FHD-GFP interferes with severity of the MZsur mutant phenotype. a–dWild-type (a, b) and MZsur mutant (c, d) embryos at 24hpf. col2a1a in-situ staining in wild-type (un-injected control (a) or injected with FHD-GFP (b)) shows wild-type notochord of expected width (white brackets in enlarged sections a′ and b′). In uninjected MZsur embryos the width is reduced (c′). Injection of FHD-GFP in MZsur mutants enhances the phenotype (d/d′; note reduced size and additionally discontinuity of staining). e–l foxa2 in-situ hybridizations show reduction of axial mesoderm formation in MZsurmutants. Injection of FHD-GFP causes a broadened axial signal in 50% of wild type embryos, but no reduction of axial cells (f), and strengthens the effect in 60% of the MZsur mutants (h). dre-miR-430 morpholinos (MOs) massively reduce axial foxa2 signals in both genotypes (l, k). Co-injection of FHD-GFP and MOs also results in a decreased staining (j, l) when compared to FHD-GFP injection (f, h). Percentage of embryos showing the same phenotype as in the image is given (upper right). m–t sox17 in-situ hybridizations show only slight reduction of endoderm after injection of MOs (q–t). Number of forerunner cells (black arrow) is reduced in MZsurcontrol and after MOs injections (o, q, s). FHD-GFP lead to complete loss of forerunner cells (black arrow) in the majority of MZsur embryos (p, t; see also Additional file : Statistical analysis of forerunner cells). Numbers of sox17-positive cells seen in dorsal view and standard deviation are given (upper right) as well as number of analyzed embryos (n) (lower right). Size bars: 200 μm. γ value was changed to 0.8 in each picture

Expression / Labeling details

Phenotype details

FoxH1 blocks maternal clearance of cd82b and jade1. a, b RT-qPCR analysis of cd82b (a) or jade1 (b) in embryos at 50% epiboly with indicated genetic background. Massive reduction of expression is shown in MZsur mutants which cannot be rescued by injection of FHD-VP16 mRNA but is rescued via injection of foxH1^WT mRNA. Error bars indicate standard error (SEM) from 2 biological replicates. Calculation of relative normalized expression, standard error, and significance was made with the Bio-Rad CFX Manager 3.1 software (***p < 0.001; n.s. p ≥ 0.05). For individual values, see also Additional file : Individual qPCR values. c–f WISH for cd82b and jade1 in wild type embryos (c, e) shows staining at sphere stage, but not at shield stage when miR-430 becomes active (c′, e′). In MZsur mutants (d, f), weak or no staining is visible for all stages indicating the negative role of FoxH1 in regulating miR-430 activity at early embryonic stages. Size bars 200 μm

Expression / Labeling details

Phenotype details