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Expression and function of an even-skipped homolog in the leech Helobdella robusta

Mi Hye Song*, Françoise Z. Huang, Gwendolen Y. Chang and David A. Weisblat{dagger}

Department of Molecular and Cell Biology, University of California, 385 LSA, Berkeley, CA 94720-3200, USA
* Present address: Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI 48109-1048, USA



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  		Fig. 1. Summary of Helobdella development. (A) Timeline (not to scale) showing selected developmental stages; all views are from the animal pole (prospective dorsal) unless indicated otherwise. Times given indicate the approximate age in hours after zygote deposition at 23°C. tb, teloblast; gb, germinal band; gp, germinal plate. (B) The formation of segmental ectoderm, focussing on the N lineage. Each N teloblast undergoes stem cell divisions to produce primary ns (red) and nf (dark blue) blast cells in exact alternation. The timing of subsequent events in each n blast cell clone is given in terms of the time elapsed since the birth of the primary blast cell (hours clonal age; timeline not to scale). Ipsilateral columns (bandlets) of primary blast cells merge to form germinal bands, which coalesce in anteroposterior progression during stage 8 into the germinal plate, from which segments arise (see A). The ns and nf blast cells undergo distinct and stereotyped lineages, beginning with unequal, obliquely anteroposterior divisions producing progeny called ns.a (red), ns.p (pink), nf.a (dark blue) and nf.p (light blue) at ~28 and ~26 hours clonal age, respectively. During subsequent development, these subclones generate approx. two-thirds of the ~200 identified neurons and glia in each hemiganglion, plus three peripheral neurons (nz1-3) and a few epidermal cells (not shown) (Bissen and Weisblat, 1987Go; Bissen and Weisblat, 1989Go; Kramer and Weisblat, 1985Go; Ramirez et al., 1995Go; Shain et al., 1998Go; Shain et al., 2000Go; Weisblat et al., 1984Go; Zackson, 1984Go).

 


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  		Fig. 2. (A) Comparison of eve-class homeodomain sequences from lophotrochozoans (Hro, Helobdella robusta; Ttr, Theromyzon trizonare), cnidarians (Afo, Acropora formosa; Nve, Nematostella vectensis), deuterostomes (Bfl, Branchiostoma floridae; Dre, Danio rerio; Mmu, Mus musculus; Hsa, Homo sapiens; Xla, Xenopus laevis) and ecdysozoans (Cel, Caenorhabditis elegans; Dme, Drosophila melanogaster; Sam, Schistocerca americana; Tca, Tribolium castaneum). Two similar non-eve-class homeodomain sequences are included for comparison (HmHD, Hirudo medicinalis homeodomain protein; Dmpb, Drosophila melanogaster proboscipedia). Consensus amino acid residues are highlighted, and amino acid residues flanking known intron sites are underlined. Values in parentheses indicate the percentage amino acid identity with Hro-eve.

 


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  		Fig. 3. Semi-quantitative RT-PCR analysis of Hro-eve expression. Ethidium bromide-stained gels (below) show Hro-eve and 18S rRNA bands from various developmental stages (0 denotes oocyte; e, early). The extent of amplification (23 cycles for 18S rRNA and 30 cycles for Hro-eve) was chosen empirically to avoid saturation of the amplified bands. The graph (above) shows the average of the intensity of the Hro-eve bands after normalizing by the intensity of the corresponding 18S rRNA band and plotting relative to stage 10, from five different experiments. Error bars indicate the standard deviation of the mean.

 


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  		Fig. 4. Early expression of Hro-eve provides no evidence for pair rule patterning. (A) Bright-field (Nomarski optics) image of a stage 7 embryo processed by in situ hybridization for Hro-eve. Staining is uniform within and among bandlets, except for punctate staining associated with apparent mitotic figures in three teloblasts; arrow indicates prophase; yoked arrows indicate telophase (left) and anaphase (right). An enlarged view (B) at a slightly different focal plane of the outlined region of the same embryo shows perinuclear staining of Hro-eve in blast cells of the bandlets (arrowheads), but no alternating intensity of staining that would be indicative of a pair-rule expression pattern. Scale bar: 100 µm in A; 30 µm in B.

