Disparities in injury response between phylogenetically related species
Previous studies on whole body regeneration (WBR) in Botryllus schlosseri concluded that there were three requirements for zooid development from isolated vasculature: (1) experimental colonies must be large, having nine or more zooids ; (2) the marginal vessel (central blood vessel that connects all zooids and ampullae; Additional file 3: Fig. S2), must be left intact following ablation of the zooids and buds for colony-wide circulation (Additional file 4: Video S2) ; and, (3) surgery required ablation of the zooids and buds when the zooids are resorbing during the takeover process [33, 34]. To make sense of the disparate requirements between this species and B. diegensis, we attempted to replicate previous experiments in B. schlosseri by carefully removing zooids and developing buds from large colonies to isolate blood vasculature and induce WBR (Fig. 1). We made detailed observations by carrying out longitudinal studies and recording timelapse videos starting immediately following surgery. While collecting data for both species, B. schlosseri and B. diegensis (Fig. 2), we never observed a zooid developing from an isolated blood vessel in B. schlosseri (Fig. 2C). In contrast, zooid development in B. diegensis was robust (Fig. 2F).
In both species, the vascular network initially reacted to colony damage by rapidly clotting up severed vessels to prevent blood loss. Next, the vasculature actively remodeled within the tunic matrix, with major differences observed between the two species. After 3 days of reorganization, the tissues in B. diegensis coalesced into a compact mass (Fig. 2E). In contrast, B. schlosseri vessels went through a characteristic global regression, followed by vessel re-extension toward the colony periphery (Additional file 5: Video S3). This retraction and re-extension process is consistent among genotypes and takes approximately 24 h.
Unremoved secondary buds migrate to vasculature and continue development
After twelve timelapse experiments with B. schlosseri we observed a WBR event following zooid ablation (Additional file 6: Video S4). However, through retrospective analyses of high-resolution images, we noticed small developing bud tissues had been inadvertently left behind following surgery. The observed tissues migrated away from their original location through the tunic, and restored contact with the peripheral blood vessel. Once fused with the circulatory system, these tissues increased in size and continued to develop as if seemingly derived from the blood vasculature.
To follow up on these results, we performed over 150 surgeries to ensure removal of all zooids and developing bodies from large, stage D colonies of B. schlosseri (Additional file 7: Fig. S3A–C). Experiments included five distinct genotypes from the Santa Barbara harbor on the Pacific coast of California (Additional file 8: Table S1). We only scored animals that restored colony-wide circulation and showed robust blood flow throughout the observation window (n = 128); therefore, in over 85% of our experiments we analyzed vascular rearrangement and blood circulation for up to 12 days following surgeries. None of these experiments provided evidence that WBR could be induced through injury. Instead, we observed characteristic vascular remodeling (described above), followed by eventual constriction of vessels, cessation of blood flow, and necrosis of remaining tissues (Additional file 7: Fig. S3D–F). If a zooid developed from the vasculature, we could visually identify its origins outside of the vasculature using stereoscope micrographs. We also carried out whole mount in situ hybridization of the vasculature following surgery to see if cell aggregates were forming. In these experiments, we used a probe for the pluripotency marker pou3 , and counterstained with an antibody to phosphohistone H3 (a mitotic marker) and the nuclear stain DAPI, which together would allow us to see aggregations of any cell type. While these markers clearly identified both aggregations and blastula-like structures consisting of proliferating pou3+ cells in B. diegensis , we never identified any clusters of pou3+ or proliferating cells in B. schlosseri, at any time point (Additional file 9: Fig. S4). Even when we performed surgeries on very large colonies, > 4× the reported minimal size requirement (n = 8), there was no indication of regeneration (Additional file 10: Fig. S5). In contrast, B. diegensis robustly and repeatedly underwent WBR from minimal vascular tissue (Fig. 2D–F).
In summary, when zooids developed after surgery in B. schlosseri, we could always retrospectively identify a previously undetected transparent tissue that was outside of the vasculature following surgery, but rapidly migrated and re-attached to the vasculature as the source of the new zooid (Table 1, Fig. 3, Additional file 6: Video S4). This tissue initially appeared near the peripheral vasculature and were most likely secondary buds that we missed during surgical ablation. At this point in the blastogenic cycle, secondary buds are small (250 × 100 µm), and lack pigmentation. It would be easy to miss ablating them, particularly since the peripheral vasculature cannot be damaged for WBR to occur; thus, one would avoid cutting close to the vessel (Fig. 2A). We found the most critical time point of these observations were the initial hours after surgery, during which we observed tissues migrating from their original position to fuse with vasculature (Fig. 3A–C). This phenotypically appeared as though a zooid developed directly from the remaining vasculature (Fig. 3D–J), but we could always predict where the zooid would arise following surgery when detailed images were scrutinized for migrating tissues.
