Science - USA (2021-11-05)

synthesis of NO, together with the NO receptor
Gucy1B2(fig. S16). In the marine demosponge
Tethya wilhelma, NO induces and modulates
whole-body contractions ( 14 ) but only affected
the osculum in the freshwater spongeEphydatia
muelleri( 13 ). During theS. lacustriscontrac-
tion cycle (fig. S18, A and B, and movie S1), the
collapse of osculum and incurrent systems
first causes excurrent canals to swell, which
subsequently shrink to expel water through
the osculum before returning to the resting
state. We tested the response to NO by treat-
ing juvenile sponges with NOC-12, a donor of NO,
and 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one
(ODQ), an inhibitor of the NO receptor (fig.
S18, C to F, and movies S2 and S3). NO release
immediately shrank the incurrent system, in-

cluding incurrent canals and tent, and expanded

the excurrent canals, as during initial stages of

the contraction cycle (fig. S18C and movie S2).

Subsequently, the incurrent system exhibited

short, pulsed movements and returned to rest-

ing state only after NO removal (fig. S18C). The

subtler effect of ODQ treatment involved ex-

pansion of incurrent, and possibly excurrent,

canal systems (fig. S18D and movie S3). Serial

combinations of NOC-12 and ODQ gave similar

results (fig. S18, E and F), establishing a key role

of NO in initiating and regulating the contrac-

tion cycle inS. lacustris, and possibly ances-

trally in demosponges.

Beyond contraction, cell type–specific expres-

sion programs indicated endymocyte roles in

photo- and mechanosensation, skeleton for-

mation, glucose metabolism, and production

of reactive oxygen for defense (fig. S15). Single

orthologs ofSixandPax( 15 ) are expressed in

a complementary manner (fig. S16), withSix+

incurrent pinacocytes enriched for genes in-

volved in circadian clock entrainment, and

the blue-light sensorcryptochrome, which

suggests light sensitivity, andPaxB+apendo-

pinacocytes expressing mechanosensory com-

ponents and exhibiting short cilia (Fig. 3D

and fig. S16). Next, we found basopinacocytes

enwrapping spicules anchored at the base of

thesponge(Fig.3Candfig.S14,FandG)and

sclerophorocytes forming clusters alongside

mature spicules (Fig. 3, C and D, and fig. S14I),

similar toSoxB+ spicule“transport cells”in

E. muelleri( 16 ). Both are enriched in genes that

SCIENCEscience.org 5 NOVEMBER 2021•VOL 374 ISSUE 6568 719

A

metazoan anc.

neuralian anc.

Porifera Cnidaria Protostomiadeuterostomia

bilaterian anc.

Ctenophora

(placement

uncertain)

Os

Ca

G T

B

5

4

3

2

12

1

33

32

41

28

26 27

40

35 3839

36

37

13

(^1415)
16 17
18
29
30
(^3119)
20
21
22
23
24 25
7
6
8
9
10
11
D
E Choanocyte
Lineage
Myopeptidocyte
Lineage
Inc. Pinac.
Lineage
Apendopinac.
Lineage
Noggin A
Eef1a1
Rsph1
V ill
Dchs B
Baiap2/L1/L2
cAMP Receptor A
Gpsm1/2
FoxL2
HPGDS
Plb1
Duox1/2 B
Tagln/2/3 A
P2rx A
Tmtc1
Alox E
Syt7 D
Tetraspanin
Fibcd1
cluster
annotation
Archaeocytes
Mesocytes 2
Neuroid
Myopeptidocytes
Mesocytes 3
Apendo-
pinacocytes
Lophocytes
Incurrent
Pinacocytes
Apopylar Cells
Choanoblasts
Mesocytes 1
Basopinacocytes
Amoebocytes
Sclerocytes
Granulocytes
Choanocytes
Sclerophoro-
cytes
Metabolocytes
1
2
3
4 5 6 7 8 9
10
11
12
13
29 14 15
16
17
18
30
31
23
24 25
26
27
28
(^3839)
40
36
34 35
19
20
21
22
41
42
32
C 33
37
Fig. 1.S. lacustriscell types from whole-body single-cell RNA sequencing.
(A) Animal phylogeny. anc., ancestor. (B)S. lacustrisoverhead view. Scale bar,
300 mm. Ca, excurrent canals; G, gemmule; Os, osculum; T, epithelial tent.
(C) tSNE plot of 10,106 cells colored by 42 clusters. (D) PAGA connectivity plot
showing connection strength (edge thickness) and log number of cells in
cluster (node diameter). (E) Scaled expression values of differentially
expressed markers for PAGA paths. Apendopinac., apendopinacocyte; Inc.
Pinac., incurrent pinacocyte.
RESEARCH | RESEARCH ARTICLES