of 21
Nature | Vol 618 | 15 June 2023 |
543
Article
A median fin derived from the lateral plate
mesoderm and the origin of paired fins
Keh-Weei Tzung
1,2
, Robert L. Lalonde
3
, Karin D. Prummel
3,4
, Harsha
Mahabaleshwar
2
,
Hannah R. Moran
3
, Jan
Stundl
5,6
, Amanda N. Cass
7
, Yao
Le
8
, Robert
Lea
9
, Karel
Dorey
9
,
Monika J. Tomecka
1
, Changqing
Zhang
2
, Eline C. Brombacher
4
, William T. White
10
,
Henry H. Roehl
11
, Frank J. Tulenko
12
, Christoph
Winkler
8
, Peter D. Currie
12,13
, Enrique
Amaya
14
,
Marcus C. Davis
15
, Marianne E. Bronner
5
, Christian
Mosimann
3,4
& Tom
J.
Carney
1,2
The development of paired appendages was a key innovation during evolution and
facilitated the aquatic to terrestrial transition of vertebrates. Largely derived from the
lateral plate mesoderm (LPM), one hypothesis for the evolution of paired fins invokes
derivation from unpaired median fins via a pair of lateral fin folds located between
pectoral and pelvic fin territories
1
. Whilst unpaired and paired fins exhibit similar
structural and molecular characteristics, no definitive evidence exists for paired
lateral fin folds in larvae or adults of any extant or extinct species. As unpaired fin core
components are regarded as exclusively derived from paraxial mesoderm, any
transition presumes both co-option of a fin developmental programme to the LPM
and bilateral duplication
2
. Here, we identify that the larval zebrafish unpaired pre-anal
fin fold (PAFF) is derived from the LPM and thus may represent a developmental
intermediate between median and paired fins. We trace the contribution of LPM to
the PAFF in both cyclostomes and gnathostomes, supporting the notion that this is
an ancient trait of vertebrates. Finally, we observe that the PAFF can be bifurcated by
increasing bone morphogenetic protein signalling, generating LPM-derived paired fin
folds. Our work provides evidence that lateral fin folds may have existed as embryonic
anlage for elaboration to paired fins.
Two alternate hypotheses have been proposed to explain the evo
-
lutionary origin of vertebrate paired appendages (fins and limbs).
Derivation from posterior gill arches was posited by Gegenbaur
3
,
whilst a number of anatomists later invoked a rival theory, the lateral
fin fold hypothesis. This proposed that paired fins derived (either
phylogenetically or ontogenetically) from longitudinal bilateral fin
folds that were then subdivided
4
,
5
. Whilst recent molecular studies
have provided some evidence in support of each hypothesis
2
,
6
,
7
, there
remains significant criticism of the lack of substantiation in the fos
-
sil record or in embryology
1
,
8
. Certain stem vertebrates, including
anaspid-related fossils, show evidence of lateral fin folds; however,
these fin folds mostly consist of soft tissue with only sporadic skeletal
elements and are thus poorly preserved. This has led to conflicting
interpretations
9
13
. The developmental programme for paired fins has
been postulated to have been first assembled in median fins, which
appear in the fossil record before the origin of paired fins
2
. A number
of studies have traced the cellular origin of median fins in lamprey,
catshark, zebrafish and
Xenopus
2
,
14
,
15
. All median fins, both larval and
adult, assessed so far have shown derivation from the paraxial meso
-
derm (PM), whilst paired fins are known to be derived from the lateral
plate mesoderm (LPM). Thus, the median fin programme was most
likely transferred to the LPM from the PM
2
, possibly before the for
-
mation of hypothesized lateral fin folds. How or when this transition
occurred is unclear. As only a subset of median fins in zebrafish has been
assayed, we expanded the characterization of the composition and
origin of median fins in zebrafish to determine if PM derivation was an
invariant characteristic.
As with most surveyed jawed vertebrates (gnathostomes), larval
zebrafish possess two median unpaired fin folds. A caudal fin fold (or
major lobe) runs continuously from the dorsal midline around the
caudal end of the larva and then ventrally to the anus (Fig.
