Developmental Cell, Volume 41

Supplemental Information

PCP and SAX-3/Robo Pathways Cooperate to Regulate

Convergent Extension-Based Nerve Cord Assembly

in C. elegans

Pavak K. Shah, Matthew R. Tanner, Ismar Kovacevic, Aysha Rankin, Teagan E.Marshall, Nathaniel Noblett, Nhan Nguyen Tran, Tony Roenspies, Jeffrey Hung, ZheqianChen, Cristina Slatculescu, Theodore J. Perkins, Zhirong Bao, and Antonio Colavita

slt-1(eh15)

wt

slt-1(ok255)

fmi-1(rh308)

wt

prkl-1(ok3182)

prkl-1(zy11)

vang-1(tm1422)

vang-1(ok1142)

sax-3(zy5)

sax-3(ky123)

DD1 DD2 DD3 DD4 DD5 DD6

Relative position along AP axis0 20 40 60 80 100%

DD1 DD2 DD3 DD4 DD5 DD6

prkl-1(ok3182)

vang-1(ok1142); prkl-1(ok3182)

vang-1(tm1422); prkl-1(ok3182)

Relative position along AP axis0 20 40 60 80 100%

vang-1(ok1142)

vang-1(ok1142); fmi-1(rh308)

vang-1(ok1142); slt-1(eh15)

prkl-1(ok3182)

prkl-1(ok3182); fmi-1(rh308)

prkl-1(ok3182); slt-1(eh15)

sax-3(zy5)

sax-3(zy5);fmi-1(rh308)

sax-3(zy5); slt-1(eh15)

A

C

B

vang-1(ok1142) sax-3(zy5)

sax-3(zy5); prkl-1(ok3182)

vang-1(zy60 [gfp::vang-1])

prkl-1(ok3182); zyIs33[gfp::prkl-1]

vang-1(ok1142) sax-3(zy5)

vang-1(ok1142) sax-3(zy5); zyIs43[sax-3::gfp]

Figure S1

Relative position along AP axis0 20 40 60 80 100%

DD1 DD2 DD3 DD4 DD5 DD6

N38

44

39

39

44

46

55

50

51

N40

33

41

45

46

53

50

48

50

50

50

52

52

50

49

48

50

50

52

56

N

0 20 40 60 80

100

zy5 [Q987stop]

TM

wt zy5 ky123

A

B C

***

0 20 40 60 80

100

% A

nte

rio

r n

erv

e r

ing

0

20

40

60

80

100

% L

eth

alit

y

% N

otc

he

d h

ea

ds

0 20 40 60 80

100

% A

VM

g

uid

an

ce d

efe

cts

0 20 40 60 80

100

%

HS

N

gu

ida

nce

de

fect

s

wt zy5 ky123 wt zy5 ky123

wt zy5 ky123 wt zy5 ky123

***

***

ns ns

D

E F

0

20

40

60

80

100

Figure S2

N=322 N=278 N=265 N=316 N=274 N=66 N=122 N=114 N=31

N=59 N=144 N=112 N=137 N=161 N=119

A

PDA

VD

VNC PAGRVG

VNC PAGRVG

wdIs6; ynIs37 sax-3(zy5); prkl-1(ok3182); wdIs6; ynIs37

VB

VA

wdIs6 sax-3(zy5); prkl-1(ok3182); wdIs6

C

D

E

gfp

rfpm

erge

B wyIs75; ynIs37 sax-3(zy5); prkl-1(ok3182); wyIs75; ynIs37

Figure S3

cp13

(NM

Y-2:

:GFP

)m

erge

cnd-

1p::P

H::R

FP

prkl-1(ok3182)vang-1(ok1142) sax-3(zy5)C D E

Contracting edge

Wild-type posterior contraction

Ectopic anterior contractionFailed posterior contraction

Ectopic anterior contractionPosterior contraction

A

-2’ 0’ -2’ 0’ -3’ 0’

-2’ 0’cp

13(N

MY-

2::G

FP)

mer

gecn

d-1p

::PH

::RFP

wt B

Figure S4

SUPPLEMENTAL INFORMATION

Supplemental Figure Legends

Figure S1. Quantification of DD neuron positions in various genetic backgrounds, related to

