Downloaded from genesdev.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press MicroRNAs prevent precocious gene expression and enable pattern formation during plant embryogenesis Michael D. Nodine1,2,3 and David P. Bartel1,2,3,4 1Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142, USA; 2Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA; 3Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA Arabidopsis embryos lacking DICER-LIKE1 (DCL1), which is required for microRNA (miRNA) biogenesis, arrest early in development. To assess the functions of embryonic miRNAs, we determined the developmental and molecular consequences of DCL1 loss. We found that DCL1 is required for cell differentiation events as early as the eight-cell stage and soon thereafter for proper division of the hypophysis and subprotoderm cells. By the early globular (~32-cell) stage, dcl1-null mutant embryos overexpress ~50 miRNA targets. In dcl1 eight-cell embryos, the two most up-regulated targets are those of miR156 and encode SPL10 and SPL11 transcription factors. SPL10 and SPL11 are derepressed >150-fold in dcl1 embryos and are redundantly required for the dcl1 early patterning defects. Moreover, as early as the eight-cell stage, miR156-mediated repression of zygotic SPL transcripts prevents premature accumulation of transcripts from genes normally induced during the embryonic maturation phase. Thus, the first perceptible molecular function of plant embryonic miRNAs is the opposite of that in vertebrates; in vertebrates, miRNAs sharpen the first developmental transition, whereas in plants, they forestall developmental transitions by repressing mRNAs that act later. We propose that, by preventing precocious expression of differentiation-promoting transcription factors, miRNAs enable proper embryonic patterning. [Keywords: MicroRNA; embryogenesis; high-throughput sequencing; plant development; post-transcriptional gene regulation; Arabidopsis thaliana] Supplemental material is available at http://www.genesdev.org. Received August 26, 2010; revised version accepted October 13, 2010. MicroRNAs (miRNAs) are ;21-nucleotide (nt) RNAs opment (Jones-Rhoades et al. 2006; Chen 2009). How- that guide the post-transcriptional regulation of target ever, relatively little is known regarding the roles of genes during plant and animal development (Bartel 2004). miRNAs in embryonic cell differentiation, and miRNA Plant miRNAs recognize nearly perfect complementary functions during embryo developmental timing have not binding sites in target mRNAs and mediate RNA cleav- been characterized. age (Llave et al. 2002; Rhoades et al. 2002; Tang et al. DICER-LIKE1 (DCL1) encodes an RNaseIII domain- 2003). The high degree of complementarity between plant containing protein that is nuclear-localized and required miRNAs and their binding sites in target mRNAs has for processing primary miRNA transcripts into mature allowed confident miRNA target predictions (Rhoades miRNAs (Park et al. 2002; Reinhart et al. 2002; Papp et al. et al. 2002; Jones-Rhoades and Bartel 2004; Fahlgren and 2003; Fang and Spector 2007). With this realization that Carrington 2010). Plant miRNA targets tend to encode DCL1 is necessary for miRNA biogenesis, the recovery key developmental regulators, including many transcrip- and analysis of dcl1 mutant alleles from previous forward tion factors (Rhoades et al. 2002; Jones-Rhoades et al. genetic screens performed using Arabidopsis thaliana 2006). Accordingly, several studies have demonstrated (Schauer et al. 2002) provide insight into the roles of that the miRNA-mediated repression of target tran- miRNAs during plant development. Null dcl1 alleles (orig- scripts is essential for correct cell differentiation and inally named emb76, then sus1) were recovered by developmental timing during post-embryonic devel- Meinke’s group (Errampalli et al. 1991; Castle et al. 1993) more than 15 years ago in screens for embryos with de- fective development. dcl1 embryos are developmentally 4Corresponding author. E-MAIL [email protected]; FAX (617) 258-6768. arrested at the globular stage of embryogenesis and ex- Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.1986710. hibit abnormal divisions throughout the extraembryonic 2678 GENES & DEVELOPMENT 24:2678–2692 Ó 2010 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/10; www.genesdev.org Downloaded from genesdev.