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Distinct stem-like cell populations facilitate functional regeneration of the Cladonema medusa tentacle [1]
['Sosuke Fujita', 'Graduate School Of Pharmaceutical Sciences', 'The University Of Tokyo', 'Tokyo', 'Graduate School Of Life Sciences', 'Tohoku University', 'Sendai', 'Mako Takahashi', 'Asamushi Research Center For Marine Biology', 'Aomori']
Date: 2024-01
Blastema formation is a crucial process that provides a cellular source for regenerating tissues and organs. While bilaterians have diversified blastema formation methods, its mechanisms in non-bilaterians remain poorly understood. Cnidarian jellyfish, or medusae, represent early-branching metazoans that exhibit complex morphology and possess defined appendage structures highlighted by tentacles with stinging cells (nematocytes). Here, we investigate the mechanisms of tentacle regeneration, using the hydrozoan jellyfish Cladonema pacificum. We show that proliferative cells accumulate at the tentacle amputation site and form a blastema composed of cells with stem cell morphology. Nucleoside pulse-chase experiments indicate that most repair-specific proliferative cells (RSPCs) in the blastema are distinct from resident stem cells. We further demonstrate that resident stem cells control nematogenesis and tentacle elongation during both homeostasis and regeneration as homeostatic stem cells, while RSPCs preferentially differentiate into epithelial cells in the newly formed tentacle, analogous to lineage-restricted stem/progenitor cells observed in salamander limbs. Taken together, our findings propose a regeneration mechanism that utilizes both resident homeostatic stem cells (RHSCs) and RSPCs, which in conjunction efficiently enable functional appendage regeneration, and provide novel insight into the diversification of blastema formation across animal evolution.
Funding: This work was supported by JSPS/MEXT KAKENHI (grant numbers JP21H05767 to G.K., JP21H05255 to E.K., JP21H04774, JP23H04766 to M.M., and JP17H06332, JP22H02762, JP23H04696 to Y.N.), JST grant (JPMJCR1852 to E.K. and JPMJSP2114 to S.F.), AMED-Aging (JP21gm5010001 to M.M.), and AMED-PRIME (JP22gm6110025 to Y.N.), and NIBB Collaborative Research Program (23NIBB202 to Y.N.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
In this study, we investigate the cellular mechanism of appendage regeneration using the Cladonema medusa tentacle. Establishing the Cladonema tentacle as an organ that can efficiently and functionally regenerate upon amputation, we show that highly proliferative cells accumulate at the injury site within 24 h to behave as blastema. Pulse-chase experiments using nucleoside analogs as well as dye labeling reveal that most blastema cells are not derived from resident stem cells but rather appear locally after damage. We further identify the role of blastema cells as the principal cellular source of the epithelium in the newly formed tentacle, while resident stem cells contribute to nematogenesis and tissue elongation during both homeostasis and regeneration. These results suggest the existence of 2 distinct proliferative cell populations: blastema as RSPCs and resident stem cells as homeostatic stem cells, both of which collectively enable functional tentacle regeneration. In a broader context, our findings highlight the diversification of blastema formation in non-bilaterian systems, providing an evolutionary insight into animal regeneration.
The hydrozoan jellyfish Cladonema pacificum is an emerging jellyfish model that has been utilized to study development and physiology [ 31 – 33 ]. Because Cladonema allows for easy lab maintenance with its high spawning rate, it enables monitoring all life cycle stages and exploring organismal responses to different stimuli. The genus Cladonema is morphologically characterized by branched tentacles at the medusa stage ( Fig 1B ) [ 31 , 34 – 36 ]. The Cladonema medusa tentacle is primarily composed of bilayered epithelial tissues (epidermis and gastrodermis) that include muscle fiber, also called epitheliomuscular cells [ 23 ]: i-cells, neurons, and nematocytes are located in the epidermal layer while neurons and gland cells are located in the gastrodermal layer. Resident stem cells, i-cells, are localized at the basal side of the tentacle (tentacle bulb), and are thought to give rise to progenitors and differentiated cells during normal development and homeostasis [ 24 , 37 ]. The continuous growth and branching potential of the medusa tentacle makes it an attractive model to understand the mechanisms of appendage morphogenesis, growth, and regeneration in cnidarians.
