plausible regulators of cell sorting during the
formation and maintenance of neural progen-
itor patterns (fig. S6, D to G).
We next investigated how the adhesion
code mediates the homotypic preference of
the three neural progenitor types.cdh11is
enriched in the pMN cells. Cdh11 belongs to
type II cadherins, a cadherin family that is
structurally incompatible to bind in trans
with type I cadherins (e.g., Cdh2) ( 25 ). Cdh11-
expressing cells and Cdh2-expressing cells
segregate in culture, indicating that Cdh11
could mediate pMN-specific homotypic adhe-
sion in a tissue dominated by Cdh2-based ad-
hesion ( 26 ). Indeed, without Cdh11, pMN cells
no longer exhibited homotypic preference
against p3 or p0 cells (Fig. 3A). Adhesion force
measurements of cell doublets further showed
that loss of Cdh11 specifically lowered pMN-
pMN homotypic adhesion without affect-
ing pMN-p3 or pMN-p0 heterotypic adhesion
(Fig. 3B).
Next,pcdh19is enriched in p3 cells. Although
Pcdh19 alone exhibits very weak adhesion, it
belongs to thed2-protocadherin family, which
is known to modulate Cdh2-based adhesion
by multiple mechanisms ( 27 , 28 ). Our data
suggested that the Pcdh19-Cdh2 interaction
creates a specific adhesion mode that is dis-
tinct from Cdh2-based adhesion without Pcdh19
(supplementary text S4). Specifically, loss of
pcdh19enrichment in the p3 cells, either by
pcdh19knockdown or addition ofpcdh19in
the pMN cells, disrupted the homotypic prefer-
ence of p3 cells in the p3-p3-pMN triplet assay
(Fig. 3C and fig. S7). Loss ofpcdh19does not
change the p3-p3 homotypic adhesion force
significantly; instead, the predominant effect
of the loss ofpcdh19is an increase of the p3-
pMN heterotypic adhesion to a level that
matches the p3-p3 homotypic adhesion
(Fig. 3D). The increase of p3-pMN adhesion
without Pcdh19 is Cdh2 dependent, con-
sistent with the role of Pcdh19 to generate
a Pcdh19-Cdh2–dependent adhesion mode that
lowers adhesion to the Cdh2-exclusive mode
in pMN cells.
Finally, Pcdh19 and Cdh2 are enriched in p0
cells relative to pMN cells. As in p3-pMN het-
erotypic adhesion, loss of Pcdh19 also led to a
Cdh2-dependent increase of pMN-p0 hetero-
typic adhesion. However, this only slightly dis-
rupted the homotypic preference of p0 cells
(Fig. 3, E and F). Instead, loss of Cdh2 com-
pletely abolished the homotypic preference of
p0 cells against pMN cells. We conclude that
the homotypic preference of p0 cells against
pMN cells is achieved primarily by higherlevels of Cdh2-based adhesion. The weaker
Pcdh19 dependence of p0 cells compared with
p3 cells is consistent with a fourfold-smaller
Pcdh19-to-Cdh2 ratio in p0 cells than p3 cells
(Fig. 2F).
To further investigate the role of the adhe-
sion code in patterning in vivo, we examined
the cohesion of pMN, p3, or p0 domains at the
neural tube stage in the mutants or morphants
ofcdh11,pcdh19, orcdh2( 21 , 29 ). We found a
significant increase of patterning errors in each
domain when the corresponding adhesion
molecule was perturbed (Fig. 3, G to I; fig.
S8; movie S7; and supplementary text S5).
Together, our results show that the adhesion
code not only mediates the adhesion specific-
ity underlying homotypic preference of neural
progenitors ex vivo but also mediates pattern-
ing robustness in vivo (Fig. 3J).
The cell type–specific adhesion code requires
coordinated regulation of cell fate specification
and the expression of adhesion molecules, pos-
sibly through a common upstream regulator.
The Shh morphogen gradient instructs spec-
ification of ventral neural progenitor fates
(e.g., p3 and pMN cells) in a dose-dependent
fashion ( 30 , 31 ). We therefore perturbed Shh
signaling and measured changes in expres-
sion patterns of both cell fate markers forSCIENCEsciencemag.org 2 OCTOBER 2020•VOL 370 ISSUE 6512 115
Fig. 3. The adhesion code
mediates homotypic preference
ex vivo and patterning robust-
ness in vivo.Green, magenta,
and blue represent p3, pMN,
and p0 cells, respectively.
(A,C, andE) Results of triplet
assays. WT, wild type; MO,
morpholino. P< 0.05; P< 0.01;
P< 0.001 (binomial test).
(B,D, andF) Adhesion force
measurements in the doublet
assays. (G,H, andI) The number of
mixed cells in the pMN (G), p3 (H),
or p0 (I) domains in control
embryos or embryos lackingcdh11
(G),pcdh19(H), orcdh2(I). For
(B), (D), and (F) to (I): P< 0.05;
P< 0.01; P< 0.001; NS, not
significant (Student’sttest).
(J) Cartoon model for molecular
mechanisms mediating homotypic
preference between p3, pMN, and
p0 cells. Cdh2-Pcdh19 denotes the
Pcdh19-regulated form of Cdh2
(supplementary text S4).
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