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press Functions of miRNAs in plant embryos suspensor (Schwartz et al. 1994). These results suggest Results that DCL1 and, by implication, miRNAs are required for embryo development and viability. Null mutations DCL1 is required for early embryonic in other genes required for normal miRNA biogenesis or pattern formation function also produce defects during embryogenesis (Lynn dcl1-null mutant embryos exhibit morphological defects et al. 1999; Lobbes et al. 2006; Grigg et al. 2009). In and arrest at the globular stage of development (Schwartz addition, specific miRNA/target interactions are required et al. 1994). To identify the earliest dcl1 morphological for proper cotyledon formation during embryo devel- defects, we systematically analyzed dcl1 embryos through- opment (Palatnik et al. 2003; Laufs et al. 2004; Mallory out early embryogenesis. Because dcl1 embryos are not et al. 2004, 2005). Moreover, several plant miRNA tar- viable, embryos from self-pollinated plants heterozygous gets are required for proper embryogenesis (Aida et al. for dcl1-5 and dcl1-10, two presumed null alleles (McElver 1997; Emery et al. 2003; Dharmasiri et al. 2005; Prigge et al. 2001; Schauer et al. 2002), were examined. et al. 2005). Collectively, these results strongly indicate Embryos developing within a single silique are approx- that miRNAs have important functions during embryo imately at the same developmental stage, which enabled development. an estimate of dcl1 embryonic stages based on the Morphogenesis is the phase of embryo development morphology of their wild-type siblings. It was reported when the basic plant body plan is established. Embryonic previously that sus1/dcl1 embryos exhibit abnormal cell cell types are specified in particular locations at precise divisions at the base of the embryo proper beginning at developmental time points. Shoot meristem precursors the globular stage and in the extraembryonic suspensor and root meristem precursors are specified at the apical beginning at the heart stage (Schwartz et al. 1994). Our and basal poles of the early embryo, respectively (Mayer morphological analysis of dcl1 embryos confirmed these et al. 1991). The post-embryonic activity of these meri- findings and revealed previously unreported phenotypes stems generates the adult plant body. Patterning along (Fig. 1A). We identified initial morphological defects in the radial axis during early embryogenesis generates the the presumptive hypophysis cells (i.e., the suspensor cell outermost protoderm layer, the innermost vascular pri- most proximal to the embryo proper) of 19% (29 of 154) of mordium, and a middle layer of ground tissue precursors dermatogen stage embryos from self-pollinated dcl1-5/+ (Laux et al. 2004). After morphogenesis, embryos tran- plants (Fig. 1A). By the early globular stage, morpholog- sition to a maturation phase, where they accumulate ical defects were observed in ;25% of embryos from storage proteins, undergo desiccation tolerance, and pre- selfed dcl1-5/+ plants (Fig. 1A). These embryos with pare to enter into a state of dormancy prior to germi- defects were presumably homozygous for dcl1-5-null nation (Gutierrez et al. 2007; Holdsworth et al. 2008). alleles, and their defects included abnormal hypophysis Although progress has been made (Weber et al. 2005; cell divisions as well as the previously unreported loss of Braybrook and Harada 2008; Park and Harada 2008), the periclinal subprotoderm cell divisions in the embryo molecular basis of early embryonic patterning and the proper (Fig. 1A). In subsequent stages, these abnormal morphogenesis-to-maturation-phase transition are not embryos did not produce cotyledons and were develop- completely understood. mentally arrested and nonviable (Fig. 1A; Schwartz et al. To assess the regulatory functions of miRNAs dur- 1994). Furthermore, dcl1-5 homozygous seedlings could ing embryogenesis, we revisited the phenotypic charac- not be recovered from selfed dcl1-5/+ plants, which terization of dcl1 embryos, with a focus on early mor- suggested that the developmentally arrested embryos phogenesis and cell specification defects. DCL1 was derived from selfed dcl1-5/+ plants were indeed homozy- required for multiple embryonic cell differentiation gous for dcl1-5 (data not shown). Because embryos from events as early as the eight-cell stage. Genome-wide selfed dcl1-5/+ and selfed dcl1-10/+ plants exhibited the transcript profiling revealed that DCL1 was required for same morphological defects at indistinguishable frequen- the early embryonic repression of nearly 50 miRNA cies, subsequent analyses focused on embryos from selfed targets. Several of the miRNA targets up-regulated in dcl1-5/+ plants (Table 1; data not shown). DCL1 tran- eight-cell dcl1 embryos encode transcription factors that scripts were detected by quantitative RT–PCT (qRT– promote differentiation during later stages of embryo- PCR) in both early globular