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We next tested whether DCL1 was required for embryonic cell differentiation by examining the expression of cell-specific markers in dcl1-5 embryos. WUSCHEL-related HOMEOBOX2 (WOX2) transcripts are localized in the apical cell lineage of wild-type preglobular embryos (Haecker et al. 2004). After the two/four-cell stage, WOX2 transcripts were not detected in about a quarter of eight-cell and dermatogen stage embryos (seven of 35 and 14 of 52, respectively) from self-pollinated dcl1-5/+ plants (Fig. 1B), which implied that dcl1-5 embryos had undetectable WOX2 expression. Similar to WOX2, PINHEAD/ZWILLE/ARGONAUTE10 transcripts, hereafter referred to as PNH, are localized in the apical cell lineage of preglobular embryos (Lynn et al. 1999). Approximately 89% (72 of 81) of eight-cell and dermatogen stage embryos from selfed dcl1-5/+ plants had detectable levels of PNH transcripts, suggesting that apical cell fate is not completely abolished in all preglobular dcl1 embryos (Supplemental Fig. 2). In contrast to WOX2 and PNH, WOX8 transcripts are localized to the basal cell lineage of preglobular wild-type embryos (Haecker et al. 2004). WOX8 transcripts were detected in 90% (62 of 69) of eight-cell and dermatogen stage embryos from selfed dcl1-5/+ plants, suggesting that basal cell lineage differentiation is not perturbed in all preglobular dcl1 embryos (Fig. 1B). Thus, dcl1 embryos had detectable defects in the differentiation of apical cell but not basal cell descendents as early as the eight-cell stage. To test whether DCL1 is required for radial patterning, we examined a series of markers that are differentially expressed along the radial axis. A. thaliana MERISTEM LAYER1 (ATML1) and PROTODERMAL FACTOR1 (PDF1) transcripts are detectable in the protoderm, but not the subprotoderm, of globular stage embryos (Lu et al. 1996; Abe et al. 1999). ATML1 and PDF1 transcripts were appropriately localized to the protoderm of globular stage dcl1-5 embryos, but were ectopically localized in the suspensors of dcl1-5 embryos whose wild-type siblings were at the early heart stage (Fig. 1B; Supplemental Fig. 2). This observation was consistent with the previously reported ectopic localization of embryo proper-specific starch grains and protein bodies in the suspensors of late stage sus1/dcl1 embryos (Schwartz et al. 1994). Consistent with the reduction/loss of subprotodermal cell divisions in dcl1 late globular/early heart stage embryos, we detected reduced levels of RPS5A transcripts in these cell types (Supplemental Fig. 2). Moreover, vascular primordium and ground tissue initial markers were absent from these cell types (Supplemental Fig. 2). In addition to the ectopic localization of protoderm markers in the suspensor, globular dcl1-5 suspensors had reduced levels of WOX8 transcripts (Fig. 1B). Furthermore, hypophysis markers were not detectable in dcl1 globular embryos (Fig. 1B; Supplemental Fig. 2). Considered together with the defective hypophysis cell divisions observed in dcl1 embryos, the lack of hypophysis markers indicates that the hypophysis is misspecified in dcl1 embryos. The plant hormone auxin is transported from the embryo proper to the presumptive hypophysis and contributes to hypophysis specification (Friml et al. 2003; Aida et al. 2004; Weijers et al. 2006). Because the auxin signaling-responsive DR5rev promoter fused to GFP (DR5rev∷GFP) is specifically active in the hypophysis cells of wild-type globular embryos (Friml et al. 2003), and hypophysis specification is perturbed in dcl1 embryos, we examined the activity of DR5rev∷GFP in dcl1-5 embryos to test whether DCL1 was required for appropriate auxin signaling. Although presumptive dcl1-5 hypophysis cells often expressed DR5rev∷GFP, cells located above the presumptive hypophysis ectopically expressed DR5rev∷GFP (Fig. 1B). Therefore, DCL1 is required for the hypophysis-specific increase in auxin signaling. Given the severe subprotodermal defects observed in dcl1 embryos, the inappropriate auxin signaling observed in dcl1 embryos was likely due to the incorrect expression of auxin transport and/or auxin signaling factors. Therefore, the auxin response defects observed in dcl1 embryos were probably secondary consequences of subprotoderm cell differentiation defects, which in turn were probably due to the apical cell lineage defects observed at the preglobular stages.

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