Expression of the neurotrophic tyrosine kinase receptors, ntrk1 and ntrk2a, precedes expression of other ntrk genes in embryonic zebrafish

View article
Loading...
Zoological Science

Main article text

 

Introduction

Materials and Methods

Fish care

Embryo collection

Embryo staging and fixation

cDNA isolation and cloning

Probe synthesis

In situ hybridization and image collection

Results

ntrk1, ntrk2a and ngfrb RNA expression

ntrk2b, ntrk3a and ntrk3b RNA expression

Discussion

Conclusions

Supplemental Information

Diagrams of zebrafish ntrk1, ntrk2a, ntrk2b, ntrk3a, ntrk3b and ngfrb transcripts marked with regions that are complementary to anti-sense probes.

Gene diagrams show 5’ and 3’ UTRs, exon-exon junctions, protein coding regions, kinase domains, and extracellular domains. On top of each gene diagram, the black line represents the region spanned by our probe. Dashed lines were hydrolyzed and solid lines were not hydrolyzed. Below each gene diagram, the red line represents the region spanned by the probe used by Nittoli et al., 2018. Information from each gene was based on the longest isoform available in Zebrafish Ensembl release 92, and diagrams were each constructed using the same scale. Protein domains were assigned using the PRINTS database.

DOI: 10.7717/peerj.10479/supp-1

Sense probe data.

(A – F) Sense probes and (A’ – F’) anti-sense probes were used for in situ hybridizations. At 24 hpf, (A) the ntrk1 anti-sense probe resulted in staining in the spinal cord and (A’) the ntrk1 sense probe did not. At 16.5 hpf, (B) the ntrk2a anti-sense probe resulted in staining in cranial ganglia and (B’) the ntrk2a sense probe did not. At 24 hpf, (C) the ngfrb anti-sense probe resulted in staining in cranial ganglia and (C’) the ngfrb sense probe did not. At 24 hpf, (D) the ntrk2b anti-sense probe resulted in staining in the forebrain and (D’) the ntrk2b sense probe did not. At 24 hpf, (E) the ntrk3a anti-sense probe resulted in staining in the forebrain and (E’) the ntrk3a sense probe did not. At 24 hpf, (F) the ntrk3b anti-sense probe resulted in staining in the forebrain, midbrain and hindbrain, while (F’) the ntrk3b sense probe did not. We think it worth noting that we noticed the most variability with our ntrk3b probe. Within a round of in situ hybridizations we saw similar staining from embryo to embryo, but from round to round, the staining could be more intense (as in Figure 6) or less intense (as in F’). We speculate that the variability could be because our ntrk3b probe recognizes the 3’ UTR (see Supplemental Figure 1).

DOI: 10.7717/peerj.10479/supp-2

Additional Information and Declarations

Competing Interests

The authors declare that they have no competing interests.

Author Contributions

Katie Hahn conceived and designed the experiments, performed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the paper, and approved the final draft.

Paul Manuel performed the experiments, analyzed the data, prepared figures and/or tables, and approved the final draft.

Cortney Bouldin conceived and designed the experiments, analyzed the data, authored or reviewed drafts of the paper, and approved the final draft.

Animal Ethics

The following information was supplied relating to ethical approvals (i.e., approving body and any reference numbers):

All zebrafish use was approved by the Appalachian State University Institutional Animal Care and Use Committee (Protocol 17-13).

Data Availability

The following information was supplied regarding data availability:

The raw data (unprocessed images of in situ staining results) are available as Figs. 16 and in the Supplemental Files.

Funding

This work was supported by the Department of Biology, the College of Arts and Sciences, and the Office of Student Research at Appalachian State University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

1 Citation 1,325 Views 322 Downloads

Your institution may have Open Access funds available for qualifying authors. See if you qualify

Publish for free

Comment on Articles or Preprints and we'll waive your author fee
Learn more

Five new journals in Chemistry

Free to publish • Peer-reviewed • From PeerJ
Find out more