In each female floret, a single diploid cell within the ovule undergoes meiosis. Four haploid megaspores result, but only one persists and enters into megagametogenesis, or embryo sac (ES) development. In early megagametogenesis, the functional megaspore enlarges gradually and undergoes three rapid free-nuclear mitoses. In the next stage of ES development, simultaneous cellularization partitions the 8-nucleate structure into 7 cells: 3 antipodal cells, a binucleate central cell, an egg cell and 2 synergid cells. Only the antipodals undergo further cell divisions as the ES enlarges, differentiates and matures. Thus, the haploid megagametophyte phase of maize begins upon completion of meiosis and ends when the egg cell and the central cell of the embryo sac (ES) are fertilized by pollen-delivered sperm cells.

In 1952, O. E. Nelson and G. B. Clary serendipitously recovered the lethal ovule2 (lo2) mutation in the course of a screen for new male steriles (J. Hered. 43:205-210). They showed that although lo2 transmits at normal frequencies through the male gametophyte (i.e., pollen), lo2 does not transmit through the female gametophyte (i.e., ES). Thus, the most straightforward interpretation of lo2 genetics suggests lo2 is a mutation in a gene which is expressed by the haploid ES and required for normal ES function. Nelson and Clary did not further pursue lo2, but their initial, brief report is the source of the contemporary description of the lo2 phenotype: "ovules containing lo2 megaspores abort."

Confocal laser scanning microscopy (CLSM) was used to observe ovules and ESs on lo2-containing plants. CLSM analysis is much more rapid than traditional sectioning techniques, is nondestructive, and allows for detailed three-dimensional interpretations of histological features. lo2 stocks were kindly provided by the Stock Center and by Ed Coe. Developing ears from lo2/+ plants were harvested and fixed, and the lower ears were testcrossed to verify the presence of lo2. Samples were stained to detect nuclei &/or cell walls with CLSM. In florets staged to contain mature ESs (as indicated by silk length), every ovule appeared to contain structurally normal sporophytic (diploid) tissues (i.e., nucellus, integuments, micropyle), and every ovule contained an ES-like structure. However, roughly 50% of the ESs appeared wild-type and mature, while the remaining 50% lagged considerably in their development, displaying slightly enlarged 2- or 4-nucleate phenotypes. The 2- and 4-nucleate stages of ES development normally transpire very rapidly, such that they are difficult to observe even in appropriately staged (younger) florets. Even in very "old" florets, 2- and 4-nucleate ESs persisted and there was no sign of ovule degeneration, abortion, or abnormality associated with the presumptive lo2-containing ESs.

These observations are consistent with the hypotheses that lo2 is a loss of function mutation, the lo2 gene function is normally expressed by the haploid ES and that the lo2 gene product is required during megagametogenesis as early as the 2-nucleate stage. Alternatively, the lo2 mutation could be a gain of function in which a novel function (sporophytic or gametophytic in origin) is expressed at a similar stage. Moreover, ovule development can apparently continue undisturbed in the presence of aberrant ES development, and ES expansion and viability can be uncoupled from nuclear division patterns and cellularization. Additional genetic experiments are underway to determine whether lo2 is transmitted at any detectable frequency through the ES, and to determine, using primary trisomic stocks, whether lo2 behaves as dominant or recessive in the megagametophyte. 

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