ZFIN ID: ZDB-FIG-181107-2
Wang et al., 2018 - Anosmin1 Shuttles Fgf to Facilitate Its Diffusion, Increase Its Local Concentration, and Induce Sensory Organs. Developmental Cell   46(6):751-766.e12 Full text @ Dev. Cell
ADDITIONAL FIGURES
EXPRESSION / LABELING:
Genes:
Antibody:
Fish:
Anatomical Terms:
Stage Range: Prim-15 to Long-pec
PHENOTYPE:
Fish:
Observed In:
Stage Range: Prim-15 to Long-pec

Fig. 3

Anos1 Enhances FGF Signaling in the Primordium

(A) Schematic of the cross-repression between Wnt and FGF signaling in the primordium.

(B) Top, anos1a and anos1b mRNA expression in the primordium outlined by membrane GFP. Bottom, anos1a and anos1b mRNA expression only (white). Scale bar represents 100 μm.

(C) dusp6:d2eGFP expression in the primordium and dusp6:d2eGFP expression only (fire heatmap) for the genotypes indicated. Scale bar represents 100 μm.

(D) Top, quantification of GFP intensity from dusp6:d2EGFP inside the primordium along the anterior-posterior axis. X axis represents distance from the front of the primordium. Mean and SEM are shown. n indicates number of embryos. Bottom, plot of p values comparing the two genotypes in the graph above for each position along the x axis.

(E) Wnt reporter readout at 36 hpf in the primordium on the left and Wnt reporter readout only (fire heatmap below). Scale bar represents 100 μm.

(F) Quantification of Wnt reporter intensity. X axis represents distance from the front of the primordium. Mean and SEM and are shown. n = number of individual embryos measured.

(G) Neuromast size and number defects in anos1 mutants. Neuromast 1 in embryos of the indicated genotype at 32 hpf (left). Scale bar represents 50 μm. Quantification of neuromast 1 volume in embryos of indicated genotype at 32 hpf (middle). Neuromast number in 42-hpf embryos of indicated genotype (right). Mean, SD, and individual data points are shown. n.s.= p > 0.05, ∗∗ = p < 0.01., and ∗∗∗∗ = p < 0.0001. ANOVA p < 0.0001.

(H) (Left) ZO-1 and GFP immunostaining in the primordium of embryos of the indicated genotype at 36 hpf. (Right) ZO-1 only (fire look-up table). Arrows indicate ZO-1 signal at the center of apical constrictions. Scale bar represents 100 μm.

(I) Quantification of microlumen volumes in forming neuromasts in embryos of indicated genotypes. Mean, SD, and individual data points are shown. n.s. = p > or = 0.05,  = p < 0.05., ∗∗ = p < 0.01. and ∗∗∗∗ = p < 0.0001. ANOVA p < 0.0001.

(J) Phenotype of embryos with reduced FGF signaling with or without mutations in anos1a and anos1b at 50 hpf. The arrow indicates the position of the primordium. Scale bar represents 500 μm.

(K) Primordium and neuromast position in embryos of indicated genotypes at 50 hpf (left). The arrow denotes the position of the primordium. The double-headed arrow indicates spacing between neuromast 1 and 2. Scale bar represents 500 μm. Box and whisker plot of the distance between neuromast 1 and 2 for the genotypes indicated (right). n is the number of individual embryos. ns = p >0.05, ∗∗ = p < 0.01. ANOVA p < 0.01.

(L) Quantification of normalized distance migrated for the genotypes indicated. Mean, SD, and individual data points are shown. n.s. = p > 0.05, ∗∗ = p < 0.01, ∗∗∗ = p < 0.001. ANOVA p < 0.0001.

See also Figure S3.

