Systemic interference with zfP95 expression results in complex morphogenetic defects during

After we cloned the full-length zebrafish P95 homolog and found that it was ubiquitously expressed during zebrafish gastrulation, we tested its functional relevance for early zebrafish development. First, we systemically reduced zfP95 expression levels by injection of different zfP95-specific Morpholinos (P95 MOs) into 1-cell stage wild- type embryos. Consistent for four independent P95 MOs (P95 MO1 & P95 MO2 blocked translation; P95 spl1 & P95 spl2 blocked splicing), systemic injection resulted in complex morphogenetic defects that accumulated in a dose-dependent manner during the first day of development (Fig. 3.3; Fig. 3.4). Strikingly, three out of four P95 MOs (P95 MO1, P95 spl1, P95 spl2) evoked these morphogenetic defects at rather low- to-medium MO concentrations (in the range of 0.5-4 ng/E). Generally summarized, the majority of P95 morphants had a much shorter body axis as compared to controls and accumulated strong dorsal-ventral (D/V) and anterior-posterior (A/P) patterning defects at 24 hpf. Furthermore, organogenesis was compromised upon P95 MO KD as shown in severely abnormal organs like smaller or not developed eyes, no visible midbrain-hindbrain boundary (MHB), a compressed trunk with defective somites, a reduced yolk extension, ventral fin formation defects and an abnormal heart.

Wild-type embryos injected with P95 MOs often accumulated apoptotic/necrotic tissue in the central nervous system, a known toxic side-effect of Morpholino injections [134]. Co-injection of P95 MOs together with 1 ng/E zfP53-specific MO (P53 MO1), to suppress the P53-mediated apoptotic response, completely prevented MO toxicity (Fig. 3.3 B). Importantly, co-injecting P95 MOs together with 1 ng/E P53 MO1 did prevent the accumulation of apoptotic/necrotic tissue in the CNS, but did not rescue any other of the major defects observed upon single injection of P95 MOs. To control for unspecific Morpholino side-effects, P95 morphants were compared to wild- type embryos (not injected) and to control-injected embryos (injected with a control Morpholino; or injected with the P53 MO1; or co-injected with Control MO and P53 MO1). None of the control-injected embryos accumulated developmental defects above the level that naturally occurred in non-injected wild-type embryos during the first day of zebrafish development (Fig. 3.4 A).

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Figure 3.3 Interference with zfP95 expression results in severe morphogenetic defects in early zebrafish larvae (24 hpf). (A) Severe and medium morphant phenotypes (see table 3.1 for description)

caused after systemic injection of 1 ng/E P95 MO1, 6 ng/E P95 MO2, 0.5 ng/E P95 spl1, and 4 ng/E P95 spl2. Control embryos (Controls) were either not injected (WT), injected with 4 ng/E Control MO, or injected with control mRNA (50 pg/E PCNA-EGFP mRNA). P95 morphants shown in (A) were co-injected with 1 ng/E P53 MO1. (B) The MO-dependent side-effect of inducing cell death in the CNS was prevented by co-injection of P95 MOs with a zfP53-specific MO (P53 MO1). Embryos co-injected with P53 MO1 did not accumulate apoptotic/necrotic tissue in the CNS (red arrow). Single injection of P53 MO1 into 1-cell stage embryos (P53 MO1 only) did not evoke abnormal phenotypes and the embryos developed like wild-types. (C) Overexpression of zfP95 (50-100 pg/E zfP95 mRNA) caused also severe developmental defects that accumulated during the first day of development. (D) The P95 MO binding sites are shown with reference to the start codon (ATG) of the zebrafish P95 gene (zgc:92087).

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Interestingly, systemic overexpression of moderate amounts of zfP95 mRNA (P95 OE; 50-100 pg/E) resulted as well in the accumulation of severe developmental defects during the first day of zebrafish development (Fig. 3.3 C). P95 OE caused a variety of strong and medium overexpression phenotypes, many of which resembled the P95 morphant phenotypes. Similar to the P95 morphant phenotypes, P95 OE caused strong D/V and A/P patterning defects, failure to extend the body axis, and multiple defects during organogenesis. Injection of only 10 pg/E zfP95 mRNA into 1-cell stage embryos was sufficient to cause the accumulation of global developmental defects and their frequency and severity increased in a dose-dependent manner (Fig. 3.4 F). The complex morphogenetic phenotypes that resulted from P95 MO KD and P95 OE, were classified into three distinct classes according to their global severity (Table 3.1).

Table 3.1: Classification of global morphogenetic phenotypes resulting from systemic MO- mediated knockdown and overexpression of zfP95 at 24 hpf.

Phenotype

Class Description

Severe

Embryos showed strong extension defects (much shorter body axis); strong D/V and A/P patterning defects; no or much shorter yolk extensions; strong somitogenesis & organogenesis defects (no or much smaller eyes, no visible MHB); patterning of ventral and posterior fates/structures was affected the most (strong defects in tail formation)

Medium

Embryos were more extended than the severe cases; the yolk extension was elongated but looked still abnormal; clearly visible eyes; the MHB was often clearly visible; patterning defects of primarily ventral and posterior fates prevailed (tail formation still compromised)

Weak

Embryos were fully elongated and looked almost WT-like; often minor patterning defects of primarily posterior structures (tail) remained still visible; Note: the classification of global morphogenetic phenotypes upon systemic interference with

zfP95 expression was done by stereo light microscopy. That allowed for only a basic

phenotypic analysis and was sufficient to conclusively identify the severe and medium phenotypes. The weak phenotypes would have required a more detailed analysis and were therefore counted as WT-like to calculate the MO/mRNA penetrance (Fig. 3.4).

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Figure 3.4 Penetrance of the complex morphogenetic phenotypes caused by systemic interference with zfP95 expression (24 hpf). Each graph shows the P95 MO/OE concentration-dependent frequency

of the complex morphogenetic phenotypes as described in table 3.1 (the severe and medium-class phenotypes were summarized as “complex morphogenetic phenotypes”). Plotted are the Mean ± SD of complex morphogenetic phenotypes as percentage of total embryos analyzed. For each MO/mRNA concentration injected, embryos were compared to non-injected wild-type (WT) embryos of the same batch (dashed lines). In addition, the respective survival rates are plotted. For each individual experiment (each concentration) n ≥ 25 embryos per condition. (A) Injection of embryos with Control MO did neither cause accumulation of developmental defects nor decrease survival rates if compared to non-injected WT fish at 24 hpf. (B-E) All four P95 MOs caused complex morphogenetic phenotypes (morphants) in a concentration-dependent manner at 24 hpf. (F) Injection of zfP95 mRNA into 1-cell stage embryos at concentrations of more than 10 pg/E resulted in the accumulation of severe developmental defects (OE phenotype).

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Systemic interference with zfP95 expression by both, P95 MO KD and P95 OE prevented 90% of embryos from surviving larval stages and resulted in death before day 6 of development (data not shown). In summary, the global accumulation of severe developmental defects in response to either systemic knockdown or overexpression of zfP95, indicate that P95 is essential for zebrafish embryogenesis. That also suggests that P95 expression levels have to be precisely regulated early during zebrafish development.

3.2.2 The P95 morphant phenotypes can be partially rescued with human P95 mRNA