Congress Abstracts - Society for Developmental Biology

activity specifically via NMDA receptors. These results suggested that the neural peptides induced by neural activity of the newly
regenerated neurons might be required for the proper phototactic behavior.
Program/Abstract # 424
Primary Cell Cultures from the Regenerating Gut of the Sea Cucumber Holothuria glaberrima
Bello, Samir A.; García-Arrarás, José E. (University of Puerto Rico, San Juan, PR, United States)
Regeneration studies in echinoderms are hampered by the lack of suitable cell culture methodologies. We have established an in vitro
model to study the cellular and molecular basis of organ regeneration in the sea cucumber Holothuria glaberrima. This organism has
the remarkable ability to regenerate its digestive tract after evisceration. Regenerating gut rudiments at 5 days post-evisceration were
dissociated and isolated cells were seeded on glass slides. Cells were incubated for 24h, 5d or 10d and their identity was established by
SEM and immunocytochemistry. The proliferative capacity was evaluated by BrdU incorporation. Among the cell phenotypes
observed in culture were: (1) Spherical (10 µm in diameter) labeled Meso-1 cells. These are dedifferentiated cells that probably give
rise to most cells of the new intestine. (2) Phalloidin labeled cells suggesting the presence of muscle cells undergoing dedifferentiation
or differentiation processes. (3) Small spherical cells (5 µm in diameter) immunoreactive for RN1 and/or calbindin (neuronal cell
markers in sea cucumbers) which correspond to neuronal precursors. (4) Ovoid and spindle shaped (5-10 µm in diameter) β-tubulin
labeled cells exhibiting cell projections. About 5% of the cultured cells incorporated BrdU at the three time points evaluated. Taken
together, our results indicate that we have established the methodology to obtain primary cultures from the regenerating gut of H.
glaberrima. This valuable tool is now being used to test the signaling events that determine regeneration-associated processes in sea
cucumbers.
Program/Abstract # 425
Transient reduction of 5-methylcytosine and 5-hydroxymethylcytosine is caused by active DNA demethylation during
regeneration of zebrafish fin
Hirose, Kentaro (Hiroshima University, Japan); Shimoda, Nobuyoshi (National Center for Geriatrics and Gerontology, Japan);
Kikuchi, Yutaka (Hiroshima University, Japan)
It is well known that dedifferentiation processes such as the loss of molecular markers for differentiated cells, re-expression of
molecular markers for progenitor cells, and restart of cell proliferation occur during regeneration in amphibians and zebrafish.
Although epigenetic modifications are thought to be critical for the dedifferentiation processes in regeneration, the status and changes
of DNA methylation during regeneration remain largely unknown. In this study, we analyzed the spatial and temporal changes of 5-
methylcytosine (5mC) or 5-hydroxymethylcytosine (5hmC) distribution during zebrafish fin regeneration by using dot blot assays and
immunohistochemical analyses. We showed that during regeneration of zebrafish fin, the levels of 5mC and 5hmC are transiently
reduced in blastema cells and cells adjacent to the amputation plane at 30 hours post-amputation (hpa), and the level of 5mC, but not
5hmC, is almost restored by 72 hpa. We observed that the dedifferentiated cells showed reduced levels of 5mC and 5hmC independent
of cell proliferation by 24 hpa, suggesting that active demethylation pathways lead to the reduction of 5mC and 5hmC levels.
Furthermore, expressions of the proposed demethylation- and DNA repair-related genes were detected during fin regeneration. Taken
together, our findings illustrate that the transient reduction of 5mC and 5hmC in dedifferentiated cells is caused by active
demethylation during regeneration of zebrafish fin.
Program/Abstract # 426
Extensive conversion of hepatic biliary epithelial cells to hepatocytes after extreme hepatocyte loss in zebrafish
Choi, Tae-Young (University of Pittsburgh, USA); Ninov, Nikolay (UC-San Francisco, USA); Stainier, Didier Y.R. (Max Planck
Institute, Germany); Shin, Donghun (University of Pittsburgh, USA)
Biliary epithelial cells (BECs) have been considered as the source of regenerating hepatocytes when hepatocyte proliferation is
compromised. However, their contribution to hepatocytes in vivo has been controversial. To resolve this issue, we established a novel
zebrafish liver regeneration model in which hepatocyte-specific ablation can be temporarily, pharmaco-genetically achieved. By
tracing the lineage of BECs, we show that BECs can extensively give rise to regenerating hepatocytes in larval and adult zebrafish.
Upon severe hepatocyte loss, BECs highly proliferated and dedifferentiated into hepatoblast-like cells and subsequently
redifferentiated into hepatocytes that were highly proliferating to restore liver mass. This BEC-driven liver regeneration was impaired
in sox9b and wnt2bb mutants: upon severe hepatocyte loss, most BECs disappeared in sox9b mutants and the proliferation of newborn
hepatocytes was greatly reduced in wnt2bb mutants. Our results demonstrate that BECs can extensively contribute to hepatocytes,
thereby leading to full liver recovery from severe liver damage.
Program/Abstract # 427
Regeneration of the adult zebrafish jaw by bone-producing chondrocytes is distinct from jaw development
Crump, Gage DeKoeyer; Paul, Sandeep; Schindler, Simone (USC Keck School of Med, USA)
A major goal in human health is to improve the ability of large fractures and skeletal wounds to heal. Here, we present a new model of
skeletal regeneration in the genetically tractable zebrafish. In a matter of just a few weeks, we find that adult zebrafish can regenerate
nearly two-thirds of their lower jawbone, and they appear to do so through an unusual chondrocyte population that directly produces
woven bone. During development, the majority of chondrocytes undergo hypertrophy and apoptosis, with bone matrix being produced
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