Abstracts - Society for Developmental Biology

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Incubation of regenerating cells with either the anesthetic MS222 or intracellular Ca2+- release inhibitors Ryanodine and
Xestospongin C, inhibits spontaneous activity by 60%. To investigate the impact of inhibiting Ca2+-mediated activity on
the success of muscle regeneration we incubated amputated tadpoles with MS222, Ryanodine, Xestospongin C, or the cellpermeant
Ca2+ chelator BAPTA-AM. Whole-mount immunostaining with muscle markers reveals that inhibiting Ca2+
transients decreases the extent of regeneration by 60-80% after 72 hpa. These findings suggest that regenerating tissues
exhibit Ca2+ transients, mediated by Ca2+ influx and release from stores, which are necessary for muscle repair.
Understanding how Ca2+-mediated electrical activity contributes to the repair of injured tissues may lead to improved
therapies for tissue regeneration.
Program/Abstract # 191
An investigation of the role of transforming growth factor beta (TGFß) during multi-tissue regeneration.
Gilbert, Richard WD; Vickaryous, Matt; Viloria-Petit, Alicia, University of Guelph, Guelph, Canada
The transforming growth factor beta (TGFb) signaling pathway has a number of well documented roles in wound healing
and is becoming increasingly appreciated as a vital component of multi-tissue regenerative processes in amphibians (e.g.,
Xenopus and axolotls). For amniotes (mammals and reptiles), less is known in part because of the lack of an appropriate
model organism capable of multi-tissue regeneration. With this in mind, we examined the localization of several key
components of the TGFb signaling pathway during tail regeneration of the leopard gecko (Eublepharis macularius). As for
many lizards, the gecko is able to spontaneously and repeatedly regenerate its tail. We focused on characterizing TGFb
1+2, TGFb 3, phosphorylated Smad2 (pSmad2), as well as target genes such as Snail, Slug and Zeb2, at various stages of
regeneration. We demonstrate that there is a sharp increase in TGFb ligand availability during both early and late
regeneration combined with a localized increase in pSmad2 in both the regenerative epidermis and blastema like structure
located basally to the epidermis. This activity has diverse effects on known Smad target genes including Snail, Slug and
Zeb2. These genes encode transcription factors that have been implicated in driving cell motility and stem cell like
features. Our characterization of the spatial and temporal expression of TGFb ligands suggests the possible role of
epithelial to mesenchymal transitions during multi-tissue regeneration.
Program/Abstract # 192
Retinal regeneration following targeted rod photoreceptor destruction
Rao, Mahesh; Patton, James, Vanderbilt University, Nashville, United States
Zebrafish have the remarkable ability to fully regenerate their retinas following damage. The genes and regulatory
mechanisms involved in retinal regeneration are largely unknown. MicroRNAs (miRNAs) are an intriguing possibility as
one possible mechanism that controls the regeneration process. Previously, deep sequencing was used to create miRNA
libraries throughout retinal regeneration following treatment with constant intense light to identify miRNAs involved in
this process. This treatment, however, non-selectively destroys all photoreceptors. A new method has been developed to
selectively destroy rod photoreceptors. Here we characterize regeneration progress in the retina following rod destruction.
Once regeneration is characterized we can perform deep sequencing analysis to identify miRNAs and mRNAs specifically
involved in rod regeneration.
Program/Abstract # 193
Analysis of gene expression in mantle and interneuromast cells reveals genes that are differentially regulated
during hair-cell regeneration
Steiner, Aaron; Kim, Taeryn; Hudspeth, A. James, The Rockefeller University and HHMI, New York, United States
Hearing loss is an increasingly common problem in modern societies. Because the majority of hearing deficits are
attributable to the death of sensory hair cells in the cochlea, therapeutic regeneration of these cells represents an attractive
avenue to recovery. Although hair cells of the adult mammalian cochlea do not regenerate, those of nonmammalian sensory
systems regenerate throughout life. In the zebrafish lateral line, for example, hair cells extirpated by chemical treatment
regenerate fully within 48 hours. Each neuromast, the sensory unit of the lateral line, comprises a cluster of hair cells
encircled by mantle cells that are connected to other neuromasts by interneuromast cells. Both of the latter cell types have
been proposed to harbor hair-cell progenitors. We have developed a transgenic line of zebrafish,Tg (tnap:mCherry), that
expressesred-fluorescent mCherry in both mantle and interneuromast cells. Fluorescence-activated cell sorting from
transgenic larvae followed by RNA-seq and microarray-based expression analyses enables us to identify genes that are
expressed more highly in these cells than in other cell types. In situ hybridization confirms that many of these genes are
specific markers of mantle and interneuromast cells. Similar analyses following hair-cell ablation reveal genes that are
differentially regulated during regeneration. Proteins encoded by these genes include cell-cycle regulators and components
of known signaling pathways as well as several novel proteins. Knockdown and overexpression of candidate genes are