DEVELOPMENTAL BIOLOGY
An adhesion code ensures robust pattern formation
during tissue morphogenesis
Tony Y.-C. Tsai^1 , Mateusz Sikora^2 *, Peng Xia^2 †, Tugba Colak-Champollion^3 , Holger Knaut^3 ,
Carl-Philipp Heisenberg^2 ‡, Sean G. Megason^1 ‡
Animal development entails the organization of specific cell types in space and time, and spatial
patterns must form in a robust manner. In the zebrafish spinal cord, neural progenitors form stereotypic
patterns despite noisy morphogen signaling and large-scale cellular rearrangements during morphogenesis
and growth. By directly measuring adhesion forces and preferences for three types of endogenous
neural progenitors, we provide evidence for the differential adhesion model in which differences in
intercellular adhesion mediate cell sorting. Cell type–specific combinatorial expression of different classes
of cadherins (N-cadherin, cadherin 11, and protocadherin 19) results in homotypic preference ex vivo
and patterning robustness in vivo. Furthermore, the differential adhesion code is regulated by the sonic
hedgehog morphogen gradient. We propose that robust patterning during tissue morphogenesis results
from interplay between adhesion-based self-organization and morphogen-directed patterning.
S
patial patterns of distinct cell types arise
reproducibly in development. The classic
French flag model posits that a morpho-
gen gradient forms across a naïve and
static field of cells to provide positional
information to specify patterned cell fates ( 1 ).
The vertebrate spinal cord has been a textbook
example of the French flag model: neural pro-
genitors interpret opposing gradients of sonic
hedgehog (Shh) and bone morphogenetic protein
and commit to 13 distinct fates to form stereo-
typic stripe-like patterns that are highly con-
served across vertebrate species ( 2 – 4 ). However,
our recent work showed that the Shh signal
is noisy, resulting in specification of neural
progenitors in a mixed pattern at the onset
of morphogenesis ( 5 , 6 ). Additionally, the ex-
tensive cell rearrangements during convergent
extension could further disrupt pattern ( 7 ).
Nevertheless, the stereotypic stripe patterns
still form reproducibly. This makes the zebra-
fish spinal cord an attractive system to study
how robust patterning can be achieved despite
imprecision in morphogen signaling and ex-
tensive cell-cell neighbor exchange during tis-
sue morphogenesis and growth.
We focused on patterning of three neural
progenitor types (p3, pMN, and p0), which are
distinguishable by individual transcription fac-
tors (nkx2.2a,olig2, anddbx1b, respectively)
(fig. S1A) ( 8 – 12 ). At the neural tube stage, these
three cell types form stereotypical stripe-like
domains, as visualized by using transgenic
zebrafish carrying fluorescent reporters of
nkx2.2a,olig2, anddbx1bor by fluorescent
in situ hybridization based on a hybridization
chain reaction (in situ HCR) (Fig. 1, A and B, and
fig. S1B) ( 13 – 16 ). Live imaging of fluorescent
reporters, together with single-cell tracking, sug-
gests that these neural progenitors form im-
precise initial patterns during morphogenesis, and
the mixed patterns are resolved predominantly
through cell sorting (figs. S1, C to O, and S2;
movies S1 to S4; and supplementary text S1).
The cell-sorting behavior suggests differ-
ences in adhesion properties among different
neural progenitor types. The differential adhe-
sion hypothesis ( 17 ) is a long-standing model
for cell sorting, but direct biophysical evidence
showing that endogenous cell types use dif-
ferential adhesion to assist patterning in vivo
is still lacking. Therefore, we set out to mea-
sure adhesion forces between different types
of endogenous neural progenitors with the
dual pipette aspiration assay (Fig. 1C, movie
S5, and methods) ( 18 , 19 ). We measured the
adhesion forces of six different doublet types
(Fig. 1D), including three homotypic contacts
(contacts between cells of the same type) and
three heterotypic contacts (contacts between
cells of different types). The average adhesion
forces at the homotypic contacts between two
pMN cells (7.6 ± 3.6 nN) and two p3 cells (4.0 ±
2.7 nN) were significantly greater than that of
the pMN-p3 heterotypic contact (2.5 ± 2.2 nN).
