ZebraShare: a new venue for rapid dissemination of zebrafish mutant data

Abstract

Background: In the past decade, the zebrafish community has widely embraced targeted mutagenesis technologies, resulting in an abundance of mutant lines. While many lines have proven to be useful for investigating gene function, many have also shown no apparent phenotype, or phenotypes not of interest to the originating lab. In order for labs to document and share information about these lines, we have created ZebraShare as a new resource offered within ZFIN.

Methods: ZebraShare involves a form-based submission process generated by ZFIN. The ZebraShare interface (https://zfin.org/action/zebrashare) can be accessed on ZFIN under "Submit Data". Users download the Submission Workbook and complete the required fields, then submit the completed workbook with associated images and captions, generating a new ZFIN publication record. ZFIN curators add the submitted phenotype and mutant information to the ZFIN database, provide mapping information about mutations, and cross reference this information across the appropriate ZFIN databases. We present here examples of ZebraShare submissions, including phf21aa, kdm1a, ctnnd1, snu13a, and snu13b mutant lines.

Results: Users can find ZebraShare submissions by searching ZFIN for specific alleles or line designations, just as for alleles submitted through the normal process. We present several potential examples of submission types to ZebraShare including a phenotypic mutants, mildly phenotypic, and early lethal mutants. Mutants for kdm1a show no apparent skeletal phenotype, and phf21aa mutants show only a mild skeletal phenotype, yet these genes have specific human disease relevance and therefore may be useful for further studies. The p120-catenin encoding gene, ctnnd1, was knocked out to investigate a potential role in brain development or function. The homozygous ctnnd1 mutant disintegrates during early somitogenesis and the heterozygote has localized defects, revealing vital roles in early development. Two snu13 genes were knocked out to investigate a role in muscle formation. The snu13a;snu13b double mutant has an early embryonic lethal phenotype, potentially related to a proposed role in the core splicing complex. In each example, the mutants submitted to ZebraShare display phenotypes that are not ideally suited to their originating lab's project directions but may be of great relevance to other researchers.

Conclusion: ZebraShare provides an opportunity for researchers to directly share information about mutant lines within ZFIN, which is widely used by the community as a central database of information about zebrafish lines. Submissions of alleles with a phenotypic or unexpected phenotypes is encouraged to promote collaborations, disseminate lines, reduce redundancy of effort and to promote efficient use of time and resources. We anticipate that as submissions to ZebraShare increase, they will help build an ultimately more complete picture of zebrafish genetics and development.

Keywords: Collaboration; Zebrafish; ctnnd1; kdm1a; lsd1; nhp2l1; phf21a; snu13.

Figure 1. Publicizing a mutant on ZebraShare in 5 steps.

Figure 2. Example of how to determine DNA coordinates for a simple deletion allele, phf21aa^aik4.

(A) Align the WT and mutant sequence. (B) Blast the aligned WT sequence and determine the base numbers altered in the mutant. (C) Transfer these coordinates to the ZebraShare submission workbook. Descriptions become more complex for combined insertion/deletions alleles (InDels) and for alleles with multiple mutation sites due to use of multiple guide RNAs.

Figure 3. Example of a mutant, phf21aa^aik4, publicized via ZebraShare.

(A) The allele description page shows information that helps researchers interpret the mutant. (B) The abstract page gives an overview of phenotypic characterization. (C) The figure associated with the abstract shows pertinent phenotypic details. For phf21aa^aik4, the mutants appeared normal, save for a mild rotation of the ceratohyal cartilage (red arrow). Fish larvae are stained with alcian blue (cartilage) and alizarin red (bone) to reveal skeletal shape. The three scalebars in C each = 200 microns.

Figure 4. Skeletal structure is normal in kdm1a maternal zygotic mutants.

Wild-type larvae (A–C) compared with kdm1a maternal zygotic mutant larvae (D–F). (A and D) Whole mount specimens, lateral view of head skeleton, (B and E) flat mount pharyngeal skeleton, pharyngeal arches 1 and 2, lateral view, (C and F) flat mount neurocranium, ventral view. Fish larvae are stained as described in Fig. 3. Scale bar = 200 microns.

Figure 5. Embryonic disintegration in ctnnd1 mutants.

(A, B) Frames from time-lapse imaging (Movie S1) illustrate disintegrating phenotype of ctnnd1 mutants. Asterisk(s) mark two of the embryos that disintegrated during the time-lapse recording. (C) DAPI staining of fixed ctnnd1 mutant embryos reveals that cells with intact nuclei dissociate from the embryo. (D–F) Phenotypes of ctnnd1 sibling embryos at the 6-somite stage. The homozygous mutant (F) has disintegrated, the heterozygous mutant (E) displays clumps of cells along the dorsal surface, and the wild-type embryo appears normal (D). The solid arrow marks a clump of cells dorsal to the midbrain and the open arrowhead marks a clump of cells dorsal to the hindbrain in the heterozygote.

Figure 6. Embryonic development arrests in snu13a;snu13b double mutants.

(A) Normal sibling and (B) snu13a;snu13b double mutant at 24 hpf. Tail region is outlined in orange, head in blue. The shown double mutant was confirmed to be homozygous by Sanger sequencing. Scale bar is 1 mm.

Figure 7. A decision tree on whether to publicize findings in ZebraShare.

(A) A ZebraShare submission immediately disseminates your information to the scientific community and also can serve as a pathway to journal publication. (B) Comparison of correct and misplaced ZebraShare submissions, with solutions for incorrect uses.