Gene Expression Details
Gene Antibody Fish Conditions Stage Anatomy Assay
anos1a zf106Tg standard conditions Prim-15 posterior lateral line primordium ISH
anos1b zf106Tg standard conditions Prim-15 posterior lateral line primordium ISH
EGFP anos1ask50/+ ; anos1bsk52/+ ; zf106Tg ; fgf3t24149/+ ; anos1ask51/+ ; fgf10atbvbo/tbvbo ; anos1bsk53/+ standard conditions Long-pec posterior lateral line neuromast IFL
Long-pec posterior lateral line primordium IFL
anos1ask50/+ ; anos1bsk53/+ ; anos1bsk52/+ ; zf106Tg ; sk89Tg ; anos1ask51/+ standard conditions Long-pec posterior lateral line neuromast IFL
Long-pec posterior lateral line primordium IFL
anos1bsk52/+ ; anos1ask51/+ ; anos1ask50/+ ; zf106Tg ; fgf3t24149/t24149 ; anos1bsk53/+ standard conditions Long-pec posterior lateral line neuromast IFL
Long-pec posterior lateral line primordium IFL
fgf3t24149/+ ; fgf10atbvbo/tbvbo ; zf106Tg standard conditions Long-pec posterior lateral line neuromast IFL
Long-pec posterior lateral line primordium IFL
fgf3t24149/t24149 ; fgf10atbvbo/+ ; anos1bsk52/+ ; anos1ask51/+ ; anos1bsk53/+ ; zf106Tg ; anos1ask50/+ standard conditions Long-pec posterior lateral line neuromast IFL
Long-pec posterior lateral line primordium IFL
zf106Tg standard conditions Prim-15 neuromast IFL
Prim-25 posterior lateral line primordium IFL
Long-pec posterior lateral line neuromast IFL
Long-pec posterior lateral line primordium IFL
zf106Tg ; anos1bsk53/+ ; anos1ask50/+ ; anos1ask51/+ ; anos1bsk52/+ standard conditions Prim-15 neuromast IFL
Prim-25 posterior lateral line primordium IFL
Long-pec posterior lateral line neuromast IFL
Long-pec posterior lateral line primordium IFL
zf106Tg ; fgf10atbvbo/+ ; fgf3t24149/t24149 standard conditions Long-pec posterior lateral line neuromast IFL
Long-pec posterior lateral line primordium IFL
GFP anos1ask50/+ ; anos1bsk52/+ ; anos1bsk53/+ ; kyu2Tg ; anos1ask51/+ ; sk85Tg standard conditions Prim-25 posterior lateral line primordium IFL
fgf3t24149/t24149 ; sk85Tg ; kyu2Tg ; fgf10atbvbo/tbvbo standard conditions Prim-25 posterior lateral line primordium IFL
kyu2Tg ; sk85Tg standard conditions Prim-25 posterior lateral line primordium IFL
mCherry anos1ask50/+ ; anos1bsk52/+ ; anos1bsk53/+ ; kyu2Tg ; anos1ask51/+ ; sk85Tg standard conditions Prim-25 posterior lateral line primordium IFL
fgf3t24149/t24149 ; sk85Tg ; kyu2Tg ; fgf10atbvbo/tbvbo standard conditions Prim-25 posterior lateral line primordium IFL
kyu2Tg ; sk85Tg standard conditions Prim-25 posterior lateral line primordium IFL
Antibody Labeling Details
Antibody Assay Fish Conditions Stage Anatomy
Ab1-tjp1 IHC zf106Tg standard conditions Prim-25 posterior lateral line primordium cell
IHC zf106Tg ; anos1bsk53/+ ; anos1ask50/+ ; anos1ask51/+ ; anos1bsk52/+ standard conditions Prim-25 posterior lateral line primordium cell
Phenotype Details
Fish Conditions Stage Phenotype
anos1ask50/+ ; anos1bsk52/+ ; anos1bsk53/+ ; kyu2Tg ; anos1ask51/+ ; sk85Tg standard conditions Prim-25 posterior lateral line primordium GFP expression increased distribution, abnormal
anos1ask50/+ ; anos1bsk53/+ ; anos1bsk52/+ ; zf106Tg ; sk89Tg ; anos1ask51/+ standard conditions Long-pec posterior lateral line neuromast increased distance posterior lateral line neuromast, ameliorated
anos1bsk52/+ ; anos1ask51/+ ; sk89Tg ; anos1ask50/+ ; anos1bsk53/+ standard conditions High-pec posterior lateral line neuromast decreased amount, abnormal
anos1bsk53/+ ; anos1bsk52/+ ; anos1ask50/+ ; anos1ask51/+ standard conditions Prim-15 neuromast decreased size, abnormal
High-pec posterior lateral line neuromast decreased amount, abnormal
fgf10atbvbo/tbvbo standard conditions Prim-15 neuromast decreased size, abnormal
fgf3t24149/t24149 standard conditions Prim-15 neuromast decreased size, abnormal
fgf3t24149/t24149 ; fgf10atbvbo/+ ; anos1bsk52/+ ; anos1ask51/+ ; anos1bsk53/+ ; zf106Tg ; anos1ask50/+ standard conditions Long-pec posterior lateral line neuromast primordium migration decreased efficacy, abnormal
fgf3t24149/t24149 ; sk85Tg ; kyu2Tg ; fgf10atbvbo/tbvbo standard conditions Prim-25 posterior lateral line primordium GFP expression increased amount, abnormal
Prim-25 posterior lateral line primordium GFP expression increased distribution, abnormal
zf106Tg ; anos1bsk53/+ ; anos1ask50/+ ; anos1ask51/+ ; anos1bsk52/+ standard conditions Prim-15 neuromast decreased size, abnormal
Long-pec posterior lateral line neuromast increased distance posterior lateral line neuromast, 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, 46(6), Wang, J., Yin, Y., Lau, S., Sankaran, J., Rothenberg, E., Wohland, T., Meier-Schellersheim, M., Knaut, H., Anosmin1 Shuttles Fgf to Facilitate Its Diffusion, Increase Its Local Concentration, and Induce Sensory Organs, 751-766.e12, Copyright (2018) with permission from Elsevier. Full text @ Dev. Cell