High resolution cell lineage tracing reveals developmental variability in leech

Abstract

Knowing the normal patterns of embryonic cell proliferation, migration, and differentiation is a cornerstone for understanding development. Yet for most species, the precision with which embryonic cell lineages can be determined is limited by technical considerations (the large numbers of cells, extended developmental times, opacity of the embryos), and these are exacerbated by the inherent variability of the lineages themselves. Here, we present an improved method of cell lineage tracing in the leech Helobdella, driving the expression of a nuclearly localized histone H2B:GFP (green fluorescent protein) fusion protein in selected lineages by microinjection of a plasmid vector. This construct generates a long lasting and minimally mosaic signal with single cell resolution, and does not disrupt the development of most lineages tested. We have validated this technique by elucidating details of cell lineages contributing to segmental and prostomial tissues that could not be observed with standard dextran lineage tracers.

Figure 1. Relevant aspects of leech development

(A) Diagramatic representations of selected stages (animal pole views unless otherwise indicated). In the 8-cell embryo (stage 4a), the D quadrant has cleaved to form micromere d′ and macromere D′, the teloblast precursor. At stage 5, macromere D′ has given rise to left and right mesodermal and ectodermal precursors (M teloblasts and NOPQ proteloblasts, respectively, the right M teloblast is out of view at the vegetal pole). At stage 7, all five pairs of teloblasts are present. At early stage 8, teloblasts have produced columns of segmental founder cells called germinal bands (gb, grey; see panel B for details); germinal bands and the territory between them are covered with a provisional epithelium generated by micromeres. At mid stage 8, the lengthening germinal bands have begun to coalesce along the prospective ventral midline to form the germinal plate (gp), from which segmental mesoderm and ectoderm arise. During stages 9 and 10, segments differentiate and the germinal plate expands from ventral to dorsal territory, displacing the micromere-derived epithelium (not shown at these stages). (B) Schematic of a stage 8 embryo, corresponding to the boxed section in panel (A), showing the relationships of teloblasts, blast cells, bandlets, and germinal band. Teloblasts mark the posterior growth zone and the older, more distal blast cells contribute to more anterior segments. (C) Arrangement of undivided ectodermal blast cells within the germinal band, corresponding to the boxed section of panel (B); a single o blast cell is highlighted with a green nucleus. The mesodermal bandlet lies beneath the ectoderm and is not shown. (D) Schematics showing the first six divisions of an o blast cell clone in the right germinal band; at each division the sister cells are named by adding a letter to indicate their relative positions at the end of cytokinesis, a = anterior, p = posterior, l = lateral, m = medial. In each panel, sister cells of the most recent division are indicated by red double arrows.

Figure 2. Expression of H2B:GFP in the DNOPQ^x and DM^x lineages

In this and all figures, anterior is up, cytoplasmic RDA lineage tracer is red, nuclear H2B:GFP is green, and images are maximum projections of stacks of confocal images, unless otherwise noted. (A) Animal pole view of an embryo whose DNOPQ^x had been injected with RDA and pEF-H2B:GFP 78 hours prior to fixation at early stage 8 (see Fig. 1A, B). This image shows blast cell bandlets in the left and right germinal bands (labeled in the right germinal band) and some teloblasts (asterisks); not all labeled cells are visible because the stack does not extend through the entire embryo. Note the reduced levels of transgene expression in the left n bandlet (arrow). (B) Animal view of a sibling embryo fixed 96 hours post injection, showing the right germinal band, comparable to the region enclosed in the dotted box in panel (A). Normal divisions of blast cells in the o bandlet are indicated by the presence of identifiable cells including o.p (white arrow), o.a (white arrowhead) and a mitotic o.a cell (open arrowhead). (C) An embryo whose DM^x was injected with RDA and pEF-H2B:GFP four days before fixation at early stage 8. Within the germinal bands, proliferation of individual m blast cell clones produce developing hemi-somites (brackets). In the region between the germinal bands, migratory, non-segmental freckle cells derived from the mesodermal lineage are evident (arrow) and additional non-segmental cells with exceptionally broad nuclei (arrowheads) are seen at the anterior of the left and right germinal bands. (D) Higher magnification image of freckle cells in a sibling embryo showing mosaic transgene expression. Scale bar: **μm in A, **μm in B, **μm in C, **μm in D.

