By using the regenerating caudal fin of zebrafish as model system, I proved a putative involvement of RA signaling in vertebrate appendage regeneration and explored its specific functions during fin regeneration.
The findings of this thesis are presented in the form of a cumulative thesis, consisting of three published research articles (chaper 1,3 and 4) and one published review article (chapter 2), and are summarized below.
Chapter 1
In chapter 1, I demonstrate that RA signaling is indispensable for adult zebrafish fin regeneration, thereby providing for the first time evidence that RA signaling has an endogenous function in adult vertebrate appendage regeneration.
By genetic and pharmacological manipulations, I investigated the cellular and molecular consequences of loss- and gain of RA signaling for fin regeneration. I show that fin amputation induces upregulation of RA synthesis in the stump mesenchyme where it controls the expression of ligands of the Fgf, Wnt/β-catenin and Igf signaling pathway, which have previously been identified as crucial regulators of blastema formation and wound epidermis maturation. Inhibition of RA signaling upon fin amputation causes suppression of blastema formation by preventing cell cycle entry of postmitotic stump cells and interferes with formation of a proper wound epidermis. In the established blastema, RA signaling remains high and drives regenerative outgrowth by supporting blastema proliferation. Besides a putative, more direct mitogenic function, RA signaling indirectly regulates blastema proliferation through the activation of growth-stimulatory signals mediated by Fgf and Wnt/β-catenin signaling, as well as by reducing signaling through the growth-inhibitory non-canonical Wnt pathway.
Cell cycle reentry of postmitotic cells and dedifferentiation are characteristics of malignant transformation, raising the question of why blastema cells are not eliminated through tumor suppressor mechanisms. My findings indicate that blastema cells evade cell death by elevated levels of the anti-apoptotic factor Bcl2, the expression of which is positively regulated by RA signaling.
Together, this study support the hypothesis that RA signaling is an essential component of vertebrate appendage regeneration and provides mechanistic insights of RA signaling in wound epidermis formation, blastema formation and blastema maintenance in the regenerating fin.
Chapter 2
In chapter 2, I integrate the gained insights of RA signaling in fin regeneration with what is known more generally about RA signaling in animal regeneration and discuss a putative involvement of RA signaling in amphibian limb regeneration.
Chapter 3
RA is a diffusible signaling factor, raising the question in which cell types RA signaling acts during fin regeneration and how cell lineage-specific programs are protected from regenerative crosstalk between neighboring fin tissues. In chapter 3, I show how bone regeneration is achieved against a background of massive RA synthesis during fin regeneration.
Osteoblasts revert from a non-cycling, mature to a cycling, immature preosteoblastic state during blastema formation. During regenerative outgrowth, preosteoblasts finally redifferentiate into mature bone matrix producing osteoblasts. My findings demonstrate that RA signaling promotes bone matrix synthesis and osteoblast proliferation while inhibiting switching between the mature and immature state. Upon fin amputation, stump osteoblasts that will participate in blastema formation, counteract raising RA levels by upregulation of the RA degrading enzyme cyp26b1. This elegant mechanism allows the establishment of an osteoblast progenitor pool in a high RA environment that is required for blastema formation.
RA dependent proliferation of preosteoblasts is ensured by downregulation of cyp26b1 upon dedifferentiation. Redifferentiation of preosteoblasts is controlled by a presumptive RA gradient, in which high RA levels towards the distal tip of the regenerate inhibit differentiation and promote proliferation. This might be achieved through repression of Bmp signaling and promotion of Wnt/β-catenin signaling. Fibroblasts-like blastema cells in more proximal regions lower local RA concentrations via Cyp26b1 activity, thereby ensuring redifferentiation of osteoblasts. This allows two processes to run in parallel: Proliferation for the continuous supply of osteoblasts in the distal part and redifferentiation of osteoblasts more proximally where the fin rays re-emerge. In addition, my findings indicate that proper hemiray regeneration requires the interplay between bone matrix-producing osteoblasts and bone-resorbing osteoclasts and
suggest that RA signaling controls formation of new bone matrix at two levels, by ensuring matrix synthesis by osteoblasts and by preventing resorption by osteoclasts.
In summary, this study reveals how RA signaling orchestrates osteoblast behavior throughout all stages of fin regeneration and unravel a so far unnoticed important role of bone resorption by osteoclasts in fin regeneration.
Chapter 4
Fin rays are separated by soft interray tissue. This pattern has to be re-established during regeneration. However, the mechanisms that confine osteoblasts to only extend the existing rays have remained unresolved. Having shown in chapter 3 that the osteoblast regenerative program depends on the tight regulation of RA levels, chapter 4 addresses a putative involvement of RA signaling in the spatial regulation of bone regeneration and ray-interray patterning.
Upon dedifferentiation, preosteoblasts migrate into the nascent blastema, where they remain restricted to proximal lateral positons. My findings show that epidermal niches of low RA levels, established by Cyp26a1, allow the spatially restricted production of a signal that pilots preosteoblasts to target regions. Disruption of these niches causes preosteoblasts to ignore ray-interray boundaries and to invade ray-interrays where they form ectopic bone. Moreover, it emerged that osteoblasts themselves exert a piloting function for non-osteoblastic blastema cells and blood vessels. During regenerative outgrowth, the cyp26a1-expressing niches remain required for the production of Shh which in turn promotes osteoblast proliferation. Finally, my data indicate that cyp26a1 expression is spatially confined by Fgf signaling.
In summary, this study uncovers the mechanism that compels osteoblasts to respect ray-interray boundaries and explain how the alternating pattern of rays and ray-interray tissue becomes re-established during fin regeneration.