Abstract
BackgroundRunx transcription factors are important regulators of metazoan development. The sea urchin Runx gene SpRunt was previously identified as a trans-activator of the CyIIIa actin gene, a differentiation marker of larval aboral ectoderm. Here we extend the functional analysis of SpRunt, using morpholino antisense oligonucleotides (morpholinos) to interfere with SpRunt expression in the embryo.ResultsThe developmental effects of four different SpRunt-specific morpholinos were evaluated. The two morpholinos most effective at knocking down SpRunt produce an identical mitotic catastrophe phenotype at late cleavage stage that is an artifact of coincidental mis-targeting to histone mRNA, providing a cautionary example of the insufficiency of two different morpholinos as a control for specificity. The other two morpholinos produce gastrula stage proliferation and differentiation defects that are rescued by exogenous SpRunt mRNA. The expression of 22 genes involved in cell proliferation and differentiation was analyzed in the latter embryos by quantitative polymerase chain reaction. Knockdown of SpRunt was found to perturb the expression of differentiation markers in all of the major tissue territories as well as the expression of cell cycle control genes, including cyclin B and cyclin D.ConclusionsSpRunt is essential for embryonic development, and is required globally to coordinate cell proliferation and differentiation.
Highlights
Runx transcription factors are important regulators of metazoan development
Mammalian genomes contain three Runx genes, each of which is essential for development of a major organ system: Runx1 is required for hematopoiesis, Runx2 is required for osteogenesis, and Runx3 is
To test the efficacy of the translation-blocking morpholinos, zygotes were injected with m1, m2, or m3 or a non-specific control morpholino, together with synthetic SpRunt mRNA (>100-fold excess over endogenous levels), and the resulting embryos analyzed by immunoblot using an antibody generated against the N-terminal peptide of SpRunt
Summary
Runx transcription factors are important regulators of metazoan development. The Runt domain (Runx) is a highly conserved, 128 amino acid sequence that defines a small family of heterodimeric transcription factors that are key regulators of animal development (reviewed in [1]). Most developmental studies of Runx gene function have been carried out in Drosophila and in mice, each of which has multiple Runx genes. The genome of Drosophila melanogaster contains four Runx genes, including the well-studied genes runt and lozenge, as well as two genes (genomic loci CG1379 and CG15455) that have not been well-characterized [2]. Runt is a primary pair rule gene involved in segmentation, sex determination and neurogenesis, whereas lozenge is a key regulator of patterning in the eye (reviewed in [3]). The multiplicity of Runx genes in insects and vertebrates reflects independent duplication events within the arthropod and chordate lineages, and the primitive condition within bilaterians appears to be possession of a single Runx gene [2]
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