1a
). A pre-anal
fin fold (PAFF; or minor lobe) runs along the underside of the yolk sac
extension, immediately anterior to the anus
16
(Fig.
1a
). The median fin
folds are resorbed during metamorphosis, and the caudal fin fold is
https://doi.org/10.1038/s41586-023-06100-w
Received: 5 August 2022
Accepted: 19 April 2023
Published online: 24 May 2023
Open access
Check for updates
1
Institute of Molecular and Cell Biology, A*STAR, Singapore, Singapore.
2
Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Singapore.
3
Department of
Pediatrics, Section of Developmental Biology, University of Colorado Anschutz Medical Campus, Aurora, CO, USA.
4
Department of Molecular Life Sciences, University of Zurich, Zurich,
Switzerland.
5
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA.
6
Faculty of Fisheries and Protection of Waters, University of South Bohemia
in Ceske Budejovice, Vodnany, Czech Republic.
7
Biology Department, Wesleyan University, Middletown, CT, USA.
8
Department of Biological Sciences, Faculty of Science, National University
of Singapore, Singapore, Singapore.
9
Division of Developmental Biology and Medicine, School of Medical Sciences, Faculty of Biology, Medicine and Health, University of Manchester,
Manchester, UK.
10
CSIRO National Research Collections Australia, Australia National Fish Collection, Hobart, Tasmania, Australia.
11
School of Biosciences, University of Sheffield, Sheffield, UK.
12
Australian Regenerative Medicine Institute, Monash University, Clayton, Victoria, Australia.
13
EMBL Australia, Victorian Node, Monash University, Clayton, Victoria, Australia.
14
Division of Cell
Matrix Biology and Regenerative Medicine, School of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK.
15
Department of Physical and
Biological Sciences, Western New England University, Springfield, MA, USA.
e-mail:
christian.mosimann@cuanschutz.edu
;
tcarney@ntu.edu.sg
544
| Nature | Vol 618 | 15 June 2023
Article
replaced by three separate PM-derived median adult fins
14
,
17
: the caudal
fin, the dorsal fin and the anal fin. No adult median fin replaces the PAFF,
which is a developmentally transient structure
16
. The mesenchyme
of the caudal fin fold can be labelled by the photoconvertible Kaede
green-to-red fluorescent protein expressed under control of the
tbx16l
promoter, confirming that this median fin is derived from the PM
18
.
In contrast, we consistently failed to observe any Kaede labelling in
the PAFF (Fig.
1b
and Extended Data Fig. 1a). This labelling difference
is not due to de novo activity of the
tbx16l
promoter specifically in
the caudal median fin fold, as we photoconverted Kaede in anterior
somites above the PAFF at 24 h post-fertilization (hpf ) and traced the
photoconverted Kaede to the mesenchyme of the dorsal portion of
the caudal fin fold; again, we never found labelled cells in the PAFF
(
n
= 11) (Extended Data Fig. 1b–d). The absence of PAFF labelling by
a PM-lineage trace is not due to absence of mesenchyme cells, as we
observed an abundance of these cells by Nomarski optics at 3 days
post-fertilization (dpf ) and in the enhancer trap line
Et(krt4:EGFP)
sqet37
(
ET37
), which labels all fin mesenchyme cells (Extended Data Fig. 1e,f ).
The
ET37
line further allowed us to visualize the morphology of the
mesenchyme cells, which showed a distally polarized stellate shape,
indistinguishable from the mesenchyme of all other fins (Fig.
1c–e
and
Extended Data Fig. 1f–h). PAFF mesenchymal cells expressed known dif
-
ferentiation markers of fin mesenchyme, including
fibulin1
(
fbln1
) and
integrin beta 3b
(
itgb3b
), and were weakly positive for
lyve1b
reporter
activity in the
lyve1b:DsRed2
transgenic line, which also expresses in
the post-anal ventral fin mesenchyme
19
(Fig.
1f–h
and Extended Data
Fig. 1i). Hence, despite their divergent developmental origin, PAFF
mesenchyme has a similar morphology and expression profile to fin
mesenchyme of the major median fin lobe.
