Systemic bud induction and retinoic acid signaling underlie whole body regeneration in the urochordate Botrylloides leachi

Abstract

Regeneration in adult chordates is confined to a few model cases and terminates in restoration of restricted tissues and organs. Here, we study the unique phenomenon of whole body regeneration (WBR) in the colonial urochordate Botrylloides leachi in which an entire adult zooid is restored from a miniscule blood vessel fragment. In contrast to all other documented cases, regeneration is induced systemically in blood vessels. Multiple buds appear simultaneously in newly established regeneration niches within vasculature fragments, stemming from composites of pluripotent blood cells and terminating in one functional zooid. We found that retinoic acid (RA) regulates diverse developmental aspects in WBR. The homologue of the RA receptor and a retinaldehyde dehydrogenase-related gene were expressed specifically in blood cells within regeneration niches and throughout bud development. The addition of RA inhibitors as well as RNA interference knockdown experiments resulted in WBR arrest and bud malformations. The administration of all-trans RA to blood vessel fragments resulted in doubly accelerated regeneration and multibud formation, leading to restored colonies with multiple zooids. The Botrylloides system differs from known regeneration model systems by several fundamental criteria, including epimorphosis without the formation of blastema and the induction of a "multifocal regeneration niche" system. This is also to our knowledge the first documented case of WBR from circulating blood cells that restores not only the soma, but also the germ line. This unique Botrylloides WBR process could serve as a new in vivo model system for regeneration, suggesting that RA signaling may have had ancestral roles in body restoration events.

Figure 1. Morphological Events during WBR in B. leachi

(A) A colony of B. leachi composed of two systems of genetically identical zooids (yellow arrowheads), each 2–3 mm long, embedded within the tunic. A network of blood vessels connects all zooids within a colony, from which pear-shaped vascular termini (ampullae) extend toward the colony margins (arrows). (B–H) Experimentally induced regeneration. The zooids and palleal buds in the center of the colony were cut out, leaving behind the marginal ampullae (B). Segments of several ampullae, 1–3 d after amputation, created a new blood vessel network (C) and (D), respectively (arrows). Isolated ampullae changed their orientations and shapes 6–7 d postseparation within the tunic matrix and coalesced into each other (E). Vasculature movements were more conspicuous when blood vessels were spaced out, creating a dense mass of vessels on one side of the fragment and a semitransparent gelatinous tunic matrix deprived of blood cells and blood vessels on the other side (F). By days 8–9, an opaque mass of blood vessels was formed, followed by the formation of an internal small transparent vesicle (G) (arrow). By days 10–14, a fully operating filter-feeding zooid equipped with functional atrial and peribranchial siphons, both facing upwards as in normal zooids, developed from the opaque mass of blood vessels (H) (arrows). The scale bar represents 1 mm.

Figure 2. Histological Characteristics of WBR in B. leachi

Regenerating vasculature fragments that were sacrificed at sequential daily intervals exhibit cellular events during the regeneration process. (A and B) Phase I events: Haemocytes are attached to vasculature epithelium (A) (arrows). Vascular detachment from the tunic (B) (arrows) enables subsequent relocation of blood vessels. (C–E) Phase II events: 2 d after dissection, aggregated haemocytes of various sizes and shapes were observed in the regeneration niches (C) (arrows). Extensive cell proliferation formed an opaque ball of cells (D) with exclusive PCNA staining (E). (F–L) Phase III events: 1 d later, continuous cell proliferation increased the size of cell aggregates and formed blastula-like structures of different shapes (F and G) (arrowheads). At this stage, multibuds developed simultaneously in various regenerating niches (G) (arrowheads and red arrows, respectively), although separation between compartments had not been completed (G) (arrow). PCNA specifically stained newly formed bud spheres and blastula-like structures inside the regeneration niches (H). As development proceeded, axial polarity was observed with the appearance of differential cell layers (I) (arrowheads). From day 5, two invaginations from both corners of the thick vesicle wall creating two elongated double-walled folds were observed (J) (arrows). Between 10–14 days postseparation, the regeneration process reached the final stages, whereby a fully functional adult zooid was developed, including the formation of palleal buds (K). Only one zooid per regenerating fragment reached this final stage, while the others degenerated (L) (arrow). (M–P) TUNEL analysis clearly shows staining in degenerating buds. Bud that was normally developed did not stain for TUNEL and exhibits a normal blastula-like structure with clear polarization: (M) and enlargement in (O). Bud that failed to develop went through a degenerating process and was stained for TUNEL: (N) and enlargement in (P). Note that it failed to develop a normal blastula structure, and cells started to fall apart. b, bud; i, intestine; ph, pharynx. Scale bar represents 100 μm. In (O) and (P) scale bars represent 30 μm.

