PNAS
2024 Vol. 121 No. 11 e2314911121
https://doi.org/10.1073/pnas.2314911121
1 of 3
BRIEF REPORT
|
Author affiliations:
a
Division of Biology and Biological
Engineering, California Institute of Technology, Pasadena,
CA 91125;
b
Emerging Model Organisms Facility, Trans
-
Scale Biology Center, National Institute for Basic Biology,
Okazaki 444
-
8585, Japan;
c
Division of Liberal Arts and
Sciences, Aichi Gakuin University, Nisshin 470
-
0195,
Japan; and
d
Laboratory of Regeneration Biology, National
Institute for Basic Biology, Okazaki 444
-
8585, Japan
Author contributions: M.S., M.E.B., and K.T.S. designed
research; M.S., A.O., A.C., Y.S., M.T., and K.T.S. performed
research; M.S., A.O., A.C., M.T., and K.T.S. contributed new
reagents/analytic tools; M.S., A.O., A.C., M.T., and K.T.S.
analyzed data; T.E., K.A., and M.E.B. supervision; K.T.S.
project administration; and M.S., A.O., A.C., Y.S., T.E., M.T.,
K.A., M.E.B., and K.T.S. wrote the paper.
The authors declare no competing interest.
Copyright © 2024 the Author(s). Published by PNAS.
This open access article is distributed under
Creative
Commons Attribution
-
NonCommercial
-
NoDerivatives
License 4.0 (CC BY
-
NC
-
ND)
.
1
To whom correspondence may be addressed. Email:
mbronner@caltech.edu or suzuk107@nibb.ac.jp.
This article contains supporting information online at
https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.
2314911121/-
/DCSupplemental
.
Published March 5, 2024.
DEVELOPMENTAL BIOLOGY
Fgf10
mutant newts regenerate normal hindlimbs despite severe
developmental defects
Miyuki Suzuki
a
, Akinori Okumura
b
, Akane Chihara
b
, Yuki Shibata
b
, Tetsuya Endo
c
, Machiko Teramoto
d
, Kiyokazu Agata
d
,
Marianne E. Bronner
a,1
, and Ken-
ichi T. Suzuki
b,1
Edited by Brigid Hogan, Duke University, Durham, NC; received August 29, 2023; accepted February 2, 2024
In amniote limbs, Fibroblast Growth Factor 10 (FGF10) is essential for limb develop-
ment, but whether this function is broadly conserved in tetrapods and/or involved in
adult limb regeneration remains unknown. To tackle this question, we established
Fgf10
mutant lines in the newt
Pleurodeles waltl
which has amazing regenerative ability. While
Fgf10
mutant forelimbs develop normally, the hindlimbs fail to develop and downreg-
ulate FGF target genes. Despite these developmental defects,
Fgf10
mutants were able
to regenerate normal hindlimbs rather than recapitulating the embryonic phenotype.
Together, our results demonstrate an important role for FGF10 in hindlimb formation,
but little or no function in regeneration, suggesting that different mechanisms operate
during limb regeneration versus development.
regeneration | development | limb | FGF10
Results and Discussion
Fibroblast Growth Factor 10 (FGF10) is well known to be essential for amniote limb
development (1, 2).
Fgf10
transcripts are expressed throughout the limb mesenchyme,
where FGF10 stimulates
Fgf8
expression in the apical ectodermal ridge (AER) (3).
Fgf10
knockout in mice is lethal and leads to severe truncation of both fore
-
and hindlimbs. In
axolotl,
Fgf8
is expressed in the limb mesenchyme rather than the epidermis (4–7), sug-
gesting that FGFs may work differently in urodele amphibians. In contrast to amniotes,
urodeles can regenerate their limbs throughout adulthood. While it has been suggested
that adult limb regeneration may recapitulate development, it is unclear whether or not
FGFs function in similar ways during development and regeneration.
To address the role of FGF10 in limb development and regeneration, we tested its
function in the newt
Pleurodeles waltl
. First, we examined the expression patterns of
Fgf8
and
Fgf10
using hybridization chain reaction (HCR) in limb buds at the early limb bud
stage and blastemas at the late bud stage. Transcripts of both were predominantly expressed
in the mesenchyme (Fig.
1
A
and
B
) (5–7), although at apparently lower levels in the
blastema than the limb bud. Bulk RNA
-
seq confirmed that
Fgf8
and
Fgf10
were expressed
in both fore
-
and hindlimbs (Fig. 1
C
).
Next, we performed
Fgf10
knockouts using CRISPR
-
Cas9. In contrast to mouse knock-
outs, the forelimb of
Fgf10
crispants developed normally; however, the hindlimb was defec-
tive with digit number reduced to one or two (Fig.
1
D
). This phenotype was reproduced
with multiple gRNAs (Fig.