Few functions of fin mesenchyme cells are defined. The zebrafish
frilly fin
(
frf
) mutants display ruffling of the caudal larval fin fold
due to mutations in
bone morphogenetic protein 1a
(
bmp1a
), which
is expressed in fin mesenchyme
20
, including in the PAFF (Extended
Data Fig. 1j). The overall morphology of the PAFF also displays ruf
-
fling in
frf
mutants (Fig.
1i,j
). Further, immunostaining of collagen II
revealed ordered parallel collagen II fibres in all median fins of the
wild type (WT), while these fibres were disorganized in both the caudal
median fin folds and the PAFF of
frf
mutants (Fig.
1i,j
), indicating that
Bmp1a in PAFF mesenchyme is also required for maturation of collagen.
Collectively, these results indicate that mesenchymal cells of the PAFF
show functional overlap with those of the caudal median fin fold yet
have a distinct developmental origin.
The transcription factor Hand2 is expressed in several LPM progeni
-
tors, including cardiac, pharyngeal, mesothelial and pectoral fin pro
-
genitors
21
,
22
. Upon examining the
TgBAC(hand2:EGFP)
transgenic line
that accurately recapitulates endogenous
hand2
gene expression
23
,
we observed the expected enhanced green fluorescent protein (eGFP)
expression in larval pectoral fins at 2–3 dpf (Fig.
2a,b
). Although we
observed no
TgBAC(hand2:EGFP)
expression in the major unpaired
larval fin lobe, the PAFF mesenchyme was strongly eGFP positive, sug
-
gesting an LPM origin (Fig.
2a,b
and Extended Data Fig. 2a,b). Addi
-
tionally, in situ hybridization at 3 and 5 dpf indicated that the PAFF
mesenchyme, but no other median fin fold, expressed
hand2
(Fig.
2c,d
and Extended Data Fig. 2c,d). If the LPM uniquely contributes to the
PAFF, then we might expect that the PAFF, but no other median fin fold,
would be affected in an LPM mutant. The zebrafish
hand2
mutant
hands
off
(
han
s6
) exhibits defects in LPM derivatives, including the heart,
pectoral fins and mesothelium
21
,
22
. Consistent with an LPM origin, the
8 dpf
frf
tp34
Collagen II
j
8 dpf WT
i
Tg(-5.2lyve1b:DsRed)
nz101
h
Post-anal n
g
Pre-anal n
5 dpf
3 dpf
fbln1
f
Pre-anal n
Et(krt4:EGFP)
sqet37
Post-anal n
Pectoral n
c
d
e
4 dpf
3 dpf
3 dpf
b
Tg(tbx16l:GAL4-VP16)
sq13
;
Tg(UAS:Kaede)
rk8
3 dpf
a
4 dpf
Fig. 1 | A non-PM-derived median f in fold.
a
, Larval 4 dpf zebrafish possess a
median PAFF (yellow arrowhead) in addition to the caudal median fin fold (cyan
arrowhead).
b
, Confocal image of a 3 dpf
Tg(tbx16l:GAL4-VP16); Tg(UAS:Kaede)
embryo with PM labelled by Kaede showing PM-derived mesenchyme in the
caudal median fin fold (cyan outline) but not the PAFF (yellow outline).
c
e
,
Confocal images of pre-anal (
c
), ventral caudal (
d
) and pectoral (
e
) fins of the
ET37
Enhancer Trap transgenic line indicating that PAFF contains
morphologically comparable mesenchyme (indicated by arrowheads) to other
larval fin folds.
f
, In situ hybridization of the fin mesenchyme marker
fbln1
in
both PAFF (yellow arrowhead) and caudal fin fold (cyan arrowhead) at 3 dpf.
g
,
h
, DsRed expression in mesenchyme of both pre-anal (
g
) and caudal (
h
) fin
folds of the 5 dpf
Tg
(-5 . 2
lyve1b:DsRed)
transgenic line.
i
,
j
, Immunostaining for
collagen II in 8 dpf
frf
mutants (
j
) shows loss of fibril organization compared
with WT (
i
). Scale bars, 200 μm (
a
); 100 μm (
b
); 20 μm (
c
,
e
); 50 μm (
f
,
g
,
j
).