Supplementary Materials for
Evolution of a chordate-specific mechanism for myoblast fusion
Haifeng Zhang
et al.
Corresponding author: Pengpeng Bi, pbi@uga.edu; Alberto Stolfi, alberto.stolfi@biosci.gatech.edu
Sci. Adv.
8
, eadd2696 (2022)
DOI: 10.1126/sciadv.add2696
The PDF file includes:
Figs. S1 to S18
Other Supplementary Material for this manuscript includes the following:
Tables S1 and S2
Supplemental Files 1 to 3
Movies S1 to S4
Fi
gure S
1
.
Muscle histolog
ical
analyses of adult amphioxus and shark.
Histology
of longitudinal sections of muscle tissu
es dissected from adult amphioxus (
A
) and shark (
Squalus acanthias
)
(
B
). Arrows point to mononucleated myocytes in
A
.
One
multinucleated myofiber
is
outlined in
B
.
Figure
S2.
Phylogenetic analysis of the Tmem8 gene family.
(
A
)
Phylogenetic tree of
Tmem8
family gene
s
inferred by a
distance
-
based method (neighbour joining). The bootstrap
percentages obtained from 1,000 replicates were shown in the cladogram
.
(
B
)
Topology and domain predictions
(SCAMPI model) for Tmem8 family transmembrane proteins. EGF domain: epidermal growth factor
like domain. (
C
)
Scenarios of two
-
round gene duplications that gave rise to various Tmem8 family gene members
in Chordata
.
Model 1
represents a
more parsimonious
scenario of evolution.
Fi
gure S
3
.
Sequence alignments
, gene structures
and
validation
of MymK ortholog
expression
.
(
A
)
Alignment of MymK proteins from eight species:
H
uman (
Homo sapiens
),
M
ouse (
Mus musculus
),
G
ar (
Lepisosteus
oculatus
),
E
lephant shark (
Callorhinchus milii
),
L
amprey (
Petromyzon marinus
),
P
hallusia
(
Phallusia mammillata
),
C
iona
(
Ciona robusta
),
S
tyela
(
Styela clava
).
S
ubstantial degree of
sequence identity
is
observed for vertebrate MymK
sequences (boxed) but no
t MymK from tunicates (
Phallusia
,
Ciona
,
Styela
).
(
B
) Exon size and structure for various
MymK orthologs.
(
C
,
D
)
Western blots confirm
ing
the expression of C
-
tagged
Ciona
MymK protein in human myoblasts
(
C
, total protein;
D
, after
membrane
fractionation).
α
-
Tubulin blot was used as a positive control of cytosolic proteins.
Insulin receptor
β
(INSR
-
β
)
blot was used as a positive control of membrane proteins.
Figure S4.
Tunicate MymK proteins induce fusion of
mouse and lizard
MymK
–
/
–
myoblasts.
(
A
)
Schematic of experiment
al
design to generate mouse
MymK
-
deficient myoblasts and test the fusogenic
activity
of
MymK
orthologs.
Expression of Cas9 and
three
single guide RNA (sgRNAs) that targets
the first three
exon
s
are
delivered by lentiviral infection. (
B
)
Western blots validated the successful depletions of MymK
protein
in
one
isolated
single clone of CRISPR treated mouse
C2C1
2
myoblasts.
(
C
)
Myosin immunostaining of mouse
MymK
–
/
–
myoblasts
transfected with MymK orthologs.
T
unicate (
Styela
and
Ciona
) MymK genes induced formation
s
of muscle syncytia
(outlined), which
are
smaller compared
to
syncytia induced by MymK proteins from jawed vertebrates; E. shark:
elephant shark. (
D
)
Measurements of myoblast fusion in
C
after
seven
days of differentiation. (
E
)
Schematic of
experiment
al design
to isolate
myogenic clone from
lizard (
Anolis sagrei
)
em
bryonic cells
, inactivate
MymK
gene and
finally test the fusogenic activity of tunicate MymK proteins. CRISPR/Cas9
-
mediated mutagenesis of lizard
MymK
gene
was performed using a pair of sgRNAs targeting exon 3
.
(
F
,
G
)
Genotyping PCR (
F
) and
S
anger sequenci
ng (
G
) analyses
of lizard
MymK
locus revealed a mutant myoblast clone where
t
w
o
alleles of
MymK
gene
are
disrupted. The predicted
sgRNAs
c
u
t
sites
are
located
b
e
t
w
e
e
n
the codons
Leu96 and Leu127
.
KO: knockout.
(
H
)
Myosin immunostaining of
lizard
MymK
-
deficient myoblasts transfected with various
MymK
orthologs. Consistent with the results in human and
mouse myoblasts, expression of tunicate MymK proteins induce fusion
of lizard myoblasts
. Cells
are
differentiated
for
nine
days before analysis.
Arro
ws point to multinucleated myotubes.
Scale bars,
100
μ
m.
Data are means ± SEM. *
P
<
0.05; ***
P
< 0.001
,
compared to control group
, one
-
way ANOVA
.