2023 Project 4

Student Projects 2023: 1 Patterning neural border and NC | 2 NPB NEUcrest | 3 EMT and NC | 4 miRNA and NC | 5 Adrenal Gland and NC | 6 Melanocyte & Melanoma | 7 Neurocristopathies | Neural Crest These projects are the sole work of undergraduate science students and may contain errors in fact or descriptions.

MicroRNAs and Neural Crest Development

MicroRNAs (miRNAs) are short RNA molecules of around 22 nucleotides that are mainly involved in the RNA interference pathway. Most miRNAs are transcribed as primary transcripts (pri-miRNAs) by the RNA polymerase II, as normal coding genes, they are then processed by the endonuclease DROSHA, that forms the pre-miRNA, exported to the cytoplasm by the protein EXPORTIN5, and further processed by another endonuclease, DICER. At this point, a 20-25 nucleotides double stranded RNA is left, the mature miRNA. The two strands are then separated and can be loaded in the RNA-Induced Silencing Complex (RISC).

The complex RISC-miRNA searches for a messenger RNA (mRNA) that has a complementary sequence and includes a crucial sequence, the so-called “seed sequence”, which is only 8 nucleotides long. Usually, the binding between the seed sequence of the miRNA and the mRNA occurs in the 3’ untranslated region (3’UTR) of the mRNA.

Once the miRNA targets an mRNA, it promotes either the stalling of the ribosomal complex, or the direct degradation of the mRNA. In either case, the resulting effect is the downregulation of the targeted gene. The action of miRNAs is also additive, meaning that if more than one miRNAs are targeting the same mRNA at the same time, the silencing effect will be stronger (Fig. 1). It has been known since 2010 that miRNAs are involved in the NC development, when Zehir and colleagues conditionally knocked out the key protein DICER in mice NC, proving embryonic lethality due to a lack of NC tissue.

Further studies followed, identifying individual miRNAs that play roles in different aspects of NC development. One of the first studies in this respect was conducted by Gessert and colleagues, who linked miR-130a, miR-219, miR-23b, miR-200b, miR-96 and miR-196a to the development of the eye and craniofacial structures of the frog Xenopus laevis. A few years later, Avellino and colleagues proved that miR-204 is important for the migration of the NC in Medaka fish, as it is able to target ankrd13A, an important modulator of NC migration.

More recently, Ward and colleagues sequenced all the miRNAs of Xenopus-derived organoids that were induced to become NC. They further showed that most of these miRNAs were expressed in those organoids. Including, miR-219, miR-196a, miR-218-2, miR-10b, miR-204a and miR-130b/c.

In chick embryos, have been shown that other miRNAs to be important at different levels of NC development, such as the maintenance of NC identity. They do so by modulating FGF and Wnt signalling pathways. In particular, miR-20a, 200a, miR-217 and miR-let-7 are involved in this process.

Many other studies have shown miRNAs to be involved in NC differentiation. For example, miR-145, miR-143 and miR-219 are important for the correct formation of craniofacial structures (8, 9), while, miR-375 and miR-124 have been proved to be involved in the differentiation of sympathoadrenal cells and chromaffin cells of the adrenal gland (10-12).

Overall, all these studies are showing that miRNAs play a key role during NC development. Further work is ongoing to include these genes in the already existing gene regulatory networks that orchestrate NC development (Fig. 2). Many of these studies linking miRNAs and NC have been recently reviewed by Antonaci and Wheeler and by Weiner (13, 14).

Fig 1. microRNAs biosynthesis: Schematic representation of the biosynthesis of microRNAs. Shortly, a primary transcript (pri-miRNA) is transcribes as a protein-coding gene, by the RNA Pol II. The endonuclease DROSHA processes a first time this transcript, producing the pre-miRNA, which is exported outside of the nucleus where a second endonuclease, DICER, generates the miRNA duplex. The RISC complex loads one of the two strands and use it to search a mRNA that pairs with the seed sequence of the miRNA. According to the type of binding between seed sequence of the miRNA and the mRNA binding site, the result is either the degradation of the mRNA, or the stalling of the ribosomal complex (Antonaci and Wheeler).

Fig 2. microRNAs involved in Neural Crest-Gene Regulatory Network: Most of the microRNAs involved in Neural Crest development, from top to bottom: Neural Crest induction and specification, epithelial-to-mesenchymal transition and migration, and differentiation to some of the Neural Crest derivatives (13).

References

1. O'Brien J, Hayder H, Zayed Y, Peng C. Overview of MicroRNA Biogenesis, Mechanisms of Actions, and Circulation. Frontiers in Endocrinology. 2018;9. PMID: 30123182

2. Zehir A, Hua LL, Maska EL, Morikawa Y, Cserjesi P. Dicer is required for survival of differentiating neural crest cells. Dev Biol. 2010;340(2):459-67. PMID: 20144605

3. Gessert S, Bugner V, Tecza A, Pinker M, Kuhl M. FMR1/FXR1 and the miRNA pathway are required for eye and neural crest development. Dev Biol. 2010;341(1):222-35. PMID: 20197067

4. Avellino R, Carrella S, Pirozzi M, Risolino M, Salierno FG, Franco P, et al. miR-204 targeting of Ankrd13A controls both mesenchymal neural crest and lens cell migration. PLoS One. 2013;8(4):e61099. PMID: 23620728

5. Ward NJ, Green D, Higgins J, Dalmay T, Munsterberg A, Moxon S, et al. microRNAs associated with early neural crest development in Xenopus laevis. BMC Genomics. 2018;19(1):59. PMID: 29347911

6. Copeland J, Simoes-Costa M. Post-transcriptional tuning of FGF signaling mediates neural crest induction. Proc Natl Acad Sci U S A. 2020;117(52):33305-16. PMID: 33376218

7. Bhattacharya D, Rothstein M, Azambuja AP, Simoes-Costa M. Control of neural crest multipotency by Wnt signaling and the Lin28/let-7 axis. Elife. 2018;7. PMID: 30520734

8. Steeman TJ, Rubiolo JA, Sanchez LE, Calcaterra NB, Weiner AMJ. Conservation of Zebrafish MicroRNA-145 and Its Role during Neural Crest Cell Development. Genes (Basel). 2021;12(7). PMID: 34209401

9. Godden AM, Antonaci M, Ward NJ, van der Lee M, Abu-Daya A, Guille M, et al. An efficient miRNA knockout approach using CRISPR-Cas9 in Xenopus. Dev Biol. 2022;483:66-75. PMID: 34968443

10. Dutt M, Wehrle CJ, Jialal I. Physiology, Adrenal Gland. StatPearls. Treasure Island (FL)2022. PMID: 30725945

11. Gai Y, Zhang J, Wei C, Cao W, Cui Y, Cui S. miR-375 negatively regulates the synthesis and secretion of catecholamines by targeting Sp1 in rat adrenal medulla. Am J Physiol Cell Physiol. 2017;312(5):C663-C72. PMID: 28356269

12. Shtukmaster S, Narasimhan P, El Faitwri T, Stubbusch J, Ernsberger U, Rohrer H, et al. MiR-124 is differentially expressed in derivatives of the sympathoadrenal cell lineage and promotes neurite elongation in chromaffin cells. Cell Tissue Res. PMID: 270944312016;365(2):225-32.

13. Antonaci M, Wheeler GN. MicroRNAs in neural crest development and neurocristopathies. Biochem Soc Trans. 2022;50(2):965-74. PMID: 35383827 Review.

14. Weiner AMJ. MicroRNAs and the neural crest: From induction to differentiation. Mech Dev. 2018;154:98-106. PMID: 29859253 Review.