1
D
), with a somatic mutation rate of more than 93% in both
fore
-
and hindlimbs (Fig.
1
E
), inferring that differences in phenotype between them were
not due to mosaicism. Furthermore, we generated
Fgf10
F2 mutants with a 2
-
bp deletion
(Fig. 1
F
and
G
). Importantly,
Fgf10
mutants exhibited either the same phenotype as crispants
(longitudinal defect, fewer digits, and absence/hypoplasia of zeugopods, Fig.
1
F
) or even
more severe phenotypes, completely missing the zeugopod and autopod (terminal transverse
defect). Even with severe hindlimb defects, forelimbs developed normally. Possible explana-
tions for forelimb versus hindlimb differences include different FGF signaling pathways and/
or functional redundancy, as previously suggested for other developmental processes (8, 9).
To investigate downstream genes, hindlimb buds from
Fgf10
mutants were individ-
ually collected and processed by RNA
-
seq.
Fgf8
(Fig.
1
H
), other FGF pathway genes,
and
Sall1
(Fig.
1
I
) were significantly downregulated in
Fgf10
mutants
. Sall1
is expressed
in distal mesenchymal cells of the limb bud in
Xenopus
(10), mouse (11), and the
regenerating hindlimb in
Xenopus
(12).
Sall1/Sall3
have redundant activity, and the
double
-
mutant mouse exhibits loss of digits in the autopod (13). In addition,
Hoxa/
d11
and
Runx1
were significantly decreased (Fig.
1
I
). Deletion of
Hoxa/d11
results in
zeugopod malformations (14), whereas the transcription factor
Runx1
is essential for
osteogenesis (15). F2 mutants had fewer digits and lacked fibula or tibia formation,
contrasting with
Fgf10
heterozygotes with proper osteogenesis (Fig.
1
J
). Based on these
OPEN ACCESS
Downloaded from https://www.pnas.org by George Porter on March 6, 2024 from IP address 131.215.225.170.
2 of 3
https://doi.org/10.1073/pnas.2314911121
pnas.org
results, we suggest that
Fgf10
plays two roles: first in autopod
patterning by coordinating FGF pathway and
Sall
genes and
second in zeugopod formation through
Hox11
and
Runx
genes.
Given that limb development and regeneration share many com
-
mon properties, we next examined the role of FGF10 in regenera-
tion. To this end, we amputated either the forelimb and hindlimb
of
Fgf10
crispants or the hindlimb of F2 mutants. As expected, the
forelimbs of
Fgf10
crispants regenerated normally. Surprisingly, the
hindlimbs of
Fgf10
crispants and mutants regenerated normal or
near
-
normal structures after amputation rather than recapitulating
their developmental morphology (Fig.
2). Whereas mutants devel-
oped severely defective hindlimbs, 61% (n
= 11/18) of mutants
regenerated completely normal zeugopods and autopods after ampu
-
tation at the stylopod (Fig.
2
A
and
B
). Overall, regeneration led to
an increase in the number of digits in 72% (n = 23/32) of mutants
(Fig. 2
C
). A higher regeneration score was obtained by stylopod
amputation, a more proximal site, suggesting that stronger interca-
lation evokes regeneration (16). The regenerated limb of
Fgf10
mutants ossified the fibula and tibia normally (Fig.
2
D
), suggesting
that the regenerated hindlimb is comparable to the wild type.
To examine downstream gene expression, we performed RNA
-
seq
in hindlimb blastemas of
Fgf10
mutants. The expression levels of
Fgf10
-
regulated genes were comparable between the wild type and
mutant, suggesting that downstream gene expression was restored
(Fig. 2
E
). Other limb development
-
related genes including FGF sign-
aling pathway genes were also similar between the wild type and mutant
(Fig. 2
F
). Thus, developing and regenerating urodele amphibian limbs
utilize differential
Fgf8
induction mechanisms. Reciprocal and feedback
loop regulation between
Fgf10
expressed in the mesenchyme and
Fgf8
expressed in the AER are essential for amniote limb development (1, 2).
However, both of these
Fgfs
are expressed in the mesenchyme of urodeles
(4–7), highlighting potential developmental differences between urodele
amphibians and amniotes.
One important difference between development and regenera-
tion is the presence of nerves. Nerve
-
derived factors are known to
play a critical role in regeneration (17, 18). These findings together
with our results raise the possibility that direct induction of
Fgf8
by regeneration cues including nerve
-
derived factor(s) rather than
FGF10 may be key to limb regeneration in urodeles (Fig. 2
G
).
