Leech Segmental Repeats Develop Normally in the Absence of Signals from either Anterior or Posterior Segments

AbstractWe have investigated whether the development of segmental repeats is autonomous in the embryo of the leech Helobdella robusta. The segmental tissues of the germinal band arise from progeny of five stem cells called teloblasts. Asymmetric divisions of the teloblasts form chains of segment founder cells (called primary blast cells) that divide in a stereotypical manner to produce differentiated descendants. Using two distinct techniques, we have looked for potential interactions between neighboring blast cell clones along the anterior–posterior axis. In one technique, we prevented the birth of primary blast cells by injection of DNase I into the teloblast, thereby depriving the last blast cell produced before the ablation of its normal posterior neighbors. We also ablated single blast cells with a laser microbeam, which allowed us to assess potential signals acting on either more anterior or more posterior primary blast cell clones. Our results suggest that interactions along the anterior–posterior axis between neighboring primary blast cell clones are not required for development of normal segmental organization within the blast cell clone. We also examined the possibility that blast cells receive redundant signals from both anterior and posterior neighboring clones and that either is sufficient for normal development. Using double blast cell laser ablations to isolate a primary blast cell clone by removal of both its anterior and its posterior neighbor, we found that the isolated clone still develops normally. These results reveal that the fundamental segmental repeat in the leech embryo, the primary blast cell clone, can develop normally in the apparent absence of signals from adjacent repeats along the anterior–posterior axis

Hau-Pax3/7A is an early marker of leech mesoderm involved in segmental morphogenesis, nephridial development, and body cavity formation

AbstractTwo genes of the Pax III subfamily, Hau-Pax3/7A and -Pax3/7B, were identified from the leech Helobdella, and the expression and function of Hau-Pax3/7A in development are described. Leech embryos undergo spiral cleavage, then produce a set of teloblastic stem cells that generate segmented mesoderm and ectoderm. Hau-Pax3/7A is present as a maternal transcript in both ectodermal and mesodermal progenitors, but this pool of early RNA disappears and is replaced by a pattern of zygotic transcription restricted to the blast cell progeny of the mesodermal M teloblasts. Each mesodermal blast cell clone goes through multiple phases of Hau-Pax3/7A expression, the last of which is associated with the organogenesis of the nephridia and other segment-specific structures. Morpholino-mediated knockdown of Hau-Pax3/7A expression causes the mesodermal blast cell clones to undergo irregular patterns of morphogenesis that disrupt the segmental organization of the germinal plate, and interferes with both the specification and morphological differentiation of the mesodermal nephridia. Knockdown of Hau-Pax3/7A in the mesoderm can also lead to abnormalities in the formation of the dorsal cavities, possibly through indirect effects of this germ layer on neighboring tissues. This is the first report of broad mesodermal Pax III expression outside of chordates, and raises the possibility that such expression may be a primitive trait inherited from the last common ancestor of the bilaterian superphyla

DEV00395P1645

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 (Hrohh) 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 Hrohh 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