Two Myristoylated Alanine‐Rich C‐Kinase Substrate (MARCKS) Paralogs are Required for Normal Development in Zebrafish

Abstract

Myristoylated alanine-rich C-kinase substrate (MARCKS) is an actin binding protein substrate of protein kinase C (PKC) and critical for mouse and Xenopus development. Herein two MARCKS paralogs, marcksa and marcksb, are identified in zebrafish and the role of these genes in zebrafish development is evaluated. Morpholino-based targeting of either MARCKS protein resulted in increased mortality and a range of gross phenotypic abnormalities. Phenotypic abnormalities were classified as mild, moderate or severe, which is characterized by a slight curve of a full-length tail, a severe curve or twist of a full-length tail and a truncated tail, respectively. All three phenotypes displayed abnormal neural architecture. Histopathology of Marcks targeted embryos revealed abnormalities in retinal layering, gill formation and skeletal muscle morphology. These results demonstrate that Marcksa and Marcksb are required for normal zebrafish development and suggest that zebrafish are a suitable model to further study MARCKS function.

Functional Diversification of the Four MARCKS Family Members in Zebrafish Neural Development

“…To better characterize the relative expression of each of these four genes along development, we performed real‐time RT‐PCR experiments, comparing them with two different housekeeping genes ( ef1a and rpl13a ) and using oocyte RNA levels as a reference. We confirmed that as described before (Ott et al., ), transcripts are present at all embryonic and larval stages. In the case of marcksl genes, they are also expressed at detectable levels between 24 and 72 hpf (Fig.…”

“…Two marcks genes ( marcksa and marcksb ) have been previously identified in the zebrafish by sequence comparison to the human marcks cDNA sequence (Ott et al., ). The analysis of the respective genome sequences showed us that while marcksb gene contains only one intron at the position described for other species (Blackshear, ), marcksa gene has two introns: the first intron is localized at the usual position coincident with the MH2 domain, and the second intron is at the 3′‐UTR (Fig.…”

“…In addition to the two previously described MARCKS‐encoding genes (Ott et al., ), we have found two MARCKSL1‐encoding genes in the zebrafish and performed a comparative characterization of their genomic, mRNA, and predicted protein primary structures, in addition to functionally testing them using specific morpholino antisense oligomers (MOs). Our phenotypic analyses of morphant embryos show that even if they have similarities, like the previously described smaller head and eyes and curved body, there are essential differences in neurulation, brain and eye morphogenesis, and neuronal differentiation.…”

Terminal Uridylyltransferases Execute Programmed Clearance of Maternal Transcriptome in Vertebrate Embryos

“…The calmodulin-binding proteins are regulated by calcium waves in the earliest cell cycles and play important roles in early development (Webb and Miller, 2000). For example, Marcks proteins, which are involved in gastrulation and neural development (Ott et al, 2011;Prieto and Zolessi, 2017), are encoded by transcripts with long poly(A) tails (69.9 and 37.9 nt for marcksb and marcksa, respectively). Some 3 0 UTR binding proteins also have long tails (40.2, 42.2, and 39.7 nt for cpeb1b, cth1, and cirbpa, respectively), which implies that they may be translated prior to the MZT and bootstrap the transcriptome to control their fates in later stages.…”

Subtractive phage display technology identifies zebrafish marcksb that is required for gastrulation

“…It has been reported that MARCKS is critical for mouse and Xenopus development (Ali et al, 1997;Shi et al, 1997;Weimer et al, 2009). Recently, two MARCKS paralogs, marcksa and marcksb, were identified in zebrafish and morpholino-based targeting of either Marcks protein resulted in increased mortality and a range of phenotypic abnormalities (Ott et al, 2011). In this study, we found that the expression level of Marcksb protein was nearly undetectable at 30%-epiboly and then significantly increased at the tailbud stage.…”