Our findings highlight both similarities and differences between
limb development and regeneration. Bryant and colleagues pro-
posed that the early phase of limb regeneration may involve
regeneration
-
specific steps. Subsequently, there is a gradual tran-
sition to an autonomous limb growth and patterning program
similar to that in development (19). We find that most of the
downstream genes required for limb development do not differ
in the blastema of
Fgf10
mutants. Accordingly, it is possible that
a limited number of genes, including
Fgf10
, have differential
effects at early stages of regeneration, whereas later events are par-
allel to development (Fig.
2
G
). Importantly, even though the
hindlimb of
Fgf10
mutants display marked abnormalities, our
results show that the hindlimb has “developmental plasticity” and
is able to regenerate a normal limb from a developmentally defec-
tive one.
Fig. 1.
Fgf10
mutants have defects only in the hindlimb. (
A
) In situ HCR of
Fgf8
and
Fgf10
in the forelimb (FL, st. 34) and hindlimb (HL, st. 40) bud. (
B
) In situ HCR of
P63
,
Fgf8,
and
Fgf10
in the HL blastema at the late bud stage.
P63
is expressed in the epidermis. The dashed line shows the epithelial and mesenchymal boundary. (
C
)
Fgf8
and
Fgf10
expression level in the FL (st. 33 to 34) and HL (st. 39 to 40). (
D
) Confirmation of phenocopy of
Fgf10
crispants using different gRNAs. (
E
) The genotype of the
FL and HL of
Fgf10
crispant (gRNA3). Mutant alleles and their occupancy rates are shown corresponding to the FL and HL of each crispant (#1
-
3). (
F
) The phenotype
of
Fgf10
mutant. The arrow and arrowhead show a normally developed FL and a defective HL, respectively. Note that the HLs are not missing above the knee, but
one digit is present and bends ventrally. The dashed line indicates the intact digit. (
G
) Sanger sequence of the wild type and
Fgf10
mutant. (
H
)
Fgf8
expression in
the HL bud of
Fgf10
mutants. (
I
) Volcano plot for differential gene expression of
Fgf10
mutant versus wild type in the HL bud. (
J
) MicroCT image of the HL of the
adult
Fgf10
mutant. (
a
) Control,
Fgf10
F1 hetero. (
b
)
Fgf10
F2 mutant. The bracket, arrowhead, and asterisk show the stylopod, fibula/tibia, and digits, respectively.
Downloaded from https://www.pnas.org by George Porter on March 6, 2024 from IP address 131.215.225.170.
PNAS
2024 Vol. 121 No. 11 e2314911121
https://doi.org/10.1073/pnas.2314911121
3 of 3
Materials and Methods
P. waltl
were obtained from a breeding colony. Experimental details are provided
in
SI Appendix
.
Data, Materials, and Software Availability.
Sequencing data have been
deposited in the NCBI BioProject (
PRJDB15083
) (20). All other data are included
in the manuscript and/or
SI Appendix
.
ACKNOWLEDGMENTS.
We thank Profs. Toshinori Hayashi and Takashi Yamamoto
in Hiroshima University and Takashi Takeuchi in Tottori University for providing
newt and their great support. We also thank Profs. Hideyo Ohuchi in Okayama
University and Yasuhiro Kamei in National Institute for Basic Biology for their
advice and help. This work was supported by Japan Science and Technology
Agency (JST), Core Research for Evolutionary Science and Technology (CREST),
(JPMJCR2025 to K.T.S.), Japan Society for the Promotion of Science (JSPS),
KAKENHI Grant
-
in
-
Aid for Scientific Research (B) (JP21H03829 to K.T.S.), Grant
-
in
-
Aid for JSPS Fellows (17J04796 to M.S.), Overseas Research Fellowships,
Human Frontier Science Program Organization (HFSPO), Human Frontier Science
Program (HFSP) Long Term Fellowship (LT0009/2022
-
L to M.S.), and NIH R35
(NS111564 to M.E.B.).
1.
K. Sekine
et al.
, Fgf10 is essential for limb and lung formation.
Nat. Genet.
21
, 138–141 (1999).
2.
H. Ohuchi
et al.
, The mesenchymal factor, FGF10, initiates and maintains the outgrowth of the
chick limb bud through interaction with FGF8, an apical ectodermal factor.
Development
124
,
2235–2244 (1997).
3.
H. Ohuchi
et al.
, Involvement of androgen
-
induced growth factor (FGF
-
8) gene in mouse
embryogenesis and morphogenesis.
Biochem. Biophys. Res. Commun.
204
, 882–888 (1994).
4.
M. J. Han, J. Y. An, W. S. Kim, Expression patterns of Fgf
-
8 during development and limb
regeneration of the axolotl.
Dev. Dyn.
220
, 40–48 (2001).
5.
G. L. Glotzer, P. Tardivo, E. M. Tanaka, Canonical Wnt signaling and the regulation of divergent
mesenchymal Fgf8 expression in axolotl limb development and regeneration.