In contrast to the sessile polyp stage, medusae, commonly called jellyfish, exhibit a more complex body structure that includes multiple types of muscle such as smooth and striated muscles, developed neural networks, and distinct organs and appendages, which together enable free swimming and avoidance behaviors [ 22 , 23 ]. While most sexually reproducing medusae do not clonally propagate like polyps, medusae can still regenerate various organs and appendages (e.g., manubrium, tentacle, umbrella, gonad, eye) after injury and reconstitute de novo structures after their removal [ 24 – 30 ]. A recent report using the hydrozoan jellyfish Clytia has suggested that, upon removal of the entire manubrium, i-cells and differentiated cells localized in neighboring organs migrate to the damage site through the canals, contributing to de novo manubrium regeneration [ 27 ]. Despite the importance of proliferating blastema cells in highly regenerative animals, the detailed mechanisms of organ and appendage regeneration in medusae, particularly the mechanism of blastema formation and its specific role are largely unknown.
Among cnidarians, polyp-type animals such as Hydra, Hydractinia, and Nematostella have been utilized as models to understand the mechanism of whole-body regeneration, including patterning, body axis formation, and mechanical responses [ 16 – 18 ]. After mid-gastric bisection in Hydra, cell proliferation of the stem cells, or i-cells, around the wound site is accelerated by Wnt3a produced from dying cells to generate blastema [ 19 ]. Upon decapitation of the colony polyp Hydractinia, i-cells remotely located in the body column migrate to the injury site to form blastema [ 20 ]. During head regeneration of the sea anemone Nematostella, 2 adult stem-like cell populations, fast-cycling cells in the body wall epithelium and slow-cycling cells in the mesenteries, migrate toward the amputation site to form blastema [ 21 ]. These studies suggest that the recruitment of resident stem cells to the injury site is a prerequisite for blastema formation after amputation of the body. However, it remains unclear whether repair-specific proliferative cells (RSPCs) constitute the cellular source of blastema during cnidarian regeneration.
(A) The phylogenetic tree of Bilateria and Cnidaria, composed of Anthozoa and Medusozoa. (B) Medusa of Cladonema pacificum. U: umbrella, M: manubrium, Ra: radial canal, Ri: ring canal, T: tentacle. (C) Tentacle of Cladonema medusa. O: ocellus, TB: tentacle bulb, Br: branch, NC: nematocyte cluster. (D) Tentacle regeneration processes after amputation while retaining the bulb. hpa: hour post-amputation. Yellow arrows indicate nematocyte clusters. n = 234/236 (tentacles). (E) Scheme of the tentacle regeneration that retains the bulb. (F) Distribution of mature nematocytes and localization of muscle fibers during tentacle regeneration. DAPI for poly-γ-glutamate (green) and nuclei (blue), and Phalloidin for F-actin (red). Yellow arrows indicate nematocyte clusters. (G) Neural morphology in the regenerating tentacle stained with the anti-FMRFamide antibody. Yellow arrows indicate neural fibers. (H) The regenerating tentacle is fully functional at 72 hpa. Image of the tentacle capturing prey, brine shrimp (white arrow). (I) The rate of functional tentacles across the regeneration time course. Intact: n = 40 (tentacles), 0–72 hpa: n = 36. (J) Tentacle regeneration process after removing the bulb from canals. Yellow arrow in (iv) indicates a nematocyte cluster. Dpa: day post-amputation. n = 117/219 (tentacles). Ra: radial canal, Ri: ring canal. (K) Scheme of tentacle regeneration without bulb. The numerical values that were used to generate the graphs in ( I ) can be found in S1 Data . Scale bars: (B–D, J) 1 mm, (F, Gi) 100 μm, (Gii) 50 μm.
Cnidarians (corals, sea anemones, hydroids, and jellyfish) are among the earliest branching metazoans, composed of 2 major groups Anthozoa and Medusozoa, forming a diverse phylum that contains over 10,000 species, and stand at a unique phylogenetic position as the sister group to bilaterians ( Fig 1A ). While cnidarians display considerably divergent morphologies and life cycles, represented by the polyp and medusa stages; their common traits are a diploblastic radially symmetric body along with tentacles as the well-defined appendages that bear the stinging cells, nematocytes (cnidocytes) [ 12 , 13 ]. Although regenerative potential may vary among the group, most documented cnidarian species are capable of regenerating lost tissues and organs, and some can even regenerate their entire body [ 14 , 15 ].
