RNA-binding protein Elavl1/HuR is required for maintenance of cranial neural crest specification
- Division of Biology and Biological Engineering, California Institute of Technology, United States
- Department of Cell and Tissue Biology, University of California, San Francisco, United States
- The Miller Institute for Basic Research in Science, University of California, Berkeley, United States
- Department of Biology, School of Math and Science, California State University Northridge, United States
Figures
Figure 1
RNA-binding proteins are differentially expressed in premigratory and migratory cranial neural crest.
(A) Schematic of early chick cranial neural crest cells at premigratory stages (HH8+/9−) in intact heads and cross-section expressing Citrine fluorescent protein under control of the FoxD3 NC1 …
Figure 2
The RNA-binding protein Elavl1 is expressed in premigratory and migratory cranial neural crest.
Representative epifluorescence images of wild-type HH8− (A), HH9 (B–D), and HH9+ (E–G) chick embryos, in whole mount (A, B, E) and cross-section (C, D; F, G) immunostained for Elavl1 (cyan) and Pax7 …
Figure 3 with 2 supplements
Elavl1 knockdown alters cranial neural crest specification and delamination.
(A) Schematic diagram illustrating experimental design. Gastrula stage chick embryos were electroporated bilaterally with a standard control and translation-blocking morpholino (MO) targeting Elavl1.…
Figure 3—figure supplement 1
Translation-blocking morpholino suppresses Elavl1 expression.
(A) Representative apotome maximum intensity projection micrograph of cross-sectioned embryo bilaterally co-electroporated with a fluorescent electroporation control construct (H2B-RFP) and control …
Figure 3—figure supplement 2
Elavl1 knockdown inhibits cranial neural crest emigration.
(A) Representative confocal maximum intensity projection micrograph of whole mount embryo bilaterally co-electroporated with a fluorescent electroporation control construct (H2B-RFP) and control …
Figure 4 with 2 supplements
Draxin mRNA is the primary target of Elavl1 during cranial neural crest specification.
(A) Schematic diagram illustrating experimental design for RNA-sequencing (RNA-seq). Gastrula stage chick embryos were electroporated bilaterally with a standard control and translation-blocking …
Figure 4—figure supplement 1
RNA-sequencing (RNA-seq) identified four neural crest genes specifically downregulated with Elavl1 knockdown.
Although RNA-seq identified four genes with established roles in neural crest development (Draxin, Axud1, Msx1, BMP4) that were significantly downregulated with Elavl1 knockdown (A), several other …
Figure 4—figure supplement 2
Putative Elavl1-binding sites within the Draxin 3′-untranslated region (UTR) predicts a direct interaction.
Representative secondary structures of the Draxin UTR (A), Axud1 3′-UTR (B), Msx1 3′-UTR (C), BMP4 3′-UTR (D), Pax7 3′-UTR (E), and Tfap2b 3′-UTR (F), predicted by ‘mfold’ analysis. Predicted …
Figure 5
Elavl1 knockdown phenotypes are rescued by Draxin.
