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dcl1-null mutant embryos exhibit morphological defects and arrest at the globular stage of development (Schwartz et al. 1994). To identify the earliest dcl1 morphological defects, we systematically analyzed dcl1 embryos throughout early embryogenesis. Because dcl1 embryos are not viable, embryos from self-pollinated plants heterozygous for dcl1-5 and dcl1-10, two presumed null alleles (McElver et al. 2001; Schauer et al. 2002), were examined.
Embryos developing within a single silique are approximately at the same developmental stage, which enabled an estimate of
dcl1 embryonic stages based on the morphology of their wild-type siblings. It was reported previously that sus1/dcl1 embryos exhibit abnormal cell divisions at the base of the embryo proper beginning at the globular stage and in the extraembryonic
suspensor beginning at the heart stage (Schwartz et al. 1994). Our morphological analysis of dcl1 embryos confirmed these findings and revealed previously unreported phenotypes (Fig. 1A). We identified initial morphological defects in the presumptive hypophysis cells (i.e., the suspensor cell most proximal
to the embryo proper) of 19% (29 of 154) of dermatogen stage embryos from self-pollinated dcl1-5/+ plants (Fig. 1A). By the early globular stage, morphological defects were observed in ∼25% of embryos from selfed dcl1-5/+ plants (Fig. 1A). These embryos with defects were presumably homozygous for dcl1-5-null alleles, and their defects included abnormal hypophysis cell divisions as well as the previously unreported loss of
periclinal subprotoderm cell divisions in the embryo proper (Fig. 1A). In subsequent stages, these abnormal embryos did not produce cotyledons and were developmentally arrested and nonviable
(Fig. 1A; Schwartz et al. 1994). Furthermore, dcl1-5 homozygous seedlings could not be recovered from selfed dcl1-5/+ plants, which suggested that the developmentally arrested embryos derived from selfed dcl1-5/+ plants were indeed homozygous for dcl1-5 (data not shown). Because embryos from selfed dcl1-5/+ and selfed dcl1-10/+ plants exhibited the same morphological defects at indistinguishable frequencies, subsequent analyses focused on embryos
from selfed dcl1-5/+ plants (Table 1; data not shown). DCL1 transcripts were detected by quantitative RT–PCT (qRT–PCR) in both early globular embryos from wild-type plants and early
globular embryos from selfed dcl1-5/+ plants that developed normally, but were not detected in abnormal early globular embryos from selfed dcl1-5/+ plants (Supplemental Fig. 1). These expression analyses further confirmed that abnormal embryos from selfed dcl1/+ plants were homozygous for dcl1-null alleles.
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