Expression of gmds and phenotypic quantification of mutants and morphants. (A) A wildtype (WT) embryo at 48 hpf (B) A gmd ^{n1002 −/−} mutant embryo at 48 hpf with a curly tail and cerebral hemorrhage. A sequencing chromatogram is shown from a wildtype embryo comparing a non-altered gmds sequence (C) to a mutant sequence (D) highlighting a missense mutation and a one base insertion at the CRISPR cut site, creating a premature ‘stop’ codon in the protein coding sequence. The rates of hemorrhage, curly tail and swimming behavior recorded at 3 dpf for gmds^n1002-/-, gmds^{towhead−/−}, and gmds morphants (E). The gmds gene is maternally deposited and ubiquitously expressed at 2 hpf (F). Low levels of ubiquitous expression are observed at 12 hpf (G), with expression increasing in the epidermis by 24 hpf (H). At 48 hpf, gmds expression is seen in the pharyngeal arches and a thin stripe in the heart (I). By 72 hpf, the pharyngeal arches and sensory neuromasts have gmds expression staining (J).

Salvage pathway genes contribute to vascular integrity. The ​fcsk ​gene is expressed ubiquitously in the head of wildtype 48 hpf embryos (A) compared to the mild background staining in the wildtype control embryos using a sense probe (B). The ​fpgt ​gene was expressed ubiquitously in the head of wildtype 48 hpf embryos (C) compared to the mild background staining in the wildtype control embryos using a sense probe (D). The recorded cerebral hemorrhaging rates of embryo clutches derived from ​gmds ​heterozygous matings and control wildtype crosses with or without ​fcsk ​morpholino (E), showing that the ​fcsk ​morpholino increase hemorrhage rates. (F), results of rescue experiments using GDP-fucose and L-fucose, demonstrating that both conditions can rescue hemorrhage rate. Statistical significance was calculated using a chi-squared test with Yate's correction. Error bars represent standard error of the mean hemorrhage rate from three independent experiments.

Manipulation of Notch signaling affects hemorrhage rates in gmds mutant embryos. A synergistic increase in hemorrhage frequency is observed when clutches of embryos derived from gmds heterozygous matings are incubated in a low dose of DAPT (A). Overexpression of the Notch intracellular domain rescues (reduces) hemorrhage frequency in clutches derived from gmds heterozygous matings (B). Hemorrhages were quantified at 50 hpf, Chi-squared analysis with Yate's correction was used for statistical testing. Data presented as mean hemorrhage frequency across three independent experiments with error bars representing standard error of the mean.

Analysis of endothelial cell organization and smooth muscle cell number due to loss of gmds. In gmds morphant embryos, ectopic branches of cerebral vessels are observed at 3 dpf (B, C, E white arrows) when compared to control injected embryos (A, D). Panel C shows merged gfp and brightfield images to show the location of cerebral hemorrhage (hindbrain ventricle) near the site of an ectopic branch as highlighted by fli1a:gfp expression. gmds mutant embryos showed decreased smooth muscle actin cells in the bulbus arteriosus and pharyngeal arch arteries (G ventral view, I, lateral view) compared to wildype siblings (F, ventral view, H, lateral view) at 5 dpf. Embryos were deemed wildtype (+/+ or +/−) based on a lack of hemorrhage and curly tail, or mutant based on the presence cerebral hemorrhage and curly tail.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.