 


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  		Fig. 5. Early Hro-eve transcripts appear to be associated with chromatin of cells in mitosis. Brightfield (Nomarski optics) images of embryos processed by in situ hybridization for Hro-eve. (A) Ventral view of a stage 7 embryo. On the right, regions of an m bandlet are in focus. The M teloblast is out of focus, but ~4 m blast cells in the proximal bandlet (one cell wide because the primary blast cells have not yet divided) are in view. Distal to this, the bandlet is in focus again, now two cells wide; in each pair of secondary m blast cells, m.l is on the left and m.m is on the right; perinuclear localization of the Hro-eve transcripts in the blast cells can be seen, as in Fig. 4 (arrowheads). Cell m.l divides prior to m.m in Helobdella (Bissen and Weisblat, 1989Go) (E. K. Schimmerling, BA Honors thesis, University of California, 1986). In this embryo, the in situ signal in successive m.l cells is punctate instead of perinuclear, which corresponds to the younger cell (arrow) in prophase, and the older cell (yoked arrows) in telophase. The other m bandlet is largely out of focus, but one m.l in that bandlet exhibits punctate staining. (B) Schematic drawing of the embryo in A. (C) In a late two-cell embryo, punctate staining (arrow) marks the metaphase chromatin of cell CD. Diffuse background staining of teloplasm is also evident. Scale bar: 100 µm.

 


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  		Fig. 6. Dynamic pattern of late Hro-eve expression in ganglionic neurons. Bright-field (Nomarski optics) images of embryos processed by in situ hybridization for Hro-eve. (A) Lateral view of a stage 9 embryo. In anterior, developmentally advanced segments, the spots of Hro-eve expression show segmental periodicity (arrows), while in posterior, less advanced segments, there are two spots per segmental repeat (yoked arrowheads and arrows). (B-D) Ventral views of anterior midbody ganglia (~M2-M5) at three different stages; note that these Hro-eve transcripts exhibit cytoplasmic localization; transcripts are excluded from nuclei. (B) At mid stage 9, there are distinct anterior and posterior spots of expression in each hemiganglion (yoked arrowheads and arrows, respectively). Inset: enlarged image of the adjacent cell shows cytoplasmic staining and unstained nucleus. (C) By late stage 9, anterior expression has ceased in anterior segments, whereas the posterior spot is still present in all three segments. (D) By late stage 10, anterior expression is completely gone, but the posterior spots (arrows) remain. Scale bar: 100 µm in A; 50 µm in B-D; 20 µm in inset.

 


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  		Fig. 7. Ganglionic cells expressing Hro-eve arise from the N lineage. Pseudo-colored confocal images of sectioned embryos that had been fixed and stained for Hro-eve transcripts (green) at stage 9 and in which 1 or more cells had been injected with RDA (red) at stage 6a. (A) A horizontal view of approx. four segmental ganglia in which the O, P and Q lineages are labeled on the left and the N lineage is labeled on the right. Anterior is upwards; the plane of section is oblique. In each hemiganglion, the posterior spot of Hro-eve expression lies just posterior to the main lobe of OPQ-derived cells (arrows) and in the anterior edge of the posterior half-ganglion (yoked arrows). The anterior spots of Hro-eve expression (yoked arrowheads) lie at the anterior edge of the ganglion. (B,C) Transverse sections through the ventral nerve cord of an embryo in which an N teloblast had been labeled; Hro-eve-positive neurons in the anterior spot (B, arrowheads) and posterior spot (C, arrows) colocalize with N-derived cells. Yolk platelets (y) exhibit background fluorescence in the RDA channel; background in situ signal is present between yolk platelets and imperfections in the sectioned material (*) also appear green in the pseudo-colored images. Scale bar: 50 µm in A; 30 µm in B,C.

 


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  		Fig. 8. Injection of AS-Hro-eve MO into N lineage perturbs gangliogenesis and neuronal differentiation (anterior is upwards). (A-C) Fluorescence images of the dissected germinal plate from a stage 10 embryo, in which one N teloblast had been co-injected with MM-Hro-eve MO and FDA (green), and the other with AS-Hro-eve MO and RDA (red) at early stage 7; nuclei are counterstained with Hoechst 33258 (blue). (B,C) Close-up views of the sections indicated by boxes in the anterior and posterior regions of A, respectively. On the control side (left), development of the N lineage is normal; peripheral nz neurons (derived from the nf blast cell clones) are present in anterior segments (arrows in B), and anterior and posterior lobes of cells (derived from ns and nf, respectively) that form the bulk of the ganglionic primordia are visible in posterior segments (C). On the experimental (right) side, the overall size and regularity of the n-derived clones are reduced and nz neurons are largely absent. (D) Fluorescence image of a preparation as in A, but processed for Hro-eve transcripts at stage 10. Two anterior ganglia, containing n blast cell clones produced prior to the injections, show bilaterally paired posterior spots of cells expressing Hro-eve (double arrows). On the control (left) side, these spots continue with segmental periodicity for a total of 14 segments (horizontal arrows; the spot in the third segment is out of focus); in the three youngest segments, the transient, anterior spots of Hro-eve expression are also visible on the control side (e.g. yoked arrowheads and arrows). On the experimental side, spots of Hro-eve expression are frequently missing, out of register, or misplaced medially with respect to those on the control side (slanted arrows). (E) Brightfield (Nomarski optics) showing approx. five ganglia from another embryo, treated as in D. Note the abnormal ganglion morphology and ectopically positioned Hro-eve spots (slanted arrows) on the experimental (right) side relative to the control side (horizontal arrows). (F) Combined bright-field and fluorescence image, showing the first five midbody ganglia (M1-M5) of an embryo injected as in A, but grown to stage 11 and processed for serotonergic neurons, which normally arise in bilateral pairs from the N teloblast lineages. No serotonergic neurons arose from the N teloblast injected with AS-Hro-eve MO and there is a gap (bracket) in the RDA-labeled lineage where N-derived neurons are missing. In M1-M5 of the control side (left), previously described serotonergic neurons (Stuart et al., 1987Go) can be identified, including the Retzius cells (Rz) and a pair of dorsolateral and ventrolateral cells (dl/vl). Ganglia M1-M3 also contain a smaller anteromedial (am) neuron. A fourth, posteromedial neuron was not detected because it develops later. (G) Digital montage fluorescence image combining several focal planes of five segments from a stage 11 embryo, in which an O teloblast had been injected with RDA and MM-Hro-eve MO at stage 7; in each segment, the O lineage generates distinct subsets of ganglionic neurons (AD, PV, CR), epidermal cells (e), plus peripheral neurons (most of which are not visible in this figure). (H) Equivalent view of a sibling embryo to that shown in G, that had been injected with AS-Hro-eve MO; clusters of undifferentiated cells are present over the ganglion (g) and in the periphery (p). Scale bar: 200 µm in A; 50 µm in B,C,F; 150 µm in D; 100 µm in E,G,H.