Similarly, the adhesion forces at the homo-
typic contacts between two pMN cells (7.6 ±
3.6 nN) and two p0 cells (7.2 ± 6.3 nN) were also
greater than those for the pMN-p0 heterotypic
contacts (4.4 ± 3.3 nN) (Fig. 1D).
To enable more direct comparison of adhe-
sion preferences, we developed a triplet com-
petition assay (Fig. 1E). The triplet is composed
oftwocellsofthesametypeandonecellofa
different type, forming one homotypic and one
heterotypic contact. When the triplet is pulledapart, it mimics the challenge faced by the cells
in vivo when they are pulled by neighboring
cells in different directions (movie S6 and
methods). All three neural progenitor cell
types showed a clear preference for homotypic
contacts, with pMN-pMN and p3-p3 homo-
typic contacts winning over pMN-p3 het-
erotypic contacts and pMN-pMN and p0-p0
homotypic contacts winning over pMN-p0
heterotypic contacts by a ratio of about 2:1
(Fig. 1F). Thus, each of the three neural pro-
genitor types exhibit a homotypic preference,
a term we use to describe the phenomenon of
cells selectively stabilizing homotypic contacts
over heterotypic contacts.
To identify the molecular mechanisms un-
derlying this adhesion specificity, we obtained
the transcriptomes of p3, pMN, and p0 cells
and used CRISPR-Cas9–mediated genome edit-
ing to knock out candidate adhesion mole-
cules to look for patterning phenotypes ( 20 ).
N-cadherin (cdh2), cadherin 11 (cdh11), and
protocadherin 19 (pcdh19) stood out as genes
with significant loss-of-function phenotypes
(fig. S3 and supplementary text S2).cdh2is
themostabundantcadherininp3,pMN,and
p0 cells (table S1) ( 21 ). Using a fluorescent re-
porter,TgBAC(cdh2:cdh2-mCherry)( 22 ), we
found that Cdh2 exhibits a protein gradient
along the ventral-dorsal (V-D) axis of the
entire spinal cord that increases by twofold
from the normalized V-D position of 0 to 0.8
(Fig. 2, A and B; fig. S4, A and B; and sup-
plementary text S3). Similar Cdh2 profiles are
also observed by antibody staining of sectioned
neural tube and in a different fluorescent re-
porter fish of Cdh2,TgBAC(cdh2:cdh2-tFT)
(fig. S4, C to F) ( 23 ). The expression ofcdh11
appears as one stripe along the entire length
of the spinal cord (fig. S5A) ( 24 ), largely over-
lapping with theolig2-positive domain but
with a wider distribution along the V-D axis
(Fig. 2, C and D).pcdh19is expressed as two
stripes along the entire spinal cord (fig. S5, C
and D), with the ventral stripe colocalizing
with the p3 domain and the medial floor plate
(Fig. 2, E and F, and fig. S5E). The twopcdh19
stripes flank the pMN domain, and expression
ofpcdh19andolig2appears to be mutually
exclusive (Fig. 2G and fig. S5F). The correlated
expressions betweencdh11andolig2and be-
tweenpcdh19andnkx2.2a,aswellasthemu-
tual exclusivity betweenolig2andpcdh19, were
verified by single cell coexpression analysis
(fig. S6, A to C; supplementary text S3; and
methods). Together, quantitative analyses of
cdh2,cdh11, andpcdh19expression revealed
an adhesion code specific to each of the three
cell types, withcdh2,cdh11, andpcdh19en-
riched in p0, pMN, and p3 cells, respectively
(Fig. 2H and supplementary text S3). The dif-
ferential expression patterns ofcdh2,cdh11,
andpcdh19are present at the onset of spi-
nal cord morphogenesis, making these genesSCIENCEsciencemag.org 2 OCTOBER 2020•VOL 370 ISSUE 6512 113
(^1) Department of Systems Biology, Harvard Medical School,
200 Longwood Avenue, Boston MA 02115, USA.^2 Institute of
Science and Technology Austria, Am Campus 1, 3400
Klosterneuberg, Austria.^3 Skirball Institute of Biomolecular
Medicine, New York University School of Medicine, 540 First
Avenue, New York, NY 10016, USA.
*Present address: Max Planck Institute of Biophysics, Frankfurt am
Main, Germany.
†Present address: Life Sciences Institute, Zhejiang University,
310058 Hangzhou, China.
‡Corresponding author. Email: [email protected]
(S.G.M.); [email protected] (C.-P.H.)
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