Lin9 Is Required for Mitosis and Cell Survival during Early Zebrafish Development

Birgit Samans,Markus A. Kleinschmidt,Stefan Gaubatz,Daniel Liedtke,Susi Spahr,Toni U. Wagner

doi:10.1074/jbc.m809635200

Birgit Samans, Markus A. Kleinschmidt + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m809635200

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Abstract

LIN9 has been described as a regulator of G(1)/S and G(2)/M progression of the cell cycle in invertebrates and human cell lines. To elucidate the in vivo function of LIN9 during vertebrate development, we took advantage of the teleost zebrafish (Danio rerio). By means of antisense morpholinos we show here that Lin9-depleted embryonic cells accumulate in mitosis. Flow cytometry and confocal microscopy data demonstrate that the delay in mitotic progression is followed by apoptosis, which strongly manifests in the developing central nervous system. In accordance with these findings, we identified a cohort of Lin9-regulated genes required for different mitotic processes, including mitotic entry, metaphase/anaphase transition, and cytokinesis. Our data establish LIN9 as an essential regulator of mitosis in vertebrate development.

Highlights

LINC/DREAM is a recently identified multiprotein complex that is required for two transcriptional processes that act on cell cycle regulation, namely repression of genes that drive G1/S transition and activation of genes required for G2/M progression [3, 5, 6]
B, semiquantitative Reverse Transcription (RT)-PCR at the indicated stages reveals that lin9, lin54, and bmyb transcripts are present during embryogenesis before and after the midblastula transition (MBT)
Apoptosis and Accumulation of Mitotic Cells in lin9 Morphant Brains—The number of cells with a sub-G1 DNA content strongly increased in lin9 morphants between 24 and 48 h after fertilization (Fig. 4C)

Summary

EXPERIMENTAL PROCEDURES

Morpholino and mRNA Injections—TU AB zebrafish were maintained and staged as described [17, 18]. For whole mount in situ hybridizations, the PCR product generated with the last pair of primers was TA-cloned into pCRII (Invitrogen). For amplification of full-length zebrafish lin, we used the following primers: forward, 5ЈTTTTGGATCCATGGCGGAGCTCGAGCAGCT-3Ј; reverse, 5Ј-TTTGCGGCCGCTCACGTTCTGTTGGTGTTGTTT-3Ј. Whole Mount in Situ Hybridization—pCRII-lin vectors (see “RNA Isolation, Reverse Transcription (RT), PCR, and Cloning”) were linearized with XhoI. In vitro transcription was performed using SP6 polymerase and the DIG RNA labeling kit (Roche Applied Science), according to the manufacturer’s instructions. Flow Cytometry—Upon removal of the chorion, 10 embryos were washed in PBS and transferred to a collection tube. Genes showing at least a 1.6-fold change were chosen

RESULTS

Findings

Summary of mopholino and mRNA injections

DISCUSSION