Figure 3. Cloning and Expression Pattern of the RA Receptor Homologue during WBR

(A) A fragment of 157 amino acid exhibiting high homology to the ligand-binding domain (marked by a red line) of hormone receptors present in all RARs. (B) An evolutionary tree of different RARs, retinoic X receptors, and thyroid hormone receptor members of this family of RA receptors in different urochordates and vertebrates. Molecular evolutionary analysis was conducted using the MEGA 2.1 program using the tree-making method Minimum evolution. The numbers along the outer side of each branch represent the bootstrap values in percentage (1,000 reps) of three different tree-making methods: the selected tree, Minimum evolution/ Neighbor-joining/ Maximum parsimony. The sign “-“ marks bootstrap values that are less than 50% or a complete different outcome of the specific branch compared to the same branch in the selected tree. (C) RT-PCR analysis was performed on regenerating ampullae at different developmental stages. Intact blood vessels (the controls) did not express the RAR transcript (lane 1) while as early as 19 h postseparation, the transcripts were detectable and showed a gradual increase in their levels along the entire process (lanes 2–12). Actin levels remained equal representing the viability of the regenerating ampullae and serve as a positive control to normalize RAR levels. (D–F) Whole-mount RNA in situ hybridization was employed on paraffin sections of regenerating ampullae at different developmental stages. Specific staining pattern was visible from day 2 (D) (arrow) in aggregates of blood cells in the regenerating niches. In cases where several regeneration niches were established and buds developed simultaneously within the ampullae, RAR expression pattern was localized specifically to all buds (E) (arrows). Later on, RAR continued to specifically stain regenerating buds through the subsequent developmental stages of spheres, invaginations and organogenesis (F). Scale bar represents 100 μm. Bl, B. leachi; Brl, Branchiostoma lanceolatum; Dr, Danio rerio; Gg, Gallus gallus; Hs, Homo sapiens; Mm, Mus musculus; Nv, Notophthalmus viridescens; Pm, P. misakiensis; Xl, Xenopus laevis.

Figure 4. Cloning and Expression Pattern of the Raldh Homologue in Intact Colonies and Regeneration Ampullae

(A) A fragment of 110 amino acid exhibiting high homology to the Aldedh domain of the budding ascidian P. misakiensis (Pm) and high sequence similarity to Aldedh2 family members from mouse (Mm), Human (Hs), Xenopus (Xl), chick (Gg), and zebrafish (Dr). A domain search revealed an Aldedh domain conserved in all Aldedh family members (98 amino acid marked by a red line). (B–E) Expression pattern of Bl-Raldh in intact colony and regenerating ampullae. Bl-Raldh is expressed in naïve ampullae, exclusively in a population of circulating macrophage cells (B) (arrows), scattered throughout vasculature. In regenerating ampullae, Bl-Raldh is observed in macrophage cells (C) (arrows) adjacent to aggregates of small cells creating morula-like structures (C) (arrowheads, rectangle), enlargement in (E). In later stages, starting from the blastula-like stage, Bl-Raldh expression appears in developing buds (D) (arrow). Note the staining along the ampullae margins represents nonspecific staining of the tunic matrix. Scale bars in (B) and (D) represent 100 μm. Scale bars in (C) and (E) represent 40 μm and 10 μm, respectively.

Figure 5. Specific RA Inhibitors, DEAB and Citral, Inhibit Bud Regeneration

Early stages of bud regeneration were blocked by different concentrations of RA inhibitors. With the administration of 100 μM of DEAB, vessel coalescence gradually slowed down and stopped completely after day 2 (A). Similar results were obtained with 60 μM of Citral (B), while with 20 μM of the same inhibitor ampullae changed orientation and migrated within the tunic but never regenerated a functional zooid (C). At the 100 μM DEAB concentration, buds had not developed, and multinucleated giant cells appeared, scattered throughout the vessel lumen (D, arrows). At the 10 μM DEAB concentration, morphologically abnormal buds were formed, which failed to develop organ structures, and retained a simple epithelial morphology (E). In severe cases, masses of undifferentiated aggregated cells occupied the interior of the vessel lumen (F). All malformed buds subsequently degenerated. PCNA immunostaining revealed distinctive proliferations at aggregated masses while no staining was detected in blood vessels (G). Scale bar in (A–C) represents 1 mm. Scale bar in (D–G) represents 100 μm.

Figure 6. Disruption of RAR Function during Regeneration Causes Bud Malformations

In order to verify the efficiency and ability of RAR siRNAs to interact and decrease RAR RNA levels, we added RAR siRNAs and control siRNAs to B. leachi colonies and compared RAR RNA levels to untreated colonies. Control siRNAs and untreated colonies exhibit approximately the same transcripts levels (A) (lanes 1 and 3), while RAR siRNAs show a significant reduction in RAR RNA levels (A) (lane 2). Actin transcript levels remain unchanged, indicating the viability of the tissue. After 6 d, control experiments using the control siRNAs completed vasculature movements, forming opaque vessel masses as specified before (B) (compare to Figure 1F). In contrast, in RNAi-affected experiments, blood vessels remained apart, situated haphazardly within the tunic matrix (C) (compare to Figure 5A). In several experiments, vessel movement halted in an intermediate state (D). A similar phenotypic morphology was observed with the RAR pan-antagonist BMS-493 (E). Histological sections reveal malformation of regenerating buds in RNAi treatments (F, arrow, compared to Figure 5E and 5F). In mild cases, buds reached progressive developmental stages but failed to invaginate properly (G, arrow). A similar histological phenotype is observed in BMS-493-treated fragments exhibiting malformed epithelial spheres (H, arrows). Scale bar in (B–E) represents 1 mm; scale bar in (F–H) represents 100 μm.

Figure 7. All-trans RA Leads to Accelerated Regeneration and Multibud Formation

RA-treated regenerating fragments that were sectioned at different final stages of the regeneration process show a remarkable accelerated regeneration. By days 6–7 postseparation, most of the regenerating buds reached these final stages (compared to 10–14 days in control experiments), exhibiting complete organogenesis. The regenerating zooids manifested fully differentiated organ systems such as stigmata with cilia (A, arrows). Different from normal blastogenic buds or control experiments, blood cells extensively colonized these regenerating buds between atrial folds and throughout internal cavities (B, arrows). The acceleration of the process was also exemplified in the subsequent blastogenesis. At day 7, the regenerating bud progressed to the stage where secondary buds were formed on the primary palleal buds (C, arrow and arrowhead, respectively). In marked contrast to control regenerating fragments, where only a single bud was developed, numerous buds at different regeneration niches simultaneously reached the final stages of organogenesis (2–5 functional zooids) (D, arrows, compare to Figure 2G). Scale bar represents 100 μm.