Cell interactions that affect axonogenesis in the leech Theromyzon rude.

The leech nervous system comprises a relatively simple network of longitudinal (connective) and transverse (segmental) nerves. We have followed the normal pattern of axon development in the glossiphoniid leech Theromyzon rude by immunostaining embryonic preparations with antibody to acetylated alpha-tubulin. The dependence of the normal pattern of axon growth on cells in the mesodermal (M) and ectodermal (N, O, P and Q) lineages was examined by selectively ablating subsets of these lineages in developing embryos. We found that ablating mesoderm severely disrupted overall axonogenesis, while various ectodermal ablations induced a range of more specific phenotypes. In particular, formation of the posterior segmental nerve (PP) was abnormal in embryos deficient in primary neuroectoderm (N lineage). More specific ablations demonstrated that a subset of N-derived cells were required for establishing the PP nerve root. Previous studies have shown that the PP nerve root is normally pioneered by an O lineage-derived neuron (PD). Our results suggest that the role of the N lineage-derived cells is to induce the migration of neuron P(D) to its normal position in the posterior compartment of the hemiganglion.

Cell cycle-dependent expression of a hairy and Enhancer of split (hes) homolog during cleavage and segmentation in leech embryos.

We have cloned genes related to hairy and Enhancer of split (hes) from glossiphoniid leeches, Helobdella robusta and Theromyzon rude. In leech, segments arise sequentially in anteroposterior progression from a posterior growth zone that consists of five bilaterally paired embryonic stem cells called teloblasts. Each teloblast gives rise to segmental founder cells (primary blast cells) that contribute iterated sets of definitive progeny in each segment. Thus, in leech, the "segmentation clock," is closely identified with the cell cycle clock of the teloblasts. We have characterized normal expression patterns of mRNA and protein for the H. robusta hes-class gene (Hro-hes). Semiquantitative RT-PCR revealed that Hro-hes mRNA levels peak while the teloblasts are actively producing primary blast cells. RT-PCR, in situ hybridization and immunostaining revealed that Hro-hes is expressed as early as the first zygotic mitosis and throughout early development. Hro-hes is expressed in macromeres, pro-teloblasts, teloblasts and primary blast cells. HRO-HES protein is localized in the nuclei of cells expressing HRO-HES during interphase; nuclear HRO-HES is reduced during mitosis. In contrast, Hro-hes is transcribed during mitosis and its transcripts are associated with mitotic apparatus (MA). Thus, Hro-hes transcription cycles in antiphase to the nuclear localization of HRO-HES protein. These results indicate that Hro-hes expression, and thus possibly its biological activity, is linked to the cell cycle.

A hedgehog homolog regulates gut formation in leech (Helobdella).

Signaling by the hedgehog (hh)-class gene pathway is essential for embryogenesis in organisms ranging from Drosophila to human. We have isolated a hh homolog (Hro-hh) from a lophotrochozoan species, the glossiphoniid leech, Helobdella robusta, and examined its expression by reverse transcription polymerase chain reaction (RT-PCR) and whole-mount in situ hybridization. The peak of Hro-hh expression occurs during organogenesis (stages 10-11). No patterned expression was detected within the segmented portion of the germinal plate during the early stages of segmentation. In stage 10-11 embryos, Hro-hh is expressed in body wall, foregut, anterior and posterior midgut, reproductive organs and in a subset of ganglionic neurons. Evidence that Hro-hh regulates gut formation was obtained using the steroidal alkaloid cyclopamine, which specifically blocks HH signaling. Cyclopamine induced malformation of both foregut and anterior midgut in Helobdella embryos, and no morphologically recognizable gonads were seen. In contrast, no gross abnormalities were observed in the posterior midgut. Segmental ectoderm developed normally, as did body wall musculature and some other mesodermal derivatives, but the mesenchymal cells that normally come to fill most of the coelomic cavities failed to develop. Taken with data from Drosophila and vertebrates, our data suggest that the role of hh-class genes in gut formation and/or neural differentiation is ancestral to the bilaterians, whereas their role in segmentation evolved secondarily within the Ecdysozoa.

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

We have identified homologs of the Drosophila pair-rule gene even-skipped in the glossiphoniid leeches Helobdella robusta and Theromyzon trizonare. In leech embryos, segments arise sequentially from five pairs of embryonic stem cells (teloblasts) that undergo iterated divisions to generate columns (bandlets) of segmental founder cells (primary blast cells), which in turn generate segmentally iterated sets of definitive progeny. In situ hybridization revealed that Hro-eve is expressed in the teloblasts and primary blast cells, and that these transcripts appear to be associated with mitotic chromatin. In more advanced embryos, Hro-eve is expressed in segmentally iterated sets of cells in the ventral nerve cord. Lineage analysis revealed that neurons expressing Hro-eve arise from the N teloblast. To assess the function of Hro-eve, we examined embryos in which selected blastomeres had been injected with antisense Hro-eve morpholino oligonucleotide (AS-Hro-eve MO), concentrating on the primary neurogenic (N teloblast) lineage. Injection of AS-Hro-eve MO perturbed the normal patterns of teloblast and blast cell divisions and disrupted gangliogenesis. These results suggest that Hro-eve is important in regulating early cell divisions through early segmentation, and that it also plays a role in neuronal differentiation.

Micromere lineages in the glossiphoniid leech Helobdella.

In leech embryos, segmental mesoderm and ectoderm arise from teloblasts by lineages that are already relatively well characterized. Here, we present data concerning the early divisions and the definitive fate maps of the micromeres, a group of 25 small cells that arise during the modified spiral cleavage in leech (Helobdella robusta) and contribute to most of the nonsegmental tissues of the adult. Three noteworthy results of this work are as follows. (1) The c"' and dm' clones (3d and 3c in traditional nomenclature) give rise to a hitherto undescribed network of fibers that run from one end of the embryo to the other. (2) The clones of micromeres b" and b"' (2b and 3b in traditional nomenclature) die in normal development; the b" clone can be rescued to assume the normal c" fate if micromere c" or its clone are ablated in early development. (3) Two qualitative differences in micromere fates are seen between H. robusta (Sacramento) and another Helobdella sp. (Galt). First, in Helobdella sp. (Galt), the clone of micromere b" does not normally die, and contributes a subset of the cells arising exclusively from c" in H. robusta (Sacramento). Second, in Helobdella sp. (Galt), micromere c"' makes no definitive contribution, whereas micromere dm' gives rise to cells equivalent to those arising from c"' and dm' in H. robusta (Sacramento).

Contribution of the geneextramacrochaetae to the precise positioning of bristles inDrosophila.

We examine the effect of mutations in theextramacrochaetae (emc) gene on the positioning of macrochaetes on the notum ofDrosophila. We show that, inemc mutants, most of the precursor cells appear earlier than in wild-type individuals, consistent with an antagonizing effect ofemc on the action of the proneural genesachaete andscute. We also show that reducingemc function affects the position of three bristles and/or of their precursors, but has no marked effect on the positioning of the other bristles.