MicroRNAs (miRNAs) are ∼21-nucleotide (nt) RNAs that guide the post-transcriptional regulation of target genes during plant
and animal development (Bartel 2004). Plant miRNAs recognize nearly perfect complementary binding sites in target mRNAs and mediate RNA cleavage (Llave et al. 2002; Rhoades et al. 2002; Tang et al. 2003). The high degree of complementarity between plant miRNAs and their binding sites in target mRNAs has allowed confident miRNA
target predictions (Rhoades et al. 2002; Jones-Rhoades and Bartel 2004; Fahlgren and Carrington 2010). Plant miRNA targets tend to encode key developmental regulators, including many transcription factors (Rhoades et al. 2002; Jones-Rhoades et al. 2006). Accordingly, several studies have demonstrated that the miRNA-mediated repression of target transcripts is essential for
correct cell differentiation and developmental timing during post-embryonic development (Jones-Rhoades et al. 2006; Chen 2009). However, relatively little is known regarding the roles of miRNAs in embryonic cell differentiation, and miRNA functions
during embryo developmental timing have not been characterized.
DICER-LIKE1 (DCL1) encodes an RNaseIII domain-containing protein that is nuclear-localized and required for processing primary miRNA transcripts
into mature miRNAs (Park et al. 2002; Reinhart et al. 2002; Papp et al. 2003; Fang and Spector 2007). With this realization that DCL1 is necessary for miRNA biogenesis, the recovery and analysis of dcl1 mutant alleles from previous forward genetic screens performed using Arabidopsis thaliana (Schauer et al. 2002) provide insight into the roles of miRNAs during plant development. Null dcl1 alleles (originally named emb76, then sus1) were recovered by Meinke's group (Errampalli et al. 1991; Castle et al. 1993) more than 15 years ago in screens for embryos with defective development. dcl1 embryos are developmentally arrested at the globular stage of embryogenesis and exhibit abnormal divisions throughout the
extraembryonic suspensor (Schwartz et al. 1994). These results suggest that DCL1 and, by implication, miRNAs are required for embryo development and viability. Null mutations in other genes required for
normal miRNA biogenesis or function also produce defects during embryogenesis (Lynn et al. 1999; Lobbes et al. 2006; Grigg et al. 2009). In addition, specific miRNA/target interactions are required for proper cotyledon formation during embryo development (Palatnik et al. 2003; Laufs et al. 2004; Mallory et al. 2004, 2005). Moreover, several plant miRNA targets are required for proper embryogenesis (Aida et al. 1997; Emery et al. 2003; Dharmasiri et al. 2005; Prigge et al. 2005). Collectively, these results strongly indicate that miRNAs have important functions during embryo development.
Morphogenesis is the phase of embryo development when the basic plant body plan is established. Embryonic cell types are specified
in particular locations at precise developmental time points. Shoot meristem precursors and root meristem precursors are specified
at the apical and basal poles of the early embryo, respectively (Mayer et al. 1991). The post-embryonic activity of these meristems generates the adult plant body. Patterning along the radial axis during
early embryogenesis generates the outermost protoderm layer, the innermost vascular primordium, and a middle layer of ground
tissue precursors (Laux et al. 2004). After morphogenesis, embryos transition to a maturation phase, where they accumulate storage proteins, undergo desiccation
tolerance, and prepare to enter into a state of dormancy prior to germination (Gutierrez et al. 2007; Holdsworth et al. 2008). Although progress has been made (Weber et al. 2005; Braybrook and Harada 2008; Park and Harada 2008), the molecular basis of early embryonic patterning and the morphogenesis-to-maturation-phase transition are not completely
understood.
To assess the regulatory functions of miRNAs during embryogenesis, we revisited the phenotypic characterization of dcl1 embryos, with a focus on early morphogenesis and cell specification defects. DCL1 was required for multiple embryonic cell differentiation events as early as the eight-cell stage. Genome-wide transcript
profiling revealed that DCL1 was required for the early embryonic repression of nearly 50 miRNA targets. Several of the miRNA targets up-regulated in
eight-cell dcl1 embryos encode transcription factors that promote differentiation during later stages of embryogenesis. Moreover, dcl1 embryos prematurely accumulated transcripts from hundreds of genes typically expressed during the maturation phase of embryo
development.