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A conserved strategy for inducing appendage regeneration
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Michael J. Abrams
1
‡
†
, Fayth Tan
1
†
, Ty Basinger
1
, Martin Heithe
1
, Yutian Li
1
,
2
Misha Raffiee
1
‡, Patrick Leahy
1
, John O. Dabiri
2
, David A. Gold
1
‡, Lea Goentoro
1
*
3
1
Division of Biology and
Biological Engineering, California Institute of Technology, Pasadena, CA 91125
4
2
Graduate Aerospace Laboratories and Mechanical Engineering, California Institute of Technology,
5
Pasadena, CA 91125
6
†
These authors contributed equally to the work.
7
‡
Current
addresses: Department of Molecular and Cell Biology, University of California at Berkeley
8
(M.J.A.), Department of Bioengineering, Stanford University (M.R.), Department of Biology and Allied
9
Health Sciences, Bloomsburg University (T.B.), Department of Ear
th and Planetary Sciences, University
10
of California at Davis (D.A.G)
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*
Correspondence: goentoro@caltech.edu
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A
lternative title:
Amino acid and sugar supplement induces appendage regeneration in cnidarian,
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insect, and mammal
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Key words:
Regeneration, appenda
ge, limb, amino acid, leucine, sugar, insulin, jellyfish,
15
Drosophila
, mouse
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Abstract
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Can limb regeneration be induced? Few have pursued this question, and an evolutionarily
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conserved strategy has yet to emerge. This study reports a strategy for inducing regenerative
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response in appendages, which works across three species that span the ani
mal phylogeny. In
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Cnidaria, the frequency of appendage regeneration in the moon jellyfish
Aurelia
was increased by
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feeding with the amino acid L
-
leucine and the growth hormone insulin. In insects, the same
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strategy induced tibia regeneration in adult
Droso
phila
. Finally, in mammals, L
-
leucine and
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sucrose administration induced digit regeneration in adult mice, including dramatically from mid
-
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phalangeal amputation. The conserved effect of L
-
leucine and insulin/sugar suggests a key role for
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energetic paramete
rs in regeneration induction. The simplicity by which nutrient supplementation
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can induce appendage regeneration provides a testable hypothesis across animals.
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Introduction
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In contrast to humans’ poor ability to regenerate, the animal world is filled with
seemingly
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Homeric tales: a creature that regrows when halved or a whole animal growing from a small body
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piece. Two views have historically prevailed as to why some animals regenerate better than others
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(Goss, 1992; Polezhaev, 1972; Morgan, 1901). Some bio
logists, including Charles Darwin and
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August Weismann, hold that regeneration is an adaptive property of a specific organ. For instance,
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some lobsters may evolve the ability to regenerate claws because they often lose them in fights
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and food foraging. Othe
r biologists, including Thomas Morgan, hold that regeneration is not an
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evolved trait of a particular organ, but inherent in all organisms. Regeneration evolving for a
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particular organ versus regeneration being organismally inherent is an important distinc
tion, as the
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latter suggests that the lack of regeneration is not due to the trait never having evolved, but rather
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due to inactivation
–
and may therefore be induced. In support of Morgan’s view, studies in past
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decades have converged on one striking insi
ght: many animal phyla have at least one or more
40
.
CC-BY-NC-ND 4.0 International license
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted November 22, 2020.
;
https://doi.org/10.1101/2020.11.21.392720
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2
species that regenerate body parts (Sanchez
-
Alvarado, 2000; Bely and Nyberg, 2010). Further,
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even in poorly regenerative lineages, many embryonic and larval stages can regenerate. In fish,
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conserved regenera
tion
-
responsive enhancers were recently identified, which are also modified in
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mice (Wang et al., 2020). These findings begin to build the case that, rather than many instances
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of convergence, the ability to regenerate is ancestral (Sanchez
-
Alvarado, 2000;
Bely and Nyberg,
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2010). Regeneration being ancestral begs the question: is there a conserved mechanism to activate
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regenerative state?
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This study explored how, and whether, limbs can be made to regenerate in animals that do
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not normally show limb regenera
tion. In frogs, studies from the early 20
th
century and few recent
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ones have induced various degrees of outgrowth in the limb using strategies including repeated
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trauma, electrical stimulation, local progesterone delivery, progenitor cell implantation, and
Wnt
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activation (Carlson, 2007; Lin et al., 2013; Kawakami et al., 2006).
Wnt
activation restored limb
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development in chick embryos (Kawakami et al., 2006), but there are no reports of postnatal
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regeneration induction. In salamanders, a wound site that nor
mally just heals can be induced to
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grow a limb by supplying nerve connection and skin graft from the contralateral limb (Endo et al.,
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2004), or by delivery of Fgf2, 8, and Bmp2 to the wound site followed by retinoic acid (Viera et
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al., 2019).
In mouse digi
ts, a model for exploring limb regeneration in mammals, bone outgrowth
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or joint
-
like structure can be induced via local implantation of Bmp2 or 9 (Yu et al., 2019).
Thus
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far, different strategies gain tractions in different species, and a common denominato
r appears
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elusive.
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However, across animal phylogeny, some physiological features show interesting correlation
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with regenerative ability (Hariharan et al., 2015; Vivien et al., 2016; Sousounis et al., 2014).
First,
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regeneration tends to decrease with age,
with juveniles and larvae more likely to regenerate than
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adults. For instance, the mammalian heart rapidly loses the ability to regenerate after birth and
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anurans cease to regenerate limbs upon metamorphosis. Second, animals that continue to grow
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througho
ut life tend to also regenerate. For instance, most annelids continue adding body segments
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and regenerate well, a striking exception of which is leeches that make exactly 32 segments and
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one of the few annelids that do not regenerate body segments. Consist
ent with the notion of
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regeneration as ancestral, indeterminate growth is thought of as the ancestral state (Hariharan et
69
al., 2015). Finally, a broad correlate of regenerative ability across animal phylogeny is thermal
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regulation. Poikilotherms, which inc
lude most invertebrates, fish, reptiles and amphibians, tend to
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have greater regenerative abilities than homeotherms
—
birds and mammals are animal lineages
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with poorest regeneration. These physiological correlates, taken together, are united by the notion
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of energy expenditure. The transition from juvenile to adult is a period of intense energy usage,
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continued growth is generally underlined by sustained anabolic processes, and regulating body
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temperature is energetically expensive compared to allowing for
fluctuation. Regeneration itself
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entails activation of anabolic processes to rebuild lost tissues (Hirose et al., 2014; Naviaux et al.,
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2009; Malandraki
-
Miller et al., 2018). These physiological correlates thus raise the notion of a key
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role of energetics
in the evolution of regeneration in animals. Specifically, we wondered whether
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energy inputs can promote regenerative state. In this study, we demonstrate that nutrient
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supplementation can induce regenerative response in appendage and limb across three va
stly
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divergent species.
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Results
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Leucine and insulin promote appendage regeneration in the moon jelly
Aurelia
84
.
CC-BY-NC-ND 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted November 22, 2020.
;
https://doi.org/10.1101/2020.11.21.392720
doi:
bioRxiv preprint