Among various regeneration contexts, appendage regeneration is widely observed in bilaterians (e.g., amphibian limbs and fish fins) and is suitable for understanding the evolution of regenerative processes [ 3 , 10 ]. The bilaterian program of blastema formation during appendage regeneration is associated with repair-specific stem/progenitor cells [ 7 , 9 , 11 ]. Yet, to elucidate the evolutionary history of blastema formation programs, their mechanisms must be studied in early-branching metazoans in addition to bilaterian models.
Blastema can be defined as an undifferentiated cellular mass that contains cells with mitotic capacity and appears after damage such as amputation [ 3 , 4 ]. Accumulating evidence has suggested that methods of blastema formation vary among animals with high regenerative abilities [ 5 ]. For example, in planarians, pluripotent stem cells called neoblasts that are distributed throughout the body are recruited to the injury site to produce blastema [ 6 ]. Salamanders can regenerate adult limbs upon amputation via blastema formation, but the underlying cellular mechanisms vary among species: in the axolotl, tissue-specific stem cells contribute to blastema, while in the newt, muscle fibers dedifferentiate into proliferative progenitor cells to behave as blastema [ 7 ]. During zebrafish caudal fin regeneration, both osteoblast-derived dedifferentiated cells and resident progenitor cells migrate to the wound site to form blastema [ 8 , 9 ]. These studies support the idea that the supply of blastema has diversified across the animal kingdom, whose members utilize resident stem/progenitor cells and/or repair-specific de novo proliferative cells to reconstruct lost body parts. While mechanisms of blastema formation have been studied extensively in a limited number of regenerative animals, little is known about their evolutionary characteristics: Which elements are acquired as lineage-specific novelties and which are widely conserved within highly regenerative species? In particular, the current understanding of blastema formation largely relies on bilaterian models, and thus the mechanisms of blastema formation outside of bilaterians remain poorly understood.
Regeneration, the phenomenon of re-forming missing body parts, is widespread among metazoans. Common regenerative processes include wound closure immediately after injury; formation of the cellular source, or blastema, that reconstitutes the lost tissue; and regrowth of the tissue that integrates different cellular behaviors such as proliferation and differentiation [ 1 ]. Among these regenerative responses, blastema formation is a critical step that can distinguish regenerative and non-regenerative systems. Indeed, most mammalian and avian species do not form blastema upon injury while wound closure occurs normally [ 2 ]. Understanding blastema formation mechanisms in highly regenerative animals may therefore help us identify the necessary elements to potentially improve our regenerative abilities.
The tentacle length ( Fig 5K and 6I ) was measured using the segment line tool of ImageJ/Fiji. The tentacle area ( S5C–S5E Fig ) was recorded as the ROI with the polygon selection tool and measured by ImageJ/Fiji. The quantification area was marked with rectangle tool, and the signal intensity was measured by ImageJ/Fiji ( S2D Fig ).
The PH3 + , EdU + , or Nanos1 + cell ratio (Figs 2D–2F , 4I , 4J , S7B , S7C, S7E and S7F ) was measured by counting the total number of cells as well as the PH3 + , EdU + , or Nanos1 + cells using ImageJ/Fiji. Each signal + cell was counted manually using the multipoint tool of ImageJ/Fiji. Total cell number quantification was performed as follows: (1) Binarize the signal of Hoechst staining using the “Threshold” command. (2) Divide continuously adjacent multiple nuclei using the “Watershed” command. (3) Measure the number of nuclei using the “Analyze Particles” command.
Tentacles were amputated at 1 day before UV exposure. At 24 hpa, the medusae were incubated with Hoechst 33342 (1:250) for 20 min and washed 3 times in ASW. After washing, the relaxed medusae were placed on slides with 7% MgCl 2 and enclosed with cover glass. UV laser was exposed to the blastema region of the amputated tentacle using the photo bleaching application (50 cycles at 100% 405 laser power) of the Zeiss LSM 880 confocal microscope. The bleached region is shown with a white dot square (400 × 400 pixels, W × H) in S11B Fig . After UV exposure, the medusae were moved to ASW in plastic containers and monitored to examine the effect on regeneration.
