Laser Microdissection of Narrow Sheath Mutant Maize Uncovers Novel Gene Expression in the Shoot Apical Meristem

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Abstract
Microarrays enable comparative analyses of gene expression on a genomic scale, however these experiments frequently identify an abundance of differentially expressed genes such that it may be difficult to identify discrete functional networks that are hidden within large microarray datasets. Microarray analyses in which mutant organisms are compared to nonmutant siblings can be especially problematic when the gene of interest is expressed in relatively few cells. Here, we describe the use of laser microdissection microarray to perform transcriptional profiling of the maize shoot apical meristem (SAM), a ~100-μm pillar of organogenic cells that is required for leaf initiation. Microarray analyses compared differential gene expression within the SAM and incipient leaf primordium of nonmutant and narrow sheath mutant plants, which harbored mutations in the duplicate genes narrow sheath1 (ns1) and narrow sheath2 (ns2). Expressed in eight to ten cells within the SAM, ns1 and ns2 encode paralogous WUSCHEL1-like homeobox (WOX) transcription factors required for recruitment of leaf initials that give rise to a large lateral domain within maize leaves. The data illustrate the utility of laser microdissection-microarray analyses to identify a relatively small number of genes that are differentially expressed within the SAM. Moreover, these analyses reveal potentially conserved WOX gene functions and implicate specific hormonal and signaling pathways during early events in maize leaf development. Unlike animals, plants exhibit a prolonged period of organogenesis, generating new leaves throughout their life cycle. This ability to maintain an embryo-like state is dependent upon the activity of shoot meristems, whose dual functions are to supply an inner core of pluripotent cells that sustain the shoot meristem while simultaneously generating new leaves derived from cells at the meristem periphery. Deciphering the complex combinations of molecular signals that transform meristematic cells into leaf primordia is a central question in plant developmental biology. In this study, we used the power of focused laser light to microdissect shoot meristems from neighboring leaf and stem tissue in the maize plant. Once isolated, we compared patterns of gene expression in normal shoot meristems to those of genetically mutant shoot meristems that form abnormal, narrow leaves. Out of more than 21,000 maize genes analyzed, 66 genes were identified as misexpressed in the mutant shoot meristems. All but one of the differentially expressed genes are previously unstudied in maize, and the majority are predicted to function during cell division, growth, or developmental signaling. Many of these novel genes are expressed in specific domains of the shoot meristem, consistent with their predicted function during maize leaf initiation.