536 | Nature | Vol 582 | 25 June 2020
Article
inflammatory response and regulates the differentiation of gastro-
intestinal cells^23. LePin and Granulin 1, which we used for ISH, have
homologues in Exaiptasia, as well as stony and soft corals. Because
LePin has an N-terminal signal peptide followed by multiple domains
(including H- and C-type lectins and a Kazal-type serine protease inhibi-
tor) (Extended Data Fig. 5b), it may confer selectivity for the Symbiod-
iniaceae. On the basis of previous studies of granulins in mammals^24 ,
Granulin 1 may modulate the immune response in Xenia endosymbiotic
cells.
Phagocytosis of the Symbiodiniaceae by gastrodermal cells (which
are of a similar size to these algal cells) requires substantial expan-
sion of the host cell, but the genes that regulate this size expansion
are unknown. Among the endosymbitoic marker genes that we found,
Plekhg5 encodes a highly conserved RhoGEF (Fig. 3g, Extended Data
Fig. 5c). In Xenopus, Plekhg5 localizes to the apical membrane of epithe-
lial cells and recruits actomyosin to induce cell elongation and apical
constriction^25. Thus, Plekhg5 is a prime candidate for regulating the
extension of the apical membrane to engulf algae of the Symbiodini-
aceae during the early stages of phagocytosis in Xenia. Upon phagocy-
tosis, algae of the Symbiodiniaceae are enclosed by the host membrane
to form symbiosomes^26. Although the symbiosome is acidified similarlyCollagen triple helix repeat containing 112345679 10 11 12 13
c Nematogalectin 1
Nematogalectin 2
Nematogalectin 3
Minicollagen 1
Minicollagen 2Xe_006064
Xe_006066
Xe_006067
Xe_028815
Xe_0012108
d e Minicollagen 1 Nematogalectin 2
Low HighXe_028815 Xe_006066f gi j Collagen 6hCnidocyte11-111-2Collagen 6a bMinicollagen 1Nematogalectin 2Low HighClusterCluster14 15 16Collagen 4-D 5
Collagen 6
Astacin-like metalloendopeptidase 1Collagen-D 2Xe_014297
Xe_009334
Xe_021684Xe_009974Meprin A
Collagen-D 1
Collagen 3-D 1Xe_011016
Xe_014383
Xe_001362
Xe_009290Astacin-like metalloendopeptidase 2Matrix metallopeptidaseXe_021685Xe_012250
Cluster2164983510 1371214111615–50–2502550–50 –25 025t-SNE 2t-SNE 1Cnidocyte1234567819101112134151612345678191011121341 5 16Fig. 2 | scRNA-seq transcriptomes suggest that there are 16 cell types in
Xenia sp. a, Transcriptomes of 19,134 individual Xenia sp. cells obtained by
scRNA-seq were grouped into 16 clusters (colour-coded) and presented in t-SNE
space. Each coloured dot represents one cell. b, Gene-expression heat map
(scale at the top) for the top 10 gene markers that define each cluster. Each
column represents one cell cluster, and each row represents one gene. Forty
cells were randomly selected from each of the 16 cell clusters for plotting.
c, Expression profiles of the indicated cluster-11 Xenia (Xe) marker genes out of
all cell clusters. d, Cluster-11 cells are subdivided into two populations (11-1,
423 cells; 11-2, 374 cells, colour-coded) and displayed in a t-SNE space. Each
coloured dot represents a cell. e, Expression levels (scale to the top right) of
two cluster-11 markers, Minicollagen 1 and Nematogalectin 2, are shown in a
t-SNE plot. n = 797 cells. f, g, Whole-mount RNA ISH of Minicollagen 1 (f) and
Nematogalectin 2 (g), showing their expression in tentacles. Arrows indicate
the expression of Minicollagen 1 at the base of pinnules. h, Expression profiles
of marker genes enriched in clusters 2, 12 and 16 out of all 16 clusters. i, j, RNA
ISH of Collagen 6. Whole-mount view of the stalk in i and cross-section image in
j. The white dashed line in i indicates the cross-section level in j. More than
12 polyps from 4 independent experiments were used for each probe. Scale
bars, 100 μm (f, g, j), 150 μm (i). Cell numbers for clusters 1–16 are 2,794; 2,704;
2,073; 1,679; 1, 511; 1,374; 1,248; 1,069; 986; 923; 797; 649; 575; 321; 246; and 185,
respectively (a, c, h).
Transporters
NPC2, Glut1,
Glut8, ChT...Alga
HostUptake
Plekhg5,
Lamp1-L...Recognition
CUZD1, CD36,
DMBT1, LePin...ga Hoechst Cy5 Merged b(^100101102103104)
101
102
103
104
DAPI channel
Cy5.5 channel
Alga-containing
Alga-free
c 1
2 3 4 5 6 7 8 9
11
12
13
10
Alga
rep 1+
Alga
rep 2+
Alga
rep 1–
Alga
rep 2–
Alga
rep +
1
Alga
rep +
2
Alga
rep –
1
Alga
rep –
2
Correlation
0
0.9
Cluster
14
15
16
e
Control
LePin Alga Merge
Alga Merge
Marker expressionLow
High
w
d f
LePinControl
5.0
2.5
0
10.0
7.5
Intensity (
×^10
3 )
Fig. 3 | Identif ication of genes specif ically expressed in Xenia sp.
endosymbiotic cells. a, The endosymbiotic algae in Xenia display
autof luorescence in the Cy5.5 far-red channel. A cross-section of Xenia, with
the Xenia and algal nuclei stained by Hoechst (blue) and alga autof luorescence
(white). b, A FACS profile of dissociated live Xenia cells using Cy5.5 and DAPI
channels. Five biological replicates (a, b). c, Pearson correlation of gene
expression between the scRNA-seq data of 16 cell clusters and the bulk RNA-seq
data of 2 biological replicates of FACS-isolated alga-containing (alga+ rep 1 and
alga+ rep 2) and alga-free (alga− rep 1 and alga− rep 2) cells. d, Heat map showing
the expression levels of the 89 marker genes for cluster-16 cells in
alga-containing and alga-free cells, isolated by FACS. e, Ultra-sensitive
f luorescence RNA ISH by RNAscope probing for LePin (red) (top) and control
(bottom). White arrows indicate the LePin signal. Hoechst staining of all nuclei
is shown in blue. Scale bars, 20 μm. f, Quantification of LePin signals. The
f luorescence signal surrounding each alga is quantified in random sections
and plotted (a dot = a section) for LePin and controls. ***P = 2 .8 4 × 10−16,
two-sided t-test. Lines in the box denote the median; the upper and lower edges
of the box represent the upper and lower quartiles, respectively. Nine polyps
from three independent experiments were used for each probe (e, f).
g, Illustration of steps through which Xenia endosymbiotic cells may recognize
and take up algae to establish endosymbiosis, with some candidate genes
shown at each step.