Total RNA was purified from the whole tentacles of 3 medusae with RNeasy Mini kits (Qiagen). Lysate was treated with Dnase I (Qiagen, 79254) for 15 min at RT. cDNA was synthesized from 200 ng of total RNA by Prime script RT master Mix (Takara, RR036A). RT-qPCR was performed using TB Green Premix Ex TaqII (Tli RnaseH Plus) (Takara, RR820L) and a Quantstudio6 Flex Real-Time PCR system (Thermo Fisher) using F-actin capping protein subunit beta (CpCapZbeta) as an internal control [ 32 ]. The qPCR primer sets are as follows: CpCapZbeta: 5′-AAAGAAAGCTGGAGACGGTTCA-3′ (Forward), 5′-GTAGTGGGCATTTCTTCCGC-3′ (Reverse), Nanos1: 5′-TTCGTCAAGTGGCAGTCGTG-3′ (Forward), 5-CAAGCCCTGGTACAAACGGA-3′ (Reverse).
The medusae were placed in a V7 plastic container with 6 to 8 ml of ASW, the minimum required for their maintenance. X-ray irradiations were performed using an X-ray machine (mediXtec, MX-160Labo). Medusae were positioned 19 cm from the X-ray source, and a dose of 30 Gy (19.3 min at 160 kV 3 mA), 50 Gy (32.2 min at 160 kV 3 mA), or 75 Gy (48.3 min at 160 kV 3 mA) was delivered. After irradiation, the medusae were moved to a plastic container with fresh ASW.
Medusae were relaxed in 7% MgCl 2 on a petri dish, and 10 mM CellTracker CM-DiI (Invitrogen, C7001) was injected into the epidermal layer of the tentacle bulb or the center of the tentacle, using a micro injector (Eppendorf, Femtojet 4i). The quartz capillary (Sutter Instrument, QF100-70-10) was pulled by Laser-Based Micropipette Puller P2000 (Sutter Instrument). After injection, injected tentacles were amputated. To identify DiI-labeled cells, medusae were incubated with 150 μm EdU for 1 h before DiI injection or antibody staining was performed after DiI injection using anti-β-catenin. To monitor DiI-labeled cells during blastema formation, at 24 hpa, the injected medusae were incubated with 150 μm EdU in ASW for 1 h. The medusae were relaxed in 7% MgCl 2 and fixed in 4% PFA. After washing in 0.1% PBT, the medusae were incubated with an EdU reaction cocktail for 30 min in the dark. After the EdU reaction, the samples were washed and incubated with Hoechst 33342 in 0.1% PBT.
For double FISH, DIG for low expression genes and FITC probes for high expression genes were used ( S3E Fig ; Nanos1-DIG and Nanos2-FITC, S3F Fig ; Mcol1-DIG and Nanos2-FITC). The samples were then washed with Tris-NaCl-Tween Buffer after incubation with anti-DIG-POD antibodies and incubated with Cy3-tyramide solution (TSA Plus Cyanine 3; AKOYA Biosciences, NEL744001KT) for 10 min. The samples were washed with 0.1% PBST and incubated with 3% H 2 O 2 for 15 min. The samples were washed with 0.1% PBST and incubated with anti-Fluorescein-POD antibodies (1:500; Roche, 11426346910) in 1% blocking buffer overnight at 4°C. The samples were then washed with Tris-NaCl-Tween Buffer and incubated with Cy5-tyramide solution for 10 min. Finally, the samples were washed with 0.1% PBST and incubated with Hoechst 33342 in 0.1% PBST for 30 min in the dark and washed 3 times.
Medusae were anesthetized 7% MgCl 2 for 5 min and fixed overnight at 4°C with 4% PFA in ASW. Briefly, fixed samples were washed 3 times with PBS containing 0.1% Tween-20 (PBST), followed by pre-hybridization in hybridization buffer (HB buffer: 5 × SSC, 50% formamide, 0.1% Tween-20, 50 μg/ml tRNA, 50 μg/ml heparin) at 55°C for 2 h. The samples were hybridized with HB buffer containing the antisense probes (final probe concentration: 0.5–1 ng/μl in HB buffer) at 55°C overnight. The samples were washed twice each with wash buffer 1 (5 × SSC, 50% formamide, 0.1% Tween-20), wash buffer 2 (2 × SSC, 50% formamide, 0.1% Tween-20), and 2 × SSC. The samples were then washed with 0.1% PBST and incubated in 1% blocking buffer (1% blocking reagent [Roche] in Maleic acid) for 1 h. The samples were incubated with anti-DIG-POD antibodies (1:500; Roche, 11207733910) in 1% blocking buffer overnight at 4°C. The samples were then washed with Tris-NaCl-Tween Buffer and incubated with Cy5-tyramide solution (TSA Plus Cyanine 5; AKOYA Biosciences, NEL745001KT) for 10 min. Finally, the samples were washed with 0.1% PBST and incubated with Hoechst 33342 in 0.1% PBST for 30 min in the dark and washed 3 times.