Representative confocal maximum intensity projection micrographs of hybridization chain reaction (HCR) processed embryos for FoxD3 (n = 7) (A), Axud1 (n = 7) (B), Msx1 (n = 4) (C), and BMP4 (n = 4) …
Tables
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Gallus gallus) | Elavl1 | GenBank | NM_204833.1 | |
| Gene (Gallus gallus) | Draxin | GenBank | AB427147.1 | |
| Gene (Gallus gallus) | Axud1 (CSRNP1) | This paper | GenBank: ON920861 | 3′-Untranslated region |
| Gene (Gallus gallus) | Msx1 | Ensembl | ENSGALT00000024209.4 | |
| Gene (Gallus gallus) | BMP4 | Ensembl | ENSGALT00000020316.7 | |
| Gene (Gallus gallus) | Tfap2b | Ensembl | ENSGALT00000026916.6 | |
| Gene (Gallus gallus) | Pax7 | Ensembl | ENSGALT00000048594.2 | |
| Strain, strain background (Gallus gallus) | Rhode Island Red | Sun State Ranch (Monrovia, CA, USA) | ||
| Antibody | Rabbit anti-Elavl1 | Abcam | Cat# ab196626 | 1:100 immunohistochemistry; 5 µg immunoprecipitation |
| Antibody | Mouse IgG1 anti-Pax7 | Developmental Studies Hybridoma Bank | Cat# pax7, RRID:AB_528428 | 1:5 |
| Antibody | Goat IgG anti-GFP | Rockland | Cat# 600-101-215, RRID:AB_218182 | 1:500 |
| Antibody | Rabbit IgG, polyclonal – Isotype Control (ChIP Grade) | Abcam | Cat# ab171870 | 5 µg immunoprecipitation |
| Recombinant DNA reagent | pCI-H2B-RFP (plasmid) | Betancur et al., 2010 | N/A | |
| Recombinant DNA reagent | pCIG (plasmid) | Megason and McMahon, 2002 | N/A | |
| Recombinant DNA reagent | Draxin-FLAG (plasmid) | Hutchins and Bronner, 2018 | N/A | |
| Recombinant DNA reagent | MCP-GFP (plasmid) | Hutchins et al., 2020 | N/A | |
| Recombinant DNA reagent | MS2-Draxin 3′-UTR (plasmid) | This paper; Hutchins et al., 2020 | N/A | |
| Sequence-based reagent | Control morpholino | GeneTools | N/A | 5′-CCTCTTACCTCAGTTACAATTTATA |
| Sequence-based reagent | Elavl1 morpholino | This paper; GeneTools | N/A | 5′-GACATCTTATAACGTATCTCGCTGC |
| Sequence-based reagent | Draxin morpholino | Hutchins and Bronner, 2018; GeneTools | N/A | 5′-AAGGTGGAAGAAGCTGCCATAATCC |
| Sequence-based reagent | Draxin qPCR primers | This paper; IDT | Custom DNA oligos | Forward: 5′-CTACGCTGTTATGCCAAATTCC; Reverse: 5′-GAATGATCCCTGCTCTCCATT |
| Sequence-based reagent | Axud1 qPCR primers | This paper; IDT | Custom DNA oligos | Forward: 5′-TCCAGTCCTTCTCGGACTATAA; Reverse: 5′-GGGAAATTAGGCAACTGAAACTG |
| Sequence-based reagent | Msx1 qPCR primers | This paper; IDT | Custom DNA oligos | Forward: 5′-AGCTGGAGAAGCTGAAGATG; Reverse: 5′-AGGCTCCGTACAGGGAT |
| Sequence-based reagent | BMP4 qPCR primers | This paper; IDT | Custom DNA oligos | Forward: 5′-TTGGTCAACTCCGTCAACTC; Reverse: 5′-CAGCACCACCTTGTCATACT |
| Sequence-based reagent | Pax7 qPCR primers | This paper; IDT | Custom DNA oligos | Forward: 5′-CAAACCAACTCGCAGCATTC; Reverse: 5′-CTGCCTCCATCTTGGGAAAT |
| Sequence-based reagent | FoxD3 qPCR primers | This paper; IDT | Custom DNA oligos | Forward: 5′-CATCTGCGAGTTCATCAGCA; Reverse: 5'-TTCACGAAGCAGTCGTTGAG |
| Sequence-based reagent | 3′-RACE QT primer | Scotto-Lavino et al., 2006; IDT | Custom DNA oligos | 5′-CCAGTGAGCAGAGTGACGAGGACTCGAGCTCAAGCTTTTTTTTTTTTTTTTT |
| Sequence-based reagent | 3′-RACE QO primer | Scotto-Lavino et al., 2006; IDT | Custom DNA oligos | 5′-CCAGTGAGCAGAGTGACG |
| Sequence-based reagent | 3′-RACE QI primer | Scotto-Lavino et al., 2006; IDT | Custom DNA oligos | 5′-GAGGACTCGAGCTCAAGC |
