Abstract
The Caenorhabditis elegans heterochronic gene pathway, which consists of a set of regulatory genes, plays an important regulatory role in the timing of stage-specific cell lineage development in nematodes. Research into the heterochronic gene pathway gave rise to landmark microRNA (miRNA) studies and showed that these genes are important in stem cell and cancer biology; however, their functions in vertebrate development are largely unknown. To elucidate the function of the heterochronic gene pathway during vertebrate development, we cloned the zebrafish homologs of the C. elegans let-7 miRNA-binding protein, Lin-28, and analyzed their function in zebrafish development. The zebrafish genome contains two Lin28-related genes, lin-28a and lin-28b. Similar to mammalian Lin28 proteins, both zebrafish Lin-28a and Lin-28b have a conserved cold-shock domain and a pair of CCHC zinc finger domains, and are ubiquitously expressed during early embryonic development. In a reciprocal fashion, the expression of downstream heterochronic genes, let-7 and lin-4/miR-125 miRNA, occurred subsequent to lin-28 expression. The knockdown of Lin-28a or Lin-28b function by morpholino microinjection into embryos resulted in severe cell proliferation defects during early morphogenesis. We found that the expression of let-7 miRNA was upregulated and its downstream target gene, lin-41, was downregulated in these embryos. Interestingly, the expression of miR-430, a key regulator of maternal mRNA decay, was downregulated in lin-28a and lin-28b morphant embryos, suggesting a role for Lin-28 in the maternal-to-zygotic transition in zebrafish. Taken together, our results suggest an evolutionarily conserved and pivotal role of the heterochronic gene pathway in early vertebrate embryogenesis.
Highlights
The spatial and temporal coordination of gene expression during development is essential for the correct morphogenesis of animals
Genome Server for sequences homologous to human Lin28a and Lin28b, which were identified in mammalian genomes as two homologs of the C. elegans heterochronic gene lin-28, Lin28a and Lin28b [30,31]
Since the cold-shock domain and retroviral-type CCHC finger regions of zebrafish Lin28A and Lin28B had 79– 90% homology at the amino acid level with corresponding regions in mouse and human LIN28A and LIN28B (Figure 1A), the function of Lin-28a and Lin-28b may be conserved across animal phylogeny
Summary
The spatial and temporal coordination of gene expression during development is essential for the correct morphogenesis of animals. Genetic studies of Caenorhabditis elegans (C. elegans) have provided significant insight into the evolutionarily conserved networks of gene regulation that underlie development [1]. The C. elegans heterochronic gene pathway, the study of which led to the landmark discovery of the first microRNA (miRNA)-mediated gene regulatory network during development, was discovered through conventional forward genetic screens in C. elegans for heterochronic (developmental timing) mutants [2,3,4,5,6]. The functional significance of miRNAs in vertebrate development has been clearly demonstrated using Dicer mutants [10,11]. The injection of miR-430, which has a crucial function in deadenylation and the clearance of maternal mRNAs, rescues these brain defects, suggesting a critical role for miR-430 in early zebrafish development [12]
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