Elife
11
, e79762 (2022).
6.
A. M. Lovely
et al.
, Wnt signaling coordinates the expression of limb patterning genes during Axolotl
forelimb development and regeneration.
Front. Cell Dev. Biol.
10
, 814250 (2022).
7.
S. Purushothaman, A. Elewa, A. W. Seifert, Fgf
-
signaling is compartmentalized within the mesenchyme
and controls proliferation during salamander limb development.
Elife
8
, e48507 (2019).
8.
I. H. Hung, G. C. Schoenwolf, M. Lewandoski, D. M. Ornitz, A combined series of Fgf9 and Fgf18 mutant
alleles identifies unique and redundant roles in skeletal development.
Dev. Biol.
411
, 72–84 (2016).
9.
L. D. Urness, S. B. Bleyl, T. J. Wright, A. M. Moon, S. L. Mansour, Redundant and dosage sensitive
requirements for Fgf3 and Fgf10 in cardiovascular development.
Dev. Biol.
356
, 383–397 (2011).
10.
D. T. Hudson
et al.
, Gene expression analysis of the Xenopus laevis early limb bud proximodistal axis.
Dev. Dyn.
251
, 1880–1896 (2022).
11.
A. Buck, A. Kispert, J. Kohlhase, Embryonic expression of the murine homologue of SALL1, the gene
mutated in Townes
-
Brocks syndrome.
Mech. Dev.
104
, 143–146 (2001).
12.
A. W. Neff, M. W. King, A. L. Mescher, Dedifferentiation and the role of sall4 in reprogramming and
patterning during amphibian limb regeneration.
Dev. Dyn.
240
, 979–989 (2011).
13.
Y. Kawakami
et al.
, Sall genes regulate region
-
specific morphogenesis in the mouse limb by
modulating Hox activities.
Development
136
, 585–594 (2009).
14.
D. M. Wellik, M. R. Capecchi, Hox10 and Hox11 genes are required to globally pattern the
mammalian skeleton.
Science
301
, 363–367 (2003).
15.
J. Tang
et al.
, Runt
-
related transcription factor 1 is required for murine osteoblast differentiation and
bone formation.
J. Biol. Chem.
295
, 11669–11681 (2020).
16.
V. French, P. J. Bryant, S. V. Bryant, Pattern regulation in epimorphic fields.
Science
193
, 969–981 (1976).
17.
A. Makanae, K. Mitogawa, A. Satoh, Co
-
operative Bmp
-
and Fgf
-
signaling inputs convert skin
wound healing to limb formation in urodele amphibians.
Dev. Biol.
396
, 57–66 (2014).
18.
E. Nacu, E. Gromberg, C. R. Oliveira, D. Drechsel, E. M. Tanaka, FGF8 and SHH substitute for
anterior
-
posterior tissue interactions to induce limb regeneration.
Nature
533
, 407–410 (2016).
19.
S. V. Bryant, T. Endo, D. M. Gardiner, Vertebrate limb regeneration and the origin of limb stem cells.
Int. J. Dev. Biol.
46
, 887–896 (2002).
20.
M. Suzuki, K. T. Suzuki, Data from “RNA
-
seq analysis of Pleurodeles waltl limb bud and limb
blastema”. NCBI BioProject. https://www.ncbi.nlm.nih.gov/bioproject/927151. Deposited
24 January 2023.
Fig. 2.
The defective hindlimb in
Fgf10
mutants was restored to nor-
mal by regeneration. (
A
) HL regen-
eration of
Fgf10
mutants amputated
at the stylopod (#1) or zeugopod
(#2). (
B
) Recovered HL defect by
regeneration (
Left
) and unamputat-
ed side (
Right
) of #1. (
C
) Summary
of restored HL in
Fgf10
mutants.
The HL which has a severely trun-
cated zeugopod was amputated
at the stylopod (first row), and the
other was amputated at the zeu-
gopod (second row). The asterisk
indicates that these newts did not
form blastemas. (
D
) MicroCT image
of regenerated HL of
Fgf10
mutant.
The bracket, arrowhead, and aster-
isk show the stylopod, recovered
fibula/tibia, and digits, respectively.
(
E
) Expression of limb development
-
related genes in the
Fgf10
mutant
blastema. (
F
) Summary of differ-
ential expression analysis of limb
development
-
related genes in the
Fgf10
mutant limb bud or blastema
compared with the wild type. The
down and right arrows indicate sig-
nificant downregulation (
p
< 0.05)
and no difference, respectively.
(
G
) A model for development and
regeneration
-
specific program in
the urodele hindlimb.
Downloaded from https://www.pnas.org by George Porter on March 6, 2024 from IP address 131.215.225.170.