaling pathways and transcription factors and contribute to our overall understanding of the genetic regulation of developmental processes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Arnott, Sampilo and Song.)

microRNAs are evolutionarily conserved non-coding RNAs that direct post-transcriptional regulation of target transcripts. In vertebrates, microRNA-1 (miR-1) is expressed in muscle and has been found to play critical regulatory roles in vertebrate angiogenesis, a process that has been proposed to be analogous to sea urchin skeletogenesis. Results indicate that both miR-1 inhibitor and miR-1 mimic-injected larvae have significantly less F-actin enriched circumpharyngeal muscle fibers and fewer gut contractions. In addition, miR-1 regulates the positioning of skeletogenic primary mesenchyme cells (PMCs) and skeletogenesis of the sea urchin embryo. Interestingly, the gain-of-function of miR-1 leads to more severe PMC patterning and skeletal branching defects than its loss-of-function. The results suggest that miR-1 directly suppresses Ets1/2, Tbr, and VegfR7 of the skeletogenic gene regulatory network, and Nodal, and Wnt1 signaling components. This study identifies potential targets of miR-1 that impacts skeletogenesis and muscle formation and contributes to a deeper understanding of miR-1's function during development., (Copyright © 2024 Elsevier Inc. All rights reserved.)

microRNAs (miRNAs) play a critical role in a variety of biological processes, including embryogenesis and the physiological functions of cells. Evolutionarily conserved microRNA-31 (miR-31) has been found to be involved in cancer, bone formation, and lymphatic development. We previously discovered that, in the sea urchin, miR-31 knockdown (KD) embryos have shortened dorsoventral connecting rods, mispatterned skeletogenic primary mesenchyme cells (PMCs) and shifted and expanded Vegf3 expression domain. Vegf3 itself does not contain miR-31 binding sites; however, we identified its upstream regulators Eve and Wnt1 to be directly suppressed by miR-31. Removal of miR-31's suppression of Eve and Wnt1 resulted in skeletal and PMC patterning defects, similar to miR-31 KD phenotypes. Additionally, removal of miR-31's suppression of Eve and Wnt1 results in an expansion and anterior shift in expression of Veg1 ectodermal genes, including Vegf3 in the blastulae. This indicates that miR-31 indirectly regulates Vegf3 expression through directly suppressing Eve and Wnt1. Furthermore, removing miR-31 suppression of Eve is sufficient to cause skeletogenic defects, revealing a novel regulatory role of Eve in skeletogenesis and PMC patterning. Overall, this study provides a proposed molecular mechanism of miR-31's regulation of skeletogenesis and PMC patterning through its cross-regulation of a Wnt signaling ligand and a transcription factor of the endodermal and ectodermal gene regulatory network., (Copyright © 2021 Elsevier Inc. All rights reserved.)

MicroRNAs (miRNAs) are small non-coding RNAs that repress the translation and reduce the stability of target mRNAs in animal cells. Post-transcriptional regulation mediated by miRNAs is a highly conserved mechanism utilized by organisms throughout phylogeny to fine tune gene expression. We document the approaches used to study the function of a single miRNA and miRNA regulation of biological pathways in the sea urchin embryo. The protocols that are described include selection of miRNA inhibitors, test of miRNA direct targets, and the use of target protector morpholinos to evaluate the impact of miRNA inhibition on its targets. Using the described techniques and strategies, the sea urchin researcher will be able to validate a miRNA's direct targets and evaluate how inhibition of the miRNA affects developmental processes. These results will contribute to our understanding of the regulatory roles of miRNAs in development., (© 2019 Elsevier Inc. All rights reserved.)

MicroRNAs (miRNAs) are highly conserved, small non-coding RNAs that regulate gene expressions by binding to the 3' untranslated region of target mRNAs thereby silencing translation. Some miRNAs are key regulators of the Wnt signaling pathways, which impact developmental processes. This study investigates miRNA regulation of different isoforms of Dishevelled ( Dvl/Dsh ), which encode a key component in the Wnt signaling pathway. The sea urchin Dvl mRNA isoforms have similar spatial distribution in early development, but one isoform is distinctively expressed in the larval ciliary band. We demonstrated that Dvl isoforms are directly suppressed by miRNAs. By blocking miRNA suppression of Dvl isoforms, we observed dose-dependent defects in spicule length, patterning of the primary mesenchyme cells, gut morphology, and cilia. These defects likely result from increased Dvl protein levels, leading to perturbation of Wnt-dependent signaling pathways and additional Dvl-mediated processes. We further demonstrated that overexpression of Dvl isoforms recapitulated some of the Dvl miRNATP-induced phenotypes. Overall, our results indicate that miRNA suppression of Dvl isoforms plays an important role in ensuring proper development and function of primary mesenchyme cells and cilia., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)