ZFIN ID: ZDB-FIG-140414-8
Miyares et al., 2013 - Long-Chain Acyl-CoA Synthetase 4A Regulates Smad Activity and Dorsoventral Patterning in the Zebrafish Embryo. Developmental Cell   27(6):635-647 Full text @ Dev. Cell
ADDITIONAL FIGURES
EXPRESSION / LABELING:
Genes:
Fish:
Knockdown Reagent:
Anatomical Terms:
Stage Range: 4-cell to 14-19 somites
PHENOTYPE:
Fish:
Knockdown Reagent:
Observed In:
Stage: Prim-5

Fig. 1

Acsl4a Is Necessary for Proper Dorsoventral Patterning

(A) acsl4a expression throughout early embryonic development. Whole-mount mRNA in situ hybridizations. Expression pattern was consistent over multiple experiments (n = 7) and between two separate riboprobes targeting acsl4a. Lateral views, animal pole to the top. e, epiboly.

(B) Morphological defects in dorsoventral patterning caused by perturbation of acsl4a expression. Phenotypic classes were assigned as described elsewhere ( Kishimoto et al., 1997). Representative bright-field images of wild-type embryos (24 hpf) that are dorsalized (class C4 phenotype shown) when injected with acsl4a MO and ventralized (class V3 phenotype shown) when injected with acsl4a mRNA. Embryos injected with a catalytically inactive version of acsl4a (acsl4aCI) mRNA and control siblings develop normally. Arrowhead indicates wound-up trunk and arrow indicates expanded blood island. 500 fmol acsl4a MO; n = 8 experiments (406 embryos); 79%C5, 8%C4, 11%C3, 1%C2, and 1%C1; 1.2ā€“1.5 ng acsl4a mRNA; n = 3 (101 embryos); 6%V4, 23%V3, 25%V2, 45%V1, 2%N; 1.2ā€“1.5 ng acsl4aCI mRNA; n = 3 (123 embryos); 100% N.

(C) Fate map of the gastrula-stage zebrafish embryo (Kimmel et al., 1990 Schier and Talbot, 2005). Blue represents ventral fates and red represents dorsal fates.

(Dā€“G) Whole-mount mRNA in situ hybridization of markers of dorsal and ventral tissues in 80% epiboly stage embryos, either control or acsl4a MO-injected. Animal pole view, dorsal to the right. (D) tbx6, a marker of dorsal presumptive paraxial mesoderm, is expanded ventrally in acsl4a MO-injected embryos. 95% of acsl4a MO-injected (750 fmol) embryos displayed expansion >180 degrees (n = 8; 323 embryos). (E) eve1, a marker of ventral mesoderm, is reduced in acsl4a MO-injected (500 fmol) embryos (97%; n = 6; 272 embryos). (F) foxb1.2, a marker of neural plate/dorsal ectoderm, is expanded ventrally in acsl4a MO-injected (500 fmol) embryos (93%; n = 4; 135 embryos). (G) gata2a, a marker for epidermis/ventral ectoderm, is reduced in acsl4a MO-injected (500 fmol) embryos (98%; n = 4; 181 embryos).

(H) pax2a and myod1 expression in four-somite stage embryos. Dorsal view, anterior to the left. pax2a marks m/h, midbrain/hindbrain boundary; o, otic placode; and p, pronephros. myod1 marks a, adaxial cells. 98% of acsl4a MO-injected (500 fmol) embryos had expansion of pax2a marking m/h (n = 7; 125 embryos). Seventy-five percent of acsl4a MO-injected (500 fmol) embryos had loss and 25% had reduced expression of pax2a marking pronephros (n = 5; 86 embryos).

(I) myod1 expression in 15-somite stage embryos. Dorsal view, anterior to the left. s, somites. Ninety-six percent of acsl4a MO-injected (500 fmol) embryos had expanded somites (n = 10; 138 embryos).

See also Figures S1 and S2.

Gene Expression Details
Gene Antibody Fish Conditions Stage Anatomy Assay
acsl4a WT standard conditions 4-cell whole organism ISH
1k-cell whole organism ISH
Germ-ring whole organism ISH
50%-epiboly whole organism ISH
50%-epiboly yolk syncytial layer ISH
Germ-ring yolk syncytial layer ISH
90%-epiboly whole organism ISH
90%-epiboly yolk syncytial layer ISH
eve1 WT control 75%-epiboly ventral mesoderm ISH
WT + MO1-acsl4a standard conditions 75%-epiboly ventral mesoderm ISH
foxb1a WT control 75%-epiboly neural plate ISH
WT + MO1-acsl4a standard conditions 75%-epiboly neural plate ISH
gata2a WT control 75%-epiboly ectoderm ISH
WT + MO1-acsl4a standard conditions 75%-epiboly ectoderm ISH
myod1 WT control 1-4 somites adaxial cell ISH
14-19 somites somite ISH
WT + MO1-acsl4a standard conditions 1-4 somites adaxial cell ISH
14-19 somites somite ISH
pax2a WT control 1-4 somites midbrain hindbrain boundary neural keel ISH
1-4 somites otic placode ISH
1-4 somites pronephric mesoderm ISH
WT + MO1-acsl4a standard conditions 1-4 somites midbrain hindbrain boundary neural keel ISH
tbx16l WT control 75%-epiboly presumptive paraxial mesoderm ISH
WT + MO1-acsl4a standard conditions 75%-epiboly presumptive paraxial mesoderm ISH
Antibody Labeling Details No data available
Phenotype Details
Fish Conditions Stage Phenotype
WT + MO1-acsl4a standard conditions Prim-5 dorsal/ventral pattern formation disrupted, abnormal
Prim-5 whole organism wholly dorsalized, abnormal
Acknowledgments:
ZFIN wishes to thank the journal Developmental Cell for permission to reproduce figures from this article. Please note that this material may be protected by copyright.

Reprinted from Developmental Cell, 27(6), Miyares, R.L., Stein, C., Renisch, B., Anderson, J.L., Hammerschmidt, M., and Farber, S.A., Long-Chain Acyl-CoA Synthetase 4A Regulates Smad Activity and Dorsoventral Patterning in the Zebrafish Embryo, 635-647, Copyright (2013) with permission from Elsevier. Full text @ Dev. Cell