Full-length zebrafish P95 is expressed during early zebrafish embryogenesis

The P95 (PPP1R21) gene is highly conserved in vertebrates but no functional characterization is published to date. The zebrafish (Danio rerio) carries one copy of the P95 gene (zgc:92087) and only one publicly available clone (NM_001007299), derived from adult zebrafish, was available at the start of my thesis project. In order to functionally characterize P95 during zebrafish development, we cloned the full-length zebrafish P95 homolog and analyzed its expression during the first day of zebrafish embryogenesis.

3.1.1 Zebrafish full-length P95 is the homolog of human full-length P95

To analyze the function of zebrafish P95 (zgc:92087) during development, we first cloned the zebrafish P95 transcript and verified that it is the actual homolog of human P95. Total RNA was isolated from wild-type zebrafish embryos (of the two major genotypes AB and TL) of three different developmental stages (tailbud stage = end of gastrulation, 3 ss = start of somitogenesis/organogenesis, 24 hpf = zebrafish larvae with all major organ systems). Reverse transcription and PCR using primer (P95P4-Fw + P95P4-Rv, see section 6.2.4) to amplify the full-length P95 coding sequence, resulted consistently in the amplification of 1 major product of 2376 nucleotides length. Practically, only 1 major P95 transcript (2.3 kb length) could be consistently amplified from total RNA extracts of all the embryonic stages tested. Sequencing of the amplified 2.3 kb product confirmed it as zebrafish P95, identical for both wild-type strains tested (AB & TL from the MPI-CBG fish facility). Sequence alignment and similarity analysis revealed that the published zebrafish P95 transcript (derived from adults; NM_001007299) was significantly shorter than the P95 version we have cloned from embryonic stages (Fig. 3.1). The annotated zebrafish P95 transcript from adult fish (NM_001007299) lacked 378 nucleotides within the middle of the transcript (Fig. 3.1 A&B).

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Mapping the two zebrafish P95 transcripts to the zebrafish P95 gene, indicated that the insertion within our cloned, longer transcript could represent an additional exon (Fig. 3.1 B). This missing sequence fragment corresponded to a peptide within the conserved C-terminal coiled-coil domain of the P95 protein. P95 contains two evolutionary conserved coiled-coil domains (with unknown function) at the N- and C- terminus of the protein, respectively. Protein domain prediction confirmed that the embryonic zebrafish P95 transcript contained the two complete conserved coiled-coil domains, while the C-terminal domain of the P95 transcript from adult zebrafish was incomplete (Fig. 3.1 C).

Figure 3.1 Full-length zebrafish P95, cloned from zebrafish embryos, is the homolog of human P95.

(A) The coding sequence (CDS) of the adult-derived zebrafish P95 transcript (NCBI; NM_001007299; ppp1r21; zgc:92087) was aligned to the full-length P95 CDS cloned from zebrafish embryos (using ClustalW). The numbers indicate the transcript length in nucleotides. The insert indicates the sequence fragment missing in the P95 transcript derived from adult zebrafish. (B) The two zebrafish P95 transcripts (adult- vs. embryonic-derived) were mapped to the NCBI zebrafish P95 gene using BLAT. The red arrow indicates that the insertion in the embryonic-derived zebrafish P95 transcript could represent an additional exon within the P95 gene. (C) The Pfam protein domains from human P95, published zebrafish P95 (NCBI; NM_001007299; adult-derived), and embryonic-derived zebrafish P95 (cloned) were predicted using the Interproscan plugin in the Geneious software. The cloned zebrafish P95 transcript (zfP95) was predicted to contain the complete, highly conserved N-terminal KLRAQ (pfam10205) and C-terminal TTKRSYEDQ (pfam10212) coiled-coil domains. Full-length zebrafish P95 was predicted to be 60% identical and 77% similar to the human full-length P95 protein (by the BLASTP and clustalW algorithms).

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Comparison of the proteins predicted from the human full-length P95 transcript and the P95 transcript we cloned from zebrafish embryos, showed that they are 60% identical (percentage of identical amino acids) and 77% similar (percentage of amino acids with similar chemical properties) (Fig. 3.1 C). That strongly suggests that the P95 transcript we have cloned from zebrafish embryos represents the full-length transcript of the zebrafish P95 gene and the functional homolog of human full-length P95. Furthermore, it indicates that the shorter P95 transcript, derived from adult wild-type fish, might be a splice variant not required during early development (it is known from the human P95 homolog that several splice isoforms exist). Only the full-length P95 transcript cloned from zebrafish embryos (zfP95) was used to analyze its expression pattern, the sub-cellular localization of its corresponding protein, and its function during zebrafish development.

3.1.2 Full-length zebrafish P95 is ubiquitously expressed during zebrafish gastrulation To analyze if zfP95 was maternally delivered and confined to specific expression domains during early zebrafish embryogenesis, we detected zfP95 mRNA levels by whole-mount in situ hybridization (WISH) throughout the first day of development.

zfP95 mRNA was maternally delivered into the zygote and ubiquitously expressed from

the 2-cell stage onwards until the end of gastrulation (tailbud stage) (Fig. 3.2). Robust

zfP95 expression was observed during blastula stages, but zfP95 mRNA levels declined

at the onset of gastrulation (germ ring) and remained low until the end of gastrulation (tailbud stage) (Fig. 3.2). During somitogenesis, enrichment of zfP95 expression was observed in anterior domains of the future nervous system and posterior domains of the tailbud region. At 24 hours post fertilization (hpf), zfP95 mRNA levels were strongly upregulated and confined to anterior regions of the central nervous system (CNS). Expression of zfP95 in posterior trunk and tail regions was very low compared to the CNS. The observed expression patterns show that zfP95 is maternally delivered, ubiquitously expressed throughout blastula and gastrula stages, but differentially expressed during organogenesis.

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Figure 3.2 zfP95 is ubiquitously expressed during zebrafish gastrulation, but differentially expressed during organogenesis. Selected developmental stages, covering the first day of zebrafish

embryogenesis, were analyzed for expression of the zebrafish full-length P95 transcript by whole-mount in situ hybridization. Detectable from the first cleavage stages, zfP95 mRNA did not show any restriction to confined expression domains until the end of gastrulation (embryo orientation: side view, animal pole to top, dorsal to the right for gastrulation stages). An upregulation in areas of the future central nervous system (CNS) could be first detected during early somitogenesis and became very pronounced at 24 hpf (black arrows). An increase in expression could also be detected in the tailbud region during early somitogenesis but was not sustained as strong as compared to the CNS at 24 hpf. The top rows of the two major panels show the specific zfP95 mRNA expression as detected by an anti-sense RNA probe against zfP95. The bottom rows show control embryos incubated with the corresponding sense RNA probe to determine background levels and specificity.