The following FISH protocol was created from the published protocol [ 38 ]. The sequences of Nanos1, Nanos2, and Piwi have been previously described [ 39 ], the presumptive sequence of Minicollagen1 (Mcol1) and Vasa1 were acquired from the annotation of an RNA-seq result [ 33 ]. Purified total RNA was reverse transcribed into cDNA by PrimeScript II 1st strand cDNA Synthesis Kit (Takara, 6210A) or SMARTer RACE cDNA Amplification Kit (Clontech). A cDNA library was used as a template for PCR (Nanos1, Nanos2, Piwi, and Mcol1) and 3′ RACE (Vasa1). The primer sets used for PCR cloning are as follows: Nanos1: 5′-AAGAGACACAGTCATTATCAAGCGA-3′ (forward) and 5′-AGCACGTAAAATTGGACACGTCG-3′ (reverse), Nanos2: 5′- ACTTCTCCAAAACCTCATGCCGAG-3′ (forward) and 5′- GAATGGCGGGCGATTTGACATCC-3′ (reverse), Piwi: 5′- CACACAAGAGTTGGACCGGA-3′ (forward) and 5′- ACCGGCTTATCGATGCAACA-3′ (reverse), Vasa1: 5′-GCCACCCAAAGAAGACAGACAGACAC-3′ (forward for 3′RACE) and 5′-CGAAACGACTTGCTGATTTTCTCGCCAG -3′ (nested forward for 3′RACE), Mcol1: 5′-CTCGTCGGTATTGCCCTCTC-3′ (forward) and 5′-CCAACCTATCGTGGACGTGT-3′ (reverse). PCR products were subcloned into the TAK101 vector (TOYOBO) and RACE product was subcloned into the pGEM-T Easy vector (Promega). The resulting plasmids were used for RNA probe synthesis with digoxigenin (DIG) labeling mix (Roche, 11277073910) or fluorescein (FITC) labeling mix (Roche, 11685619910), and T7 (Roche, 10881767001) or T3 RNA polymerase (Roche, 11031163001) was used, according to the insert direction.
The medusae were anesthetized with 7% MgCl 2 for 5 min and fixed with 4% PFA in ASW. The samples were washed 3 times with 0.1% PBT and incubated with an EdU reaction cocktail for 30 min in dark. The samples were washed 3 times and treated with 2N HCl for 30 min and then washed 3 times and incubated in primary antibodies in 0.1% PBT overnight at 4°C. The primary antibody used was mouse anti-BrdU (1:100; BD, 347580). The samples were washed 3 times and incubated with secondary antibodies (1:500; ALEXA FLUOR 555 Goat anti-mouse IgG) and Hoechst 33342 in 0.1% PBT for 1 h in the dark and washed 3 times.
The medusae were incubated with 150 μm EdU in ASW for 1 h or 24 h before amputation, depending on the experiments. Just after incorporation of EdU, the medusae were washed in ASW 3 times, and tentacles were amputated. At 24 hpa, the medusae were incubated with 2.5 mM BrdU (abcam, ab142567) in ASW for 1 h. The medusae were washed out in ASW 3 times.
The medusae were incubated with 150 μm 5-ethynyl-2′-deoxyuridine (EdU) (EdU kit; Invitrogen, C10337) in ASW for 1 h or 24 h. In chase labeling, the medusae were washed with ASW at least twice (up to 3 times, depending on amount). In pulse labeling, after EdU treatment, the medusae were anesthetized with 7% MgCl 2 in deionized water and fixed 4% PFA in ASW. After fixation, the samples were washed and incubated with an EdU reaction cocktail (1× reaction buffer, CuSO 4 , Alexa Fluor azide 488 or 647, and 1× reaction buffer additive; all included in EdU kit; Invitrogen, C10337 or C10340) for 30 min in the dark. After the EdU reaction, the samples were washed and incubated with Hoechst 33342 in 0.1% PBT for 30 min.