 


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  		Fig. 9. Embryos with severe AS-Hro-eve MO phenotype produce hairpin loops in germinal plate. (A) Bright-field image showing a lateral view (ventral is downwards, anterior towards the left) of the posterior portion of an embryo in which an N teloblast had been injected with AS-Hro-eve MO and RDA at early stage 7; the embryo was processed for ganglionic Hro-eve expression at stage 10 (inset shows the whole embryo). Note that the pattern of Hro-eve positive spots demonstrates a prominent dorsal excursion (arrow). (B) Fluorescence image of the same specimen shows that the labeled bandlet contains a dorsally directed hairpin loop in this region (arrow indicates same point as in A). Scale bar: 50 µm in A,B; 175 µm in the inset.

 


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  		Fig. 10. Embryos with severe AS-Hro-eve MO phenotype produce kinked germinal bands. Fluorescence images of an embryo in which the left N teloblast had been injected with AS-Hro-eve MO and RDA (red), and the right N teloblast with generic control MO and RDA, at early stage 7. The resultant embryo was fixed and counterstained with Hoechst 33258 (pseudo-colored green in A and D); the animal pole (prospective dorsal) is upwards. (A) A roughly posterior (P) view of the embryo reveals the labeled n bandlets at the leading edges of the left and right germinal bands. While the control germinal band (circle, arrowhead) projects equatorially, the experimental germinal band (square, arrow) makes a marked dorsal deflection. (B) Lateral view of the right germinal band shows that it projects along the equator of the embryo. (C) Lateral view of the left germinal band shows the dorsally directed kink. (D) An obliquely ventral view shows the anterior (A) ends of the labeled bandlets within the partially formed germinal plate. The lineage tracer is brighter within the control bandlet, suggesting that the volume of the control injection was greater than that of the AS-Hro-eve MO injection. Scale bar: 100 µm.

 


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  		Fig. 11. Progeny of teloblasts injected with AS-Hro-eve MO exhibit dramatic variability in the brightness of inherited lineage tracer. Digital fluorescence images of an intact embryo (A) and a sectioned one (B,C) in which one N teloblast had been injected with MM-Hro-eve MO and FDA (green) and the other with AS-Hro-eve MO and RDA (red) at stage 7; the injected embryos were fixed and counterstained with Hoechst 33258 (blue) ~24 hours after the injections. Blast cells derived from teloblasts injected with MM-Hro-eve MO are uniformly labeled (A,B), whereas those derived from teloblasts injected with AS-Hro-eve MO exhibit marked cell-to-cell differences in the intensity (A,C). Scale bar: 50 µm.

 


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  		Fig. 12. Injection of AS-Hro-eve MO disrupts the normal pattern of teloblast and blast cell divisions. Schematic summary of the analysis of labeled bandlets in sectioned embryos such as those depicted in Fig. 11. Each bandlet is represented by a row of rectangles, the color of which corresponds to the lineage tracer used (green, FDA; red, RDA). The parent teloblast (tb) would lie at the left end of each row; approximate ages of the blast cell progeny are indicated. Differences in lineage tracer intensity are indicated by brighter and darker shading. Blast cells that have undergone their first division are indicated by diagonal lines; question marks indicate a case in which we could not determine whether the cells indicated were two primary blast cells or sister cells derived from a single primary blast cell.

 

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© The Company of Biologists Ltd 2002