Figure 3. Expression of pEF-H2B:GFP in the O lineage

(A) Frames from a time-lapse video (one image per minute, 199 minutes total duration) showing the division of a primary o blast cell (white arrow) to form sister cells o.a (white arrowhead) and o.p (red arrowhead). In this field of view, two pairs of o.a and o.p sister cells are visible (see Fig. 1C), slightly out of focus, anterior to the dividing cell, and undivided primary blast cells lie posterior to it. Elapsed time (minutes since the start of imaging) is indicated in bottom left hand corner. (B-E) Maximal projections of confocal stacks of O lineages labeled with RDA and pEF-H2B:GFP in embryos fixed at various time points post injection. (B) 72 hours post injection; anterior portion of the o bandlet. Early progeny of the o clones are indicated, with distinct colors for each clone (cell nomenclature as in Fig. 1C). (C) 144 hours post injection; anterior portion of a dissected germinal plate. Bracket marks progeny of first labeled blast cell clone, which is not expressing the transgene. Differentiated epidermal cells can be distinguished by their broadened nuclei (arrowheads) and by their superficial location. (D) 206 hours post injection; anterior portion of the germinal plate. By this time, GFP transgene expression was no longer visible by direct fluorescence. This embryo was visualized by fluorescent immunostaining against GFP (green). (E) Labeled O pattern elements. Views of six segments from the anterior portion of a germinal plate dissected from an embryo fixed 144 hours post injection. The two left-hand panels are maximal projections from the complete stack of confocal images; the second panel shows GFP signal only, to better visualize the nuclei corresponding to the secondary lateral dopaminergic cell (LD2), the o.aa-derived lateral skin dot (LSD) and the ganglionic crescent (CR). The three right-hand panels show partial stacks corresponding to superficial (ventral surface), medial and deep optical sections, highlighting o.app–derived epidermal cells (epi), O-derived peripheral neurons (oz1 and oz2) and a group of anteriordorsal ganglionic cells (AD), respectively. Note that in the anteriormost segment the o.app-derived epidermis, oz1 and AD neurons are not labeled, because they are contributed to by the next anterior, unlabeled o blast cell clone (Weisblat and Shankland, 1985). Scale bar 10 μm in A,B; 50 μm C-E.

Figure 4. Variability in the o.appl/m lineage

(A) Partial confocal stack showing the ventral-most aspect of a dissected germinal plate, to highlight epidermal cells derived from o.aa (arrowheads) and o.aap (arrows) in an embryo fixed 144 hours after injection of the parent teloblast with RDA and pEF-H2B:GFP. In this partial stack, o.aa-derived epidermal cells are not visible in posterior segments due to the curvature of the embryo. Numbers refer to count of o.app progeny per segment. Asterisks indicate the merger of o.app-derived epidermal clusters between neighboring segments. Proliferation of morphologically differentiated epidermal cells is evidenced by cells dividing in the plane of the epithelium (bracket and middle asterisk). (B) Graphical representation of segment-by-segment o.app-derived epidermal cell counts vs. clonal age in 21 embryos fixed at various time points. Grey and black lines indicate the maximum, minimum and mean values obtained for each clonal age for which multiple specimens were examined. We did not interpolate between batches. Colored lines and dots represent data obtained from four individual specimens spanning various age ranges. Scale bar: 25 μm.

Figure 5. Plasmid-driven transgene expression in a micromere lineage

Images of embryos in which micromere d′ was injected at approximately 15 minutes after its birth with RDA and pEF-H2B:GFP; injected embryos were fixed at a range of time points post injection (48-168 hours, as indicated below each panel). In each panel, the topmost images are a low power epifluorescence image of the entire embryo (RDA, red; H2B:GFP, green; DAPI counterstain, blue, except that in panel (A), the blue color is autofluorescence) and a cartoon representation of that image to show the location of the labeled d′ clone. The two lower images in each panel are maximum projection confocal stacks as in other figures. (A) The d′ lineage undergoes early stem-cell like divisions, and in this embryo the parental “stem cell” was captured completing mitosis (white bracket). (B-F) At later time points, the d′ comes to comprise a mixed population of cells including provisional epithelial cells (arrows) and prostomial cells (arrowheads) that are readily distinguished based on differences in location, nuclear size, and cell morphology. Scale bar: 100 μm in upper panels; 20 μm in lower panels.

Figure 6. Comparison of transcript distributions resulting from plasmid and mRNA injections

Right N teloblasts of stage 6a embryos were injected with RDA and either mRNA or plasmid encoding the same transcript, and examined at various time points post injection (1-168 hours). (A) Brightfield images of embryos injected with ngfp mRNA or pEF-nGFP plasmid, fixed at the times indicated (hours post injection) and processed for GFP in situ hybridization; animal pole views are shown for embryos fixed 1-51 hours post injection and lateral views for embryos fixed 71-168 hours post injection. (B) High power image of a portion of a bandlet in the posterior germinal plate of an embryo processed for nGFP in situ 96 hours after plasmid injection shows non-uniform distribution of transcript. (C) Epifluorescent images of embryos injected with RDA and either h2b:gfp mRNA or pEF-H2B:GFP plasmid, and fixed 144 hours post injection. RDA signals are comparable, but the GFP signal has largely disappeared from the mRNA-injected embryos. Scale bar: A, C 100 μm; B 20 μm.