Abstracts - Society for Developmental Biology

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currently underway to determine how they affect regeneration. This approach will help elucidate the molecular
mechanisms of hair-cell regeneration.
Program/Abstract # 194
Expression of stem pluripotency-inducing factors during RPE reprogramming
Luz-Madrigal, Agustin; Grajales-Esquivel, Erika; Di-Lorenzo, Ashley; Dannenfelser, Janessa; Del Rio-Tsonis, Katia,
Miami University, Oxford, United States
The embryonic chicken can regenerate its retina by the reprogramming of the retinal pigmented epithelium (RPE) and by
the activation of stem/progenitor cells present in the ciliary margin (CM) in the presence of fibroblast growth factor
2(FGF2). Recently, it has been demonstrated that somatic mammalian cells can be reprogrammed in vitro to generate
induced pluripotent stem cells (iPSC) by the ectopic expression of Oct4, Nanog, Sox2, Klf4, c-Myc or Lin28 (ONSKCL).
However, there is limited information concerning the reprogramming during the process of retina regeneration in vivo.
Here, we test the hypothesis that reprogramming of the RPE can share similarities to the reprogramming of somatic cells
that generate iPSC. Therefore, we analyzed the expression of stem cell pluripotency factors during chick development and
RPE reprogramming. The analysis of ONSKCL expression showed that only Sox2, c-Myc and Klf4 mRNAs were detected
by RT-PCR in the CM. Furthermore, Sox2 was detected by immunofluorescence in the CM and central retina but not in the
RPE ofdeveloping embryos at day 4-7. Upon retina removal, while Sox2, c-Myc and Klf4 remained expressed in the CM,
these genes were induced in the RPE. Their expression was maintained up to day 3, and at that time, FGF2 was required to
keep the expression. These results suggest that the injury itself is sufficient to induce the expression of stem cell
pluripotency-inducing factors. However, FGF2 is required to maintain the expression during RPE reprogramming. We also
observed up-regulation of Lin28 and the pro-neural transcriptional factor Ascl1a in the RPE post-retinectomy suggesting
that these two factors might also contribute to the process of RPE reprogramming. Finally our results demonstrate that
Oct4 and Nanog are dispensable during the process of chick retina regeneration and indicate that reprogrammed RPE cells
do not generate pluripotent cells. However, our results do suggest that retina regeneration through RPE reprogramming
share similar mechanisms to the generation of iPSC cells.
Program/Abstract # 195
The role of microRNAs as downstream effectors of RARß-mediated retinoid signalling during spinal cord
regeneration in the adult newt.
Lepp, Amanda C.; Carlone, Robert, Brock University, St. Catharines, Canada
Urodele amphibians possess the unique ability to regenerate lost structures, including spinal cord, following tail
amputation. Little is known regarding the coordination of molecular pathways that control the formation of the tail
blastema, as well as the outgrowth and patterning of the regenerating spinal cord. Our lab has been involved in studies
examining the role of retinoic acid signalling in this epimorphic regenerative phenomenon. We have previously
demonstrated that inhibition of RARβ-mediated retinoic acid signalling using a specific antagonist, LE135, significantly
inhibits tail and spinal cord regeneration by delaying the formation and outgrowth of an ependymal tube caudal to the
amputation plane. Our current focus is to identify the downstream effectors of RARβ-mediated retinoid signalling and
elucidate the mechanism by which they contribute to regeneration. We have utilized a microarray approach with
microRNA-based profiling to identify 18 highly conserved microRNAs that display significant changes in expression in
tail regenerates treated with LE135 compared to DMSO control regenerates during the first 48 hours post amputation.
Initially we have chosen seven of these microRNAs (miR-133a, miR-1, miR-26a,miR-145, miR-223, miR-1306 and let7c)
for further investigation. miR-133a is expressed in the ependymal cells surrounding the central canal of the spinal cord in
adult newt tail tissues. This miRNA is significantly down regulated in these cells during the first three weeks following tail
amputation. Moreover, the spatial and temporal pattern of expression of miR-133a is consistent with a role for this micro
RNA as a mediator of RARβ signalling in this process. Analysis of the patterns of expression and putative functions of the
other microRNAs as downstream regulators of retinoid signalling during caudal spinal cord regeneration are currently
underway.
Program/Abstract # 196
Two-Photon microscopy to capture live cell behavior in the hair follicle stem cell niche
Greco, Valentina; Rompolas, Pantelis, Yale School of Medicine, New Haven, United States
Stem cells and niche components are responsible for the timely orchestration of the regeneration process that leads to
highly organized tissues. Despite recent progress in our understanding of stem cell biology, the dynamic interaction
between stem cells and the niche is not well understood. A current challenge in the field is having access to a well-defined
stem cell niche in which the orderly development of stem cells can be observed, characterized and manipulated in vivo. To