An in situ sequencing approach maps PLASTOCHRON1 at the boundary between indeterminate and determinate cells

Abstract

The plant shoot apex houses the shoot apical meristem, a highly organized and active stem-cell tissue where molecular signaling in discrete cells determines when and where leaves are initiated. We optimized a spatial transcriptomics approach, in situ sequencing (ISS), to colocalize the transcripts of 90 genes simultaneously on the same section of tissue from the maize (Zea mays) shoot apex. The RNA ISS technology reported expression profiles that were highly comparable with those obtained by in situ hybridizations (ISHs) and allowed the discrimination between tissue domains. Furthermore, the application of spatial transcriptomics to the shoot apex, which inherently comprised phytomers that are in gradual developmental stages, provided a spatiotemporal sequence of transcriptional events. We illustrate the power of the technology through PLASTOCHRON1 (PLA1), which was specifically expressed at the boundary between indeterminate and determinate cells and partially overlapped with ROUGH SHEATH1 and OUTER CELL LAYER4 transcripts. Also, in the inflorescence, PLA1 transcripts localized in cells subtending the lateral primordia or bordering the newly established meristematic region, suggesting a more general role of PLA1 in signaling between indeterminate and determinate cells during the formation of lateral organs. Spatial transcriptomics builds on RNA ISH, which assays relatively few transcripts at a time and provides a powerful complement to single-cell transcriptomics that inherently removes cells from their native spatial context. Further improvements in resolution and sensitivity will greatly advance research in plant developmental biology.

Figure 1

Individual and combined expression patterns visualized by ISS in longitudinal sections through the maize SAM. A, Annotation of the tissue on a longitudinal section through the maize shoot apex, stained by DAPI, on which the expression domains of (B–F) are assayed. Expression patterns of (B) RS1; (C) OCL4; (D) PIN1a; and (E) DRP4a. F, Composite image of RS1 (vermillion), OCL4 (yellow), and DRP4a (blue) expression patterns. G, Zoomed inset of RS1, OCL4, and DRP4a in the maize SAM. ISS images generated using the third biological repeat of the longitudinal sections.

Figure 2

Validation of the ISS data with single gene mRNA ISHs. A and B, PIN1a is expressed in vascular tissue; (C and D) AN3/GIF1 is expressed in the meristem and developing leaves; (E and –F) GRF1 is expressed in young leaves and in vascular tissue; (G and H) CUC2 is expressed at the leaf-meristem boundaries; (I and J) BE1 is expressed in leaf primordia and at the base of young developing leaves; (K and L) LAX2 is expressed in developing vascular bundles; (M and N) DRP4a is expressed at the tip of the meristem; and (O and P) H4 expression marks actively dividing cells. Background signal in ISS pictures are DAPI stained nuclei. All ISS images are generated using the third biological repeat of the longitudinal sections, except the ISS image depicting BE1 expression, which was generated using an image from the first biological repeat.

Figure 3

Combined expression patterns obtained by ISS in longitudinal section through the maize shoot apex. A, Composite image of LOG7 (vermillion), DRP4a (yellow), AGO18a (pink), YAB9 (cyan), and YAB14 (purple). B, Composite image of boundary genes BA1 (purple), BAF1 (pink), CUC2 (yellow), CUC3 (cyan), and D11-LIKE (vermillion). C, Zoomed inset of B. D, Image of vascular markers BIF1 (yellow), GRXC8 (red), LAX2 (white), NAS3 (purple) PIN1a (blue), and ZCN1/3 (pink). E, Zoomed inset of (D). Background signal are DAPI stained nuclei. ISS images generated were obtained from the first biological repeat for (A), and from the third biological repeat for (B–D).

Figure 4

Expression patterns of PLA1 obtained through ISH and ISS in the maize shoot apex. A, Expression pattern of PLA1 obtained by RNA ISH in a longitudinal section of a B104 SAM. B, Expression pattern of PLA1 obtained by ISS in a longitudinal section of a B104 SAM. C, Expression pattern of PLA1 obtained through RNA ISH on a transverse section of a B104 SAM. D, PLA1 expression pattern in transverse sections of a B104 SAM using ISS. E, Heat-map of cells expressing PLA1 in a B104 shoot apex. F, PLA1 is more broadly expressed in the stem, vascular bundles and in the leaves (black arrowheads) in the GA2ox::PLA1 overexpression line as compared with the expression in B104. Background signal in ISS pictures are either DAPI or CW stained cells. ISS images obtained from biological repeat three (longitudinal) and a transverse section of the SAM.

Figure 5

ISS on segmented cells expressing indeterminate and determinate cell types. A, Cells expressing RS1 are depicted in purple. B, Determinate leaf epidermal cells expressing OCL4 are depicted in cyan. C, Combination of cells expressing RS1 (purple) and OCL4 (cyan) illustrate the sharp demarcation of indeterminate to determinate tissues. D and E, Expression of PLA1 (yellow) is located at the boundary between indeterminate (purple) and determinate tissues (cyan). Brown cells depict expression of both PLA1 and RS1, cells expressing both PLA1 and OCL4 are depicted in green, cells expressing both RS1, PLA1 and OCL4 are depicted in dark green. Background signal is CW stained cells. Segmented images obtained from biological repeat three.

Figure 6

Comparison of multi-gene expression patterns obtained by ISS. Expression patterns obtained in the shoot apex from: A, PLA1 (yellow), CUC2 (blue), and D11-LIKE (white). B, PLA1 (yellow), CUC2 (blue), D11-LIKE (white), BA1 (pink), and BAF1 (vermillion). C, AN3/GIF1 (green), PLA1 (yellow), D11-LIKE (white), and CUC2 (blue). D, Zoomed-in region marked in (C). E, PLA1 (yellow) and PIN1a (blue). Background signals are DAPI stained nuclei. ISS images obtained from biological repeat three.