The medusae were anesthetized with 7% MgCl 2 in deionized water for 5 min and fixed at room temperature (RT) for 1 h or overnight at 4°C with 4% paraformaldehyde (PFA) in ASW. After fixation, the samples were washed 3 times (10 min each) with PBS containing 0.1% Triton X-100 (0.1% PBT). The samples were incubated in primary antibodies in 0.1% PBT overnight at 4°C. The antibodies used were mouse anti-acetylated tubulin (1:500; Sigma-Aldrich, T6793), rabbit anti-FMRFamide (1:1,000; ImmunoStar, 20091), rabbit anti-Phospho-Histone H3 (Ser10) (1:500; Upstate, 06–570), mouse anti-β-catenin (8E4) (1:100; Enzo, ALX-804-260-C100), and rabbit anti-PKC ζ C20 (1:100; Santa Cruz, sc-216). After primary antibody incubation, the samples were washed 3 times (10 min each) with 0.1% PBT. The samples were incubated with secondary antibodies (1:500; ALEXA FLUOR 488, 555, 647 Donkey or Goat anti-mouse IgG, ALEXA FLUOR 488, 555, 647 Donkey or Goat anti-rabbit IgG; Thermo Fisher Scientific) and Hoechst 33342 (1:500; Thermo Fisher Scientific, H1399) in 0.1% PBT for 1 h in the dark. After 3 washes (10 min each) in 0.1% PBT, the samples were mounted on slides with 70% glycerol. Nuclear and poly-γ-glutamate were stained with DAPI (1:250; Invitrogen, D1306), and actin fibers were stained with Alexa 546 phalloidin (1:400; Invitrogen, A22283) in 0.1% PBT for 1 h. Confocal images were collected through a Zeiss LSM 880 confocal microscope. Image processing (color display) and quantification were performed using ImageJ/Fiji software.
Before surgical manipulation, the medusae were anesthetized with 7% MgCl 2 in deionized water for 2 min in a petri dish (Falcon, 351008). Surgical manipulations were performed with micro scissors (Teraoka, NY33408). After manipulations, the medusae were quickly returned to ASW. When the tentacle was amputated with the bulb left intact, the medusae were fed 1 day before manipulation and were starved after amputation. When the tentacle was amputated along with the bulb, the medusae were fed 1 day before manipulation and were fed 3 times a week afterward.
Cladonema pacificum (female strain 6W) medusae were used for this research. The medusae were maintained in plastic containers (V-type container, V-7, V-8, and V-9; AS ONE) at 22°C in artificial sea water (ASW). ASW was prepared using SEA LIFE (Marin Tech) dissolved in tap water with chlorine neutralizer (Coroline off; GEX Co.) (24 p.p.t). The medusae were fed Vietnamese brine shrimp (A&A Marine LLC).
Results
For cnidarians, tentacles are essential and common appendages for capturing prey and defending against predators. Cnidarian polyps such as Hydra, Hydractinia, and Nematostella have tentacles in the head region, near the hypostome, which can restore functional tentacles during head regeneration [18,20,40]. Similarly, studies using hydrozoan jellyfish have shown that medusae tentacles can regenerate upon amputation [24,27]. Although cnidarians commonly exhibit high regenerative capacity for tentacles, the exact cellular processes and the limitation of regeneration are poorly understood.
To monitor the process of tentacle regeneration and to explore its responses to different dissections, we utilized the medusa tentacle of the hydrozoan jellyfish Cladonema (Fig 1). In Cladonema tentacles, proliferative cells including stem cells, or i-cells, are localized on the basal side, called the tentacle bulb, while nematocytes are distributed as clusters on the distal side, similar to the jellyfish Clytia (Figs 1C, 2Ai, 3Ci, S1A, S3A–S3D, S4Ai, and S4Bi) [24,37,39]. The existence of localized stem cells at the bulb prompted us to examine tentacle regeneration as a model for appendage regeneration in non-bilaterians.
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