3. RESULTS
3.5 P95 is required to maintain activity of developmental signaling
So far the results of this study suggest that well-balanced P95 protein levels are crucial for patterning and morphogenetic movements, as well as maintaining WNT/β- catenin signaling during zebrafish gastrulation. Given that P95 is a novel protein with a specific function in early endosomal trafficking, we were interested to clarify the functional specificity of the WNT signaling phenotype. To assess if P95 was required to maintain signaling activity of other signaling pathways that control development, we analyzed its effects on BMP, FGF and Shh signaling during zebrafish gastrulation.
As we could show that P95 MO KD resulted in an early D/V patterning defect with ectopic expression of chordin (expansion towards ventral; Figs. 3.7 B & 3.8 B), a negative regulator of BMP signaling, we first analyzed BMP signaling activity at the onset of gastrulation. Using the shield-stage imaging assay, we detected the nuclear accumulation of phosphorylated Smad1/5/8 (P-Smad1/5/8), an established marker to directly test for BMP signaling activity [110]. We found a gradient of nuclear P- Smad1/5/8 intensity across the whole D/V axis of flat-mounted shield-stage embryos (Fig. 3.17). In agreement with the literature, P-Smad1/5/8 intensities were highest at the ventral side (highest BMP ligand expression, opposite of shield) and lowest at the dorsal pole of the D/V axis (shield). Quantification of the nuclear P-Smad1/5/8 intensity profile across the D/V axis of whole shield-stage embryos, revealed a significant reduction of global P-Smad1/5/8 intensity in P95 morphants (Fig. 3.17 A). More specifically, the P-Smad1/5/8 intensity profile was flattened on the ventral half in P95 morphants, indicating an altered gradient of BMP signaling activity (Fig. 3.17 B). This data shows that systemic knockdown of zfP95 causes reduced BMP signaling activity on the ventral side of the embryo and is in agreement with the corresponding ventro-lateral expansion of the chordin expression domain.
Next, we tested FGF signaling activity at the end of gastrulation using the transgenic FGF reporter line Tg(Dusp6:d2EGFP) [142], in which EGFP expression is driven by the Dusp6 promoter region. Expression of the dual specificity phosphatase 6 (Dusp6) is transcriptionally regulated by FGF signaling in zebrafish [142]. We found that reporter gene expression was unaffected in most of the embryos injected with P95 MO1, with a trend towards reduced EGFP expression levels (Fig. 3.18). That indicates no critical alteration of FGF signaling upon P95 MO KD.
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Figure 3.17 P95 MO1 KD alters the gradient of BMP signaling activity at the onset of gastrulation.
(A) The representative image shows a flat-mounted shield-stage control embryo in which P-Smad1/5/8 is detected by immunofluorescence in one confocal plane of deep cells across the whole embryonic D/V axis (v: ventral, d: dorsal). Quantification of total nuclear P-Smad1/5/8 intensity showed a global reduction upon P95 MO1 KD. The scatter plot shows the averaged total P-Smad1/5/8 intensities (Mean ± SD) per embryo, normalized to the Mean of the control of each independent experiment. *** P value ≤ 0.001; unpaired t test with Welch’s correction. (B) Quantification of nuclear P-Smad1/5/8 intensities along a rectangular scan area across the D/V axis (intensity profile, indicated by the yellow rectangle in A). The P-Smad1/5/8 intensity profile corresponds to the D/V gradient of BMP activity (highest ventral; lowest dorsal) and precisely detects signaling strength downstream of activated BMP receptors in wild- type (WT) and Control MO-injected embryos. Compared to controls, P95 MO1 KD resulted in a flattened P-Smad1/5/8 intensity gradient (Mean ± SD for each scan position along the rectangular scan area are plotted from several embryos per condition). iExp: independent experiment; n: number of embryos analyzed; Control MO: 1 ng/E Control MO + 1 ng/E P53 MO1; P95 MO1 was co-injected with 1 ng/E P53 MO1.
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Figure 3.18 P95 MO KD does not compromise FGF signaling at the end of gastrulation (tailbud stage).
Systemic knockdown of zfP95 expression had only minor effects on reporter gene (EGFP) expression in the FGF reporter line Tg(dusp6:d2EGFP). The majority of P95 morphants had control-like expression levels (medium and strong expression) of the reporter gene. iExp: independent experiment; n: number of embryos analyzed; Control MO: 1 ng/E Control MO + 1 ng/E P53 MO1; P95 MO1 was co-injected with 1 ng/E P53 MO1.
Shh signaling was assessed by the expression levels of sonic hedgehog (shha) and its receptor and direct target gene patched (ptc1) [127]. The broader and shorter axial
shha expression domain confirmed the moderate C/E defects resulting from P95 MO
KD. Especially in the most anterior part of the axial domain, shha expression was often reduced (Fig. 3.19). A more pronounced reduction of expression levels was found for
ptc1. The majority of P95 morphants showed considerable reduced ptc1 expression
(Fig. 3.19) at the end of gastrulation. That showed that P95 negatively affected the transcriptional outcome of Shh signaling in zebrafish development. In summary, this evidence suggests that P95 is required for maintaining signaling activity of distinct signaling pathways that govern vertebrate embryogenesis.
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Figure 3.19 P95 MO KD results in a reduction of ptc1 expression at the end of zebrafish gastrulation.
WISH for shha showed reduced expression levels in the anterior dorsal axis and also shorter axial domains of P95 morphants at tailbud stage. Ptc1 expression was reduced in the majority of embryos upon P95 MO KD, consistent for two independent P95 MOs (P95 MO1, P95 spl2). The graph shows the quantification of P95 morphants with reduced ptc1 expression as percentage of total embryos analyzed. iExp: independent experiment; n: number of embryos analyzed; Control MO: 1 ng/E Control MO + 1 ng/E P53 MO1; all P95 MOs were co-injected with 1 ng/E P53 MO1.