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. 2017 Oct;207(2):609-623.
doi: 10.1534/genetics.117.300187. Epub 2017 Aug 23.

Genetic Screen for Postembryonic Development in the Zebrafish ( Danio rerio): Dominant Mutations Affecting Adult Form

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Free PMC article

Genetic Screen for Postembryonic Development in the Zebrafish ( Danio rerio): Dominant Mutations Affecting Adult Form

Katrin Henke et al. Genetics. .
Free PMC article

Abstract

Large-scale forward genetic screens have been instrumental for identifying genes that regulate development, homeostasis, and regeneration, as well as the mechanisms of disease. The zebrafish, Danio rerio, is an established genetic and developmental model used in genetic screens to uncover genes necessary for early development. However, the regulation of postembryonic development has received less attention as these screens are more labor intensive and require extensive resources. The lack of systematic interrogation of late development leaves large aspects of the genetic regulation of adult form and physiology unresolved. To understand the genetic control of postembryonic development, we performed a dominant screen for phenotypes affecting the adult zebrafish. In our screen, we identified 72 adult viable mutants showing changes in the shape of the skeleton as well as defects in pigmentation. For efficient mapping of these mutants and mutation identification, we devised a new mapping strategy based on identification of mutant-specific haplotypes. Using this method in combination with a candidate gene approach, we were able to identify linked mutations for 22 out of 25 mutants analyzed. Broadly, our mutational analysis suggests that there are key genes and pathways associated with late development. Many of these pathways are shared with humans and are affected in various disease conditions, suggesting constraint in the genetic pathways that can lead to change in adult form. Taken together, these results show that dominant screens are a feasible and productive means to identify mutations that can further our understanding of gene function during postembryonic development and in disease.

Keywords: dominant screen; mapping; pigment; postembryonic; skeletogenesis; zebrafish.

Figures

Figure 1
Figure 1
Screen and mapping strategy (A). Wild-type males were mutagenized with ENU to induce random mutations in the genome. Mutagenized males were outcrossed to wild-type females, and the subsequent generation (F1) was screened for dominant mutations affecting adult form. Isolated mutant founders were outcrossed, and the phenotype and penetrance was assessed in their progeny (F2). (B) For mapping and candidate mutation identification, one mutant and a pool of three to six wild-type siblings from the same cross were sequenced. Sequence data were analyzed in three steps: (1) all SNPs in the mutant and sibling pool were identified (blue and red lines; gray bars represent individual chromosomes); (2) to identify the chromosome carrying the mutation (M), mutant specific SNPs (blue bars) were identified by removing all SNPs present in the wild-type siblings from the set of previously identified SNPs; (3) candidate mutations are then identified by excluding all background SNPs from wild-type strains or other mutants/siblings sequenced. The remaining SNPs are classified as synonymous or nonsynonymous, and ranked by their predicted consequence on gene function (deleterious/tolerated). Blue dots indicate nonsynonymous changes on the linked chromosome.
Figure 2
Figure 2
Mapping and candidate gene identification in dmh35. To identify mutant specific haplotypes, first all SNPs in the genome are identified. After exclusion of all SNPs that are present in the sibling pool, only mutant specific SNPs are left. The highest number of mutant specific SNPs on a chromosome should indicate the chromosome carrying the mutation. (A) The red bars in the diagram on the left represent SNPs identified in the mutant and sibling pool on each of the 25 chromosomes. Blue bars in the diagram on the right represent mutant-specific SNPs on each of the 25 chromosomes. (B) Summary of the number of total and mutant-specific SNPs per chromosome. (C) In a second step, all nonsynonymous changes are identified in the genome. Blue circles indicate missense mutations; green circles mark nonsense mutations; black asterisks mark mutations predicted to be deleterious; the red asterisk indicates the position of the G138D missense mutation in connexin 43, shown to be linked to the dmh35 mutant phenotype.
Figure 3
Figure 3
Mutants with vertebral defects. Representative photographs (left) and μCT images of the vertebral column (right) of the five different subgroups of mutants with vertebral defects. (A) wild-type zebrafish, regularly patterned and shaped vertebrae, and vertebral spines. (B–D) Heterozygous dmh13, dmh29 and dmh15 mutants have a shorter trunk, and show strongly deformed vertebral bodies and spines with excessive bone formation. (E) Heterozygous dmh27 fish are also shorter than wild-type fish, but show mostly normal patterned vertebral bodies with the exception of a couple of fused vertebrae. Vertebral spines show slight changes in angle. (F) In addition to a shorter body, heterozygous dmh16 mutants have strong deformation of their vertebrae. In addition to fusion of vertebral bodies, some vertebrae are split up into multiple hemi-segments. (G) The body of dmh31 mutants seems to be proportionally normal until the region in front of the caudal peduncle. Here, vertebrae are strongly deformed, leading to a bend in the body axis. (H) Strong curvature of the spine in dorsal, ventral, and lateral direction leads to an overall shorter body length of the dmh4 mutant. The vertebral bodies seem to be mostly normally patterned.
Figure 4
Figure 4
Larval phenotypes of mutants with vertebral defects. Representative photographs of wild-type (A) and mutant (B–D) phenotypes at 5 dpf. Mutants show a shorter body length when compared to wild-type larvae. A fraction of heterozygous dmh28 and dmh31 mutants, in addition show deformations of the notochord (red arrows). Inset in (D) shows a dorsal view of a dmh31/+ mutant larva, illustrating the kink in the trunk coinciding with deformations in the notochord.
Figure 5
Figure 5
Mutants with defects of the dermal skeleton. Representative photographs of wild type and mutants (left), with close ups of the flank stained with alizarin red to visualize scales (right). For better visualization, two rows of scales are outlined in red. (A) In wild-type fish, the scales are similar in size and are very regularly patterned. (B–H) All mutants in this class show differences in size, pattern, and some even in orientation of scales. (D, F, H) These differences are even more obvious in homozygous mutants. In addition, all mutants show changes in the patterning and length of their fin rays to varying degree.
Figure 6
Figure 6
Mutants with fin deformations. Representative photographs of mutants with wild type (A), short (B), and deformed (C) fins. (B) Both the median and paired fins of dmh35 heterozygous mutants are shorter. Alizarin red stained pelvic fins of wild type (D) and dmh33/+ mutant (E) fish. Dmh33 heterozygous mutants show shorter fin rays with deformed segments of unequal length.
Figure 7
Figure 7
Pigment mutants. Representative photographs of wild type (A) or pigment mutants on the left panels, and a close-up of their flank on the right, highlighting the differences in pigmentation. Homozygous phenotypes are shown for dmh1 (C) and dmh11 (F). (B) dmh1 mutants are characterized by a reduced number of melanophore stripes on the fins and wider stripes on the trunk. (C) Homozygous dmh1 mutants have only a single xanthophore stripe on their trunks. (D) Stripes in dmh7 mutants are disrupted, and show a spot-like pattern. (E) Heterozygous dmh11 mutants show mild disruptions of the melanophore stripes, leading to a wave appearance. (F) In contrast, homozygous dmh11 mutants show a strong reduction in melanophore number, while mostly maintaining a stripe like pattern.

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