Development 128, 1629-1641 (2001) 1629 Printed in Great Britain © The Company of Biologists Limited 2001 DEV3380 Establishment of segment polarity in the ectoderm of the leech Helobdella Elaine C. Seaver* and Marty Shankland‡ Section of Molecular Cell and Developmental Biology, Institute of Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA *Present address: Kewalo Marine Lab, PBRC/University of Hawaii, 41 Ahui St, Honolulu, HI 96813, USA ‡Author for correspondence (e-mail: [email protected]) Accepted 31 January; published on WWW 5 April 2001 SUMMARY The segmented ectoderm and mesoderm of the leech arise and in every case the ablation eliminated the normal via a stereotyped cell lineage from embryonic stem cells descendants of the ablated cell while having little or no called teloblasts. Each teloblast gives rise to a column of detectable effect on the developmental fate of the remaining primary blast cell daughters, and the blast cells generate cells. This included experiments in which we specifically descendant clones that serve as the segmental repeats of ablated those blast cell progeny that are known to express their particular teloblast lineage. We have examined the the engrailed gene, or their lineal precursors. These mechanism by which the leech primary blast cell clones findings confirm and extend a previous study by showing acquire segment polarity – i.e. a fixed sequence of positional that the establishment of segment polarity in the leech values ordered along the anteroposterior axis of the ectoderm is largely independent of cell interactions segmental repeat. In the O and P teloblast lineages, the conveyed along the anteroposterior axis. Both intercellular earliest divisions of the primary blast cell segregate signaling and engrailed expression play an important role anterior and posterior cell fates along the anteroposterior in the segment polarity specification of the Drosophila axis. Using a laser microbeam, we ablated single cells from embryo, and our findings suggest that there may be little both o and p blast cell clones at stages when the clone was or no conservation of this developmental mechanism two to four cells in length. The developmental fate of the between those two organisms. remaining cells was characterized with rhodamine-dextran lineage tracer. Twelve different progeny cells were ablated, Key words: Segmentation, Cell lineage, Leech INTRODUCTION stem cells called teloblasts (for an overview, see Shankland and Savage, 1997). Each of the five teloblasts undergoes an iterated Segmentation of the anteroposterior (AP) body axis is a sequence of highly asymmetric cell divisions, and produces a structural motif shared by a number of animal phyla (Brusca linear column of much smaller primary blast cell daughters. and Brusca, 1990). Some authors argue that the last common The five ipsilateral columns of blast cells come together in ancestor of bilaterian animals was segmented, and hence that parallel to form a germinal band, and the right and left bands the developmental mechanism underlying segmentation is then fuse to produce a bilaterally symmetric germinal plate that homologous between different segmented phyla (De Robertis, differentiates into the 32 body segments of the adult leech. 1997; Kimmel, 1996). But others propose that segmentation Primary blast cells function as segmental founder cells for originated multiple times within specific bilaterian clades their respective teloblast lineages. The mesodermal teloblast M (Patel et al., 1989a; Brusca and Brusca, 1990), a scenario that and two of the four ectodermal teloblasts, O and P, each more readily explains the disparate location of the segmented generate a single class of primary blast cell that is designated taxa within the metazoan tree (Aguinaldo et al., 1997; de by the same letter in lower case, i.e. m, o and p. The O and P Rosa et al., 1999). One reason for this uncertainty is that teloblasts are equivalent in developmental potential (Weisblat the developmental mechanisms that generate body axis and Blair, 1984), but their blast cell daughters become segmentation are poorly understood outside of a few model committed to distinct O or P developmental pathways in systems, most notably the fruitfly Drosophila. Much less is accordance with dorsoventral positioning in the germinal band known about the mechanistic basis of segmentation in other (Shankland and Weisblat, 1984; Huang and Weisblat, 1996). arthropods or vertebrates, and even less in the annelids. To Within each of these three lineages, all of the primary blast address the latter deficiency, we have investigated the sequence cells undergo a very similar and teloblast-specific sequence of of cellular events that give rise to segment polarity in an development (Zackson, 1984) to produce descendant clones annelid, the leech Helobdella robusta. that serve as segmental repeats (Shankland, 1987a; Shankland, The Helobdella embryo generates its segmented ectoderm 1987b). The two remaining ectodermal teloblasts, N and Q, and mesoderm from a bilaterally symmetric set of embryonic develop in much the same fashion, except that they produce 1630 E. C. Seaver and M. Shankland alternating pairs of primary blast cells that give rise to distinct – is homologous between arthropods such as the fly and anterior and posterior halves of their segmental repeats annelids such as the leech (Wedeen and Weisblat, 1991; (Zackson, 1984; Weisblat and Shankland, 1985). Shankland, 1994; Ramirez et al., 1995). However, at least some We have investigated the process of segment polarity aspects of leech segmentation are not under the direct control specification in primary blast cell clones of the O and P of en expression, as en-expressing cells appear to play little or teloblast lineages. By analogy with Drosophila, we use the no role in the formation of intersegmental fissures in the term ‘segment polarity’ to refer to the establishment of nervous system of the leech Theromyzon rude (Shain et al., subsegmental positional values along the AP axis of the repeat 2000). (Nüsslein-Volhard and Wieschaus, 1980). In the leech embryo, In this paper we more closely examine the developmental segment polarity is first evident when the primary blast cells mechanism that specifies segment polarity in embryos of the initiate their subsidiary divisions. For example, a primary p leech H. robusta. In contrast to many other segmented animals, blast cell undergoes two rounds of division parallel to the the leech embryo displays a lineal stereotypy that allows us to AP axis to generate grand-daughter cells p.aa, p.ap, p.pa and uniquely identify the individual founder cells that comprise a p.pp (Fig. 1C), thereafter switching to transverse divisions nascent segment, and to precisely characterize the descendant (Shankland, 1987b). A single p blast cell clone will eventually fate of those cells in both normal (Shankland, 1987a; give rise to about 70 differentiated descendants (Shankland and Shankland, 1987b) and experimentally manipulated embryos. Weisblat, 1984), and the relative AP positions of the four We here use a laser microbeam to ablate various blast cell grand-daughter cells in the germinal band predicts the overall progeny from either the O or the P teloblast lineage, and find AP disposition of their descendants within the differentiated no evidence that the primary blast cell clone requires cell blast cell clone (Fig. 1D). It is not currently known how the interactions along its AP axis in order to specify anterior and anterior and posterior blast cell progeny acquire their differing posterior cell fates. In particular, our results show that the en- fates, but the specification of those differences is a crucial step expressing cells do not serve as a source of required in establishing the segment polarity of each individual primary intercellular signals for the normal patterning of the remainder blast cell clone. of the segmental repeat. Thus, the developmental mechanism The primary o blast cell clone develops in much the same that establishes and maintains segment polarity in the leech way as the p blast cell clone, although it differs with respect to embryo appears to be quite different from that characterized the details of cleavage pattern (Shankland, 1987a) and the exact in fruitflies, a conclusion that raises doubt as to whether set of descendants produced (Shankland and Weisblat, 1984). the annelids and arthropods could have inherited their The earliest o blast cell divisions are also parallel to the AP segmentation mechanisms from a segmented common axis (Fig. 1A), and its progeny cells o.aa, o.apa, and o.app are ancestor. arrayed within the germinal band in an order that predicts the AP disposition of their differentiated descendants (Fig. 1B). MATERIALS AND METHODS Progeny cell o.p has proven too small for lineage tracer injection (Shankland, 1987a), and its relationship to segment Animals polarity remains unclear. There is some evidence that the leech Helobdella employs H. robusta embryos were taken from a breeding colony founded with individuals collected from the mouth of Shoal Creek in Austin, Texas. a genetic mechanism of segment polarity specification similar H. robusta is closely related to H. triserialis, and displays essentially to that found in the fruitfly Drosophila. In the fly embryo, identical patterns of cell lineage and morphological development zygotic expression of the engrailed (en) gene occurs at the (Shankland et al., 1992; Huang and Weisblat, 1996; Seaver and cellular blastoderm