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

Rinkevich Y, Paz G, Rinkevich B, Reshef R - PLoS Biol. (2007)

Bottom Line: We found that retinoic acid (RA) regulates diverse developmental aspects in WBR.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.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.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Biology, Technion-Israel Institute of Technology, Haifa, Israel.

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.

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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.
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pbio-0050071-g003: 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.

Mentions: A homologue of RAR (Bl-RAR) was cloned from the cDNA of regenerating blood vessels. An 808-bp fragment was sequenced, revealing 87% identity to RAR of the budding tunicate Polyandrocarpa misakiensis. A domain search revealed a conserved 157-amino acid fragment, corresponding to the ligand-binding domain of hormone receptors present in all RARs [34], (Figure 3A, red line). Furthermore, Bl-RAR is phylogenetically clustered with other urochordate RAR family members in a general glade of other vertebrate RARs and is significantly distinguished from other RA receptor families (Figure 3B).


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

Rinkevich Y, Paz G, Rinkevich B, Reshef R - PLoS Biol. (2007)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1808485&req=5

pbio-0050071-g003: 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.
Mentions: A homologue of RAR (Bl-RAR) was cloned from the cDNA of regenerating blood vessels. An 808-bp fragment was sequenced, revealing 87% identity to RAR of the budding tunicate Polyandrocarpa misakiensis. A domain search revealed a conserved 157-amino acid fragment, corresponding to the ligand-binding domain of hormone receptors present in all RARs [34], (Figure 3A, red line). Furthermore, Bl-RAR is phylogenetically clustered with other urochordate RAR family members in a general glade of other vertebrate RARs and is significantly distinguished from other RA receptor families (Figure 3B).

Bottom Line: We found that retinoic acid (RA) regulates diverse developmental aspects in WBR.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.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.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Biology, Technion-Israel Institute of Technology, Haifa, Israel.

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.

Show MeSH
Related in: MedlinePlus