| Sequence-based reagent | 3′-RACE Axud1 Gene Specific Primer 1 | This paper; IDT | Custom DNA oligos | 5′-CGTGTTCCAAGAGCTATGCC |
| Sequence-based reagent | 3′-RACE Axud1 Gene Specific Primer 2 | This paper; IDT | Custom DNA oligos | 5′-GGTTTCCCGCAAGCTGG |
| Commercial assay or kit | RNAqueous-Micro Total RNA isolation kit | Ambion | Cat# AM1931 | |
| Commercial assay or kit | SMART-Seq v4 Ultra Low Input cDNA kit | Takara Bio | Cat# 634889 | |
| Commercial assay or kit | Agencourt AMPure XP beads | Beckman Coulter | Cat# A63880 | |
| Commercial assay or kit | Endofree maxi prep kit | Qiagen | Cat# 12362 | |
| Commercial assay or kit | Duolink In Situ PLA Probe Anti-Rabbit MINUS | Millipore/Sigma | Cat# DUO92005 | |
| Commercial assay or kit | Duolink In Situ PLA Probe Anti-Goat PLUS | Millipore/Sigma | Cat# DUO92003 | |
| Commercial assay or kit | Duolink In Situ Detection Reagents FarRed | Millipore/Sigma | Cat# DUO92013 | |
| Commercial assay or kit | Duolink In Situ Wash Buffers, Fluorescence | Millipore/Sigma | Cat# DUO82049 | |
| Chemical compound, drug | Duolink In Situ Mounting Medium with DAPI | Millipore/Sigma | Cat# DUO82040 | |
| Chemical compound, drug | Fluoromount-G | SouthernBiotech | Cat# 0100-01 | |
| Chemical compound, drug | DAPI | Thermo Fisher | Cat# D1306 | 1:5000 |
| Chemical compound, drug | FastStart Universal SYBR Green Master (Rox) | Millipore/Sigma | Cat# FSUSGMMRO | |
| Chemical compound, drug | SuperScript III Reverse Transcriptase | Thermo Fisher | Cat# 18080044 | |
| Chemical compound, drug | cOmplete, Mini, EDTA-free Protease Inhibitor | Millipore/Sigma | Cat# 11836170001 | |
| Chemical compound, drug | RNaseOUT Recombinant Ribonuclease Inhibitor | Thermo Fisher | Cat# 10777019 | |
| Chemical compound, drug | Accumax | Innovative Cell Technologies, Inc | Cat# AM105 | |
| Chemical compound, drug | Protein G Dynabeads | Thermo Fisher | Cat# 10003D | |
| Chemical compound, drug | Actinomycin D | Millipore/Sigma | Cat# A9415 | 10 µg/ml |
| Software, algorithm | Fiji | Schindelin et al., 2012 | RRID:SCR_002285 | https://imagej.net/Fiji |
| Software, algorithm | Seurat | Butler et al., 2018 | RRID:SCR_007322 | https://satijalab.org/seurat/ |
| Software, algorithm | QuantStudio Design & Analysis software, version 2.4 | Life Technologies | RRID:SCR_018712 | |
| Software, algorithm | Zen 2 Blue | Zeiss | N/A | |
| Software, algorithm | Zen Black | Zeiss | N/A | |
| Software, algorithm | Photoshop CC | Adobe | N/A | |
| Software, algorithm | Prism8, Prism9 | GraphPad | N/A | |
| Software, algorithm | FastQC | Andrews, 2014 | RRID:SCR_014583 | |
| Software, algorithm | Cutadapt | Martin, 2011 | RRID:SCR_011841 | |
| Software, algorithm | Bowtie2 | Langmead and Salzberg, 2012 | RRID:SCR_016368 | |
| Software, algorithm | HTSeq-Count | Anders et al., 2015 | RRID:SCR_011867 | |
| Software, algorithm | biomaRt package | Durinck et al., 2009 | RRID:SCR_019214 | |
| Software, algorithm | mfold | Zuker, 2003 | RRID:SCR_008543 | |
| Software, algorithm | G*Power | Faul et al., 2007 | RRID:SCR_013726 | |
| Other, deposited data | sc-RNA (SmartSeq2) | Williams et al., 2019 | GEO: GSE130500 | Single-cell RNA-sequencing datasets; see Figure 1 and Data Availability Statement |
| Other, deposited data | RNA-seq data (bulk) | This paper | NCBI Bioproject: PRJNA861325 | Single-end RNA-sequencing datasets; see Figure 4 and Data Availability Statement |