Injury and characterizing development of remaining tissues
Developing buds can be near the marginal vessel or situated partially underneath the zooid; thus, it is possible to leave fragments of primary buds with secondary buds after surgery. We followed up on previous observations by removing all zooids at stage D and purposefully leaving combinations of primary and secondary bud tissues to characterize the response. We initially carried out two experiments, leaving only intact secondary buds or leaving fragments of the primary bud coupled to the secondary bud. In both cases, the remaining tissue migrated from its original location, re-attached to the peripheral vasculature, and then completed development into a zooid exactly as we had seen previously. When part of the primary bud was left, it was resorbed by the developing bud, and the zooid developed in 89% of the cases (Additional file 11: Fig. S6). When only a secondary bud was isolated without any anterior primary bud tissue, this decreased survival to open siphon down to 48% (Table 1). Finally, when only a secondary bud was left, in some cases we observed that the resulting zooid had an abnormal phenotype, including being shifted sideways in the tunic, such that the siphons pointed to the left or right, rather than dorsally (Additional file 12: Fig. S7) .
One interesting observation regarded differences in the timing of development following these two surgeries. During normal peribranchial budding in lab-reared colonies, stage D secondary buds which are 6 days old will form a pumping heart 3 days later (day 9) and the siphon will open 5 days after the heartbeat initiates (day 14). When we observed development following surgeries in which the secondary bud remained attached to the primary bud anterior region (Fig. 4A), it required on average 3 days for heart formation and 6 days to open a siphon (Fig. 4C). Thus, secondary buds developed at a normal pace when remaining primary bud tissues were present. When we performed surgeries to leave only the secondary bud (Fig. 4D, E), it required 6 days for hearts to pump, and 12 days for siphon opening (Fig. 4F), an approximately twofold delay vs unmanipulated peribranchial budding (Fig. 4G, Additional file 13: Table S2). These are similar ranges described for heart beat initiation and siphon opening to occur in previous WBR studies [14, 25, 33, 34].
If WBR in B. schlosseri is due to tissues leftover by accident, it would not be in the controlled fashion utilized in the previous experiments. We next characterized the level of damage that could occur to a secondary bud and still result in zooid development. We removed all but one intact secondary bud from a large colony at stage D, then injured that bud and observed the results. Our experimental injury applied pressure to the tunic above the bud without tearing into the tunic, until gross morphology was perturbed (Additional file 14: Fig. S8A, B). While the cells do aggregate prior to migration, they do not form the same tight association as that of an undamaged secondary bud (Fig. 4D, E). The reason for this method was because secondary buds did not survive direct surgical cuts. Interestingly, 25% of damaged secondary buds developed a pumping heart, but only in 1/16 cases did we observe the damaged bud develop into a mature zooid with open siphons. While this injury model is not replicating what may have happened in previous studies, it does suggest that a relatively undamaged secondary bud is required to generate a zooid. These experiments also provide strong evidence that WBR does not occur in B. schlosseri: a single secondary bud was purposefully left and damaged prior to revascularization, but no WBR event was observed under these controlled conditions. Importantly, in the case where development did occur, it did so from the damaged bud.
Isolated secondary bud survival has a vascular tissue size requirement
Previous studies reported that a continuous marginal blood vessel and approximately 10× more vascular area was required for WBR in B. schlosseri versus B. diegensis. Additionally, the ablation must take place during takeover, when adult zooids are dying and being phagocytosed in stage D (Additional file 2: Video S1, 155–166 h). Taken together, this suggested that B. schlosseri required more energy via catabolism of the remaining tissue versus B. diegensis, where only a small portion of the vasculature is required to support WBR (Fig. 2).
To address this potential difference in energetic demand, we determined the minimal size of remaining vasculature that was required to facilitate successful post-surgery zooid development of a single secondary bud (Table 1). Secondary buds left with ≤ 3.4 mm2 of total tissue area did not survive (Fig. 5A–F), whereas isolated secondary buds (Fig. 5G) with tissue area ≥ 6 mm2 (Fig. 5H) developed and continued asexual budding (Fig. 5I; Additional file 15: Video S5). Surprisingly, the time to complete development was equivalent whether we used an entire vascular network (Fig. 1), or only a 6 mm2 section (Fig. 5): in other words, an area of vasculature larger than the minimum size did not expedite the developmental process. These data show that there is a vascular tissue size requirement for secondary bud survival, but it is < 10% of the size required for successful WBR as described previously .