Figure 2

(A) Quantification of relative DD position in wt and various mutant worms grown at 20°C. The

anterior displacement of DD neurons in vang-1, prkl-1 and sax-3 single mutants is generally

more severe when worms are grown at 25°C (Fig 2F) compared to 20°C (this plot). All DD2-6

neurons in mutants were compared to the corresponding wt neuron. (B) Quantification of slt-1

and fmi-1 single and double mutant combinations with vang-1, prkl-1 and sax-3 and

confirmation that prkl-1 and sax-3 GFP fusions and zy60(gfp::vang-1) are functional. DD

neurons display normal positioning in slt-1 mutants and relatively mild position defects in fmi-1

mutants. The DD anterior displacement phenotype in vang-1, prkl-1 and (F) sax-3 single

mutants is not enhanced in slt-1 and fmi-1 double mutants. DD positioning in the CRSPR/cas9

vang-1(zy60[gfp::vang-1]) allele resembles wt worms indicating the GFP knock-in does not

disrupt protein function. Transgenes expressing PRKL-1 (zyIs33) and SAX-3 (zyIs43) GFP fusions

used for localization studies rescue the DD position phenotypes in the corresponding mutant

background. DD2-6 neurons in each strain were compared to the corresponding wt, single or

double mutant neuron. (C) vang-1; prkl-1 double mutants display a less severe DD anterior

displacement phenotype compared to prkl-1 single mutants. DD2-6 neurons in double mutants

were compared to the corresponding prkl-1(ok3182) neuron. For all plots, DD neuron

quantification and color scheme is as described in Fig 2F (N=33-56 worms). Means and 95%

confidence intervals are shown for each DD neuron. *p≤0.01; **p≤0.001, using the two-tailed t-

test. Worms quantified in (B and C) were grown at 25°C.

Figure S2. Characterization of sax-3(zy5) phenotypes, related to Figure 2

(A) Schematic of SAX-3 protein structure and location of zy5 mutation. TM indicates position of

transmembrane domain. (B-F) Quantification of (B) embryonic lethality (N=265-322 embryos),

(C) notched head (N=66-316 worms), (D) anterior nerve ring (N=31-122), (E) AVM (N=59-144)

and (F) HSN (N=119-161) axon guidance phenotypes in wt, sax-3(zy5), and sax-3(ky123) null

mutants. Error bars indicate standard error of proportion. Significance was assessed using

Fisher exact tests, ***<0.001, ns, not significant.

Figure S3. VNC positioning of post-embryonically-derived VD, VA and VB neurons in sax-

3(zy5); prkl-1(ok3182) double mutants, related to Figure 2

(A and B) Characterization of post-embryonically derived VD neurons. (A) Schematic showing

VD and PDA neuron position in C. elegans. (B) Representative images of wt and sax-3(zy5); prkl-

1(ok3182) L4 stage worms in which DD (RFP) and VD (RFP and red arrowheads) neurons are

labeled with a wyIs75[unc-47p::mCherry; exp-1p::gfp] and DD (GFP and green arrowheads)

neurons with ynIs37 reporters. DD neurons are identified by their overlap when GFP and RFP

channels are merged. The embryonically-derived PDA (GFP and yellow arrow) neuron is also

labeled by exp-1p::gfp in wyIs75. VD neurons in sax-3(zy5); prkl-1(ok3182) mutants display

relatively minor positioning errors compared to the severe anterior displacement of DD

neurons. The position of the PDA neuron which is located in the preanal ganglion (PAG) is not

affected in sax-3(zy5); prkl-1(ok3182) mutants (0% position defect, N=50 worms). (C-E)

Characterization of post-embryonically derived VA and VB neurons. (C) Schematic showing VA

and VB neuron position in C. elegans. (D) Representative images of wt and sax-3(zy5); prkl-

1(ok3182) L4 stage worms in which VA and VB neurons are labeled with a wdIs6[del-1p::gfp]

reporter. The posterior most del-1p::gfp labeled neurons (VB9, VB10, VB11, and VA11) are

marked with white arrows. (E) Representative images of wt and sax-3(zy5); prkl-1(ok3182) L4

stage worms in which VA, VB and DD neurons are labeled with wdIs6 and ynIs37 reporters. VB9,

VB10, VB11, and VA11 (white arrows) neurons in sax-3(zy5); prkl-1(ok3182) mutants display

relatively minor positioning errors compared to the severe anterior displacement of DD (green

arrowheads) neurons. DD neurons were identified by their brighter GFP compared to VA and

VB neurons. All scale bars are 50 μm.

Figure S4. NMY-2 Localization During Contraction of Cell Contacts, related to Figure 4

(A) Single slices showing NMY-2::GFP localization in a representative wt embryo during the

contraction of the cell contact between posterior DA and DD neurons (dashed oval) and a non-

contracting cell contact (dashed box). (B) Schematics illustrating wt and mutant patterns of

intercalation. Single slices showing NMY-2 localization during ectopic cell contact contraction

(dashed oval) and failed contraction (dashed box) in (C) vang-1(ok1142), (D) prkl-1(ok3182) and

(E) sax-3(zy5) mutants. Scale bars are 2 μm. Red dot marks DA5, blue dot marks DD4 and purple

dot marks DD6.