Secondary buds compete for sole-survivor
Previous studies on WBR in B. leachii have revealed that while multiple vascular buds are initiated following surgery, only a single zooid completes development, and this observation was consistent over a large range of vascular tissue [26, 28]. A single zooid can develop from a 3 mm2 fragment (Fig. 2D), so larger fragments around 40 mm2 (Additional file 16: Fig. S9) could theoretically support the development of multiple zooids, but that is not observed. This suggests that buds compete for resources during WBR, and previous studies suggested competition occurs at the blastula-like stage .
If previous results documenting WBR in B. schlosseri were actually due to ectopic development of remaining secondary buds, we wondered why these experiments also resulted in the development of only a single zooid [14, 25, 33, 34]. We next asked if competition exists between developing secondary buds in B. schlosseri. To assess the presence of interbud competition, we left two or three secondary buds after surgically removing all zooids and primary buds and observed the outcome. When two isolated secondary buds in B. schlosseri were left after surgery, the result was a single surviving zooid (N = 9). We next examined the outcome when three secondary buds were left (N = 3) (Fig. 6). Seven days after surgery, hearts pumped in all three buds, but their sizes varied (Fig. 6C–F). By day 13, only one persisted and opened its siphons to become an active filter-feeding zooid (Fig. 6G). The other two developing secondary buds, which were always sharing blood with the winner, ended up dying and resorbing into the vasculature (Fig. 6H). Because these experiments were done on buds derived from a single system, we next checked if secondary buds originating from separate systems within the same colony could compete, as these larger distances would be more representative of other WBR studies (Fig. 7A). The surgery performed in these experiments left behind two developing buds spaced approximately 1 cm apart (N = 3) (Fig. 7B). Both buds developed pumping hearts by day 6 (Fig. 7C) but on day 13 only a single zooid developed, while the other was resorbed (Fig. 7D). Whatever is mediating the interactions between the buds can operate at this distance.
Competition provides winner secondary bud with more resources
A zooid that develops from a single secondary bud (Fig. 8A, B) is significantly smaller (Fig. 8C, D) than a control zooid derived from peribranchial budding (Fig. 8E). However, leaving behind two secondary buds (Fig. 8F, G) gives rise to a single zooid (Fig. 8H, I) that is quantitatively similar in size to the control (Fig. 8J, Additional file 17: Table S3). Interestingly, when multiple secondary buds are left, they commensurately increase in size until the heart begins beating (Fig. 8J). At this point, the non-competitive buds stop growing, begin regressing, and are eventually resorbed. This demonstrates that competition is causing the resorption of loser buds, reallocating those resources to the winner, and that competition is not visually apparent until after the heart has completed development.
Growth inhibition is due to circulatory factors and is reversible
To narrow down the tissues mediating competition, we did the same experiments leaving two secondary buds, but this time severed the blood vessels 48 h later, when the two buds were approximately equal in size, but prior to the appearance of a functional heart. The shared tunic was left partially intact so that we only disrupted the vascular connections (Additional file 18: Fig. S10B). In this case, secondary buds sharing tunic but not blood both developed to zooids (Additional file 18: Fig. S10D). These findings show that factors in the blood are responsible for the signals driving competition between secondary buds.
To characterize the timing and mechanisms of competition, the circulation was severed following visual changes in growth rates between the two buds. In these experiments, colonies with two secondary buds (Fig. 9B) were left to develop following completion of heart development, and until one bud (presumed to be the winner) was growing steadily, and the other (presumed to be the loser) was not increasing in size. By day 8 we observed that the smaller secondary bud had started shrinking, indicating it was beginning to die (Fig. 9C, right side), and at that point the vasculature was severed. Within 4 days, the loser secondary bud had substantially increased in size, opened its siphons, and started to bud (Fig. 9D, right side).
We repeated this experiment, but this time allowed the smaller bud to decrease in size to a point where we could observe accumulation of pigmented cells in the body, which is characteristic of the later stages of apoptosis and phagocyte resorption, and at that point severed the vascular connection between them (Fig. 10). During the next few days, the loser bud increased in size, decreased in pigmentation, and eventually opened its siphons. These data indicate that although both buds have the potential to develop, signaling from a winner secondary bud creates a continuously repressive environment for the loser. Importantly, this also shows that a partially resorbed bud can reverse its fate and complete development.
In summary, both WBR in B. diegensis  and bud competition in B. schlosseri clearly show that a botryllid colony can shift metabolic resources within an individual to support development of a feeding zooid. In B. schlosseri, remnants of the peribranchial budding process can detect and respond to injury via migrating and reconnecting to the vasculature. If multiple developing zooids are attached to the common vascular network, a competitive situation arises in which only a single zooid completes development. This competition likely exists to increase the chances that a single bud reaches maturity. Afterward, the colony can feed and resume normal peribranchial budding.