paper

Article https://doi.org/10.
1038/s41467-023-37947-2
Molecular mechanisms of tubulogenesis

revealed in the sea star hydro-vascular organ
1,3
Received: 14 September 2022 Margherita Perillo , S. Zachary Swartz2,3, Cosmo Pieplow1 &
Gary M. Wessel 1
Accepted: 6 April 2023
A fundamental goal in the organogenesis field is to understand how cells

Check for updates organize into tubular shapes. Toward this aim, we have established the hydro-
vascular organ in the sea star Patiria miniata as a model for tubulogenesis. In
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this animal, bilateral tubes grow out from the tip of the developing gut, and
precisely extend to specific sites in the larva. This growth involves cell
migration coupled with mitosis in distinct zones. Cell proliferation requires
FGF signaling, whereas the three-dimensional orientation of the organ
depends on Wnt signaling. Specification and maintenance of tube cell fate
requires Delta/Notch signaling. Moreover, we identify target genes of the FGF
pathway that contribute to tube morphology, revealing molecular mechan-
isms for tube outgrowth. Finally, we report that FGF activates the Six1/2
transcription factor, which serves as an evolutionarily ancient regulator of
branching morphogenesis. This study uncovers distinct mechanisms of
tubulogenesis in vivo and we propose that cellular dynamics in the sea star
hydro-vascular organ represents a key comparison for understanding the
evolution of vertebrate organs.
The coordinated organization of cells into a precise three-dimensional elegans, C. intestinalis, zebrafish, mice, cell culture, and organoids3–7.
architecture is critical for establishing functional organs in develop- However, there are some key differences amongst these systems. For
ment. Tubulogenesis, the process by which hollow tubes form, is an example, while it is broadly true that cells within tubular epithelia must
important transient step in the development of many organs, includ- become polarized, an essential step for lumen formation, polarization
ing the neural tube, heart and pancreas1. In addition, many vital organs is acquired differently in diverse organs and species4. Similarly, cell
are ultimately organized into tubular shapes with the essential scope of migration, morphogenetic movements, and mitotic division are
transporting fluids, gasses, or cells, as in the case of kidneys, mammary important for forming the three-dimensional structures. However,
glands, and vasculature2. each of these parameters contribute to different extents in different
A great diversity of mechanisms are used to form hollow tubes in species. For instance, while in Drosophila tubular organs cells undergo
animals, including how cells reach their final position and distinct massive proliferation before migration, in vertebrates cell proliferation
external cues that guide the proper organ shape1,2. Abnormalities in and migration are coupled6,8–10. From an evolutionary perspective, it is
these processes can cause congenital disorders, dysfunctional or dis- therefore an important challenge to determine whether these different
placed organs, and loss of organ symmetry. Yet, because of the great cell mechanics in organogenesis reflect the evolution of organs, and
diversity of tubular structures, the general and conserved mechanisms whether a basal tubulogenesis toolkit may exist.
of tube formation in organogenesis are not completely understood1,2. Similarly, it remains unclear whether there exists a core and
Our knowledge of tube formation is largely derived from specific conserved network of signaling pathways that guide cell movements
organs within selected model systems, including D. melanogaster, C. and fate decisions in tubulogenesis. For example, in angiogenesis and
1
Department of Molecular, Cellular Biology and Biochemistry, BioMed Division, Brown University, 185 Meeting Street, Providence, RI 02912, USA. 2Whitehead
Institute for Biomedical Research, 455 Main Street, Cambridge, MA 02142, USA. 3Present address: Marine Biological Laboratory, 7 MBL Street, Woods Hole,
MA 02543, USA. e-mail: mperillo@mbl.edu; rhet@brown.edu
Nature Communications | (2023)14:2402 1

Article https://doi.org/10.1038/s41467-023-37947-2
vasculogenesis, Notch signaling regulates the differentiation of vas- luminal side (Fig. 1e insert 1). Although the basal lamina was generally
cular endothelial cells11. However, whether Notch participates in tube contiguous with the tube epithelium, we observed a laminin protru-
formation in other contexts remains poorly understood. In addition, sion on the anterior side of the tube that was devoid of cells (Fig. 1e–e”,
Wnt signaling controls the development of branching organs with f arrows). This region where cells are temporarily lacking was also
complex architectures in both vertebrates and invertebrates12,13. observed in live samples (Fig. 1f). A closer examination showed that
However, because of the complexity of these organs, other morpho- some cells were within this laminin layer (Fig. 1e insert 2). With live
logical aspects besides branching have been difficult to investigate. imaging of actin and nuclei, we observed the dynamic movements of
Furthermore, FGF signaling is important for tubulogenesis across epithelial cells on the anterior side of the growing tubes that migrate
species, including the mouse lungs and kidney14,15. Nevertheless, many out of the epithelium towards the blastocoel (Fig. 1g and Supplemen-
critical aspects of FGF action are still poorly understood, including the tary Movie 3), suggesting that cells left the laminin network on the
downstream targets of FGF signaling, and the conservation of these anterior side. Similarly, areas devoid of laminin on the anterior region
target genes across organ types and species. were observed until the late gastrulae (Fig. 1h–h”). In larvae, the two
The canonical model systems from which most of our knowledge tubes are fully separated from the gut and the laminin layer fully
of tubulogenesis derives generally belong to two main phylogenetic covered the tubes (Fig. 1i, j). In the fully-formed tubes, we detected cilia
groups: ecdysozoans (e.g. worms and flies) and chordates (e.g. tuni- beating (Supplementary Movie 4) suggesting that fluids are circulating
cates and vertebrates) (Fig. 1a). Echinoderms, which include sea inside the lumen of the tubes and that these are not primary cilia.
urchins and sea stars, are part of the sister group to chordates and as These results overall indicate that the progenitors of the hydro-
such occupy a critical phylogenetic position to understand evolution vascular organ are polarized early in the development of this structure
of vertebrate organs, while offering a series of experimental advan- and form a dynamic epithelium.
tages. Here, we take advantage of the sea star Patiria miniata as a
powerful system to study tubulogenesis within an intact organism. The Tubulogenesis involves directional cell migration
sea star larva is genetically tractable, optically transparent, and forms a By live imaging, we determined that the tubes elongate from cells at
tubular coelom organ called the anterior coeloms, coelomic sacs, or the top of the growing gut (Supplementary Movie 1). We tracked
the hydro-axocoel (hydro-vascular organ from here on)16. The hydro- single-cell movements from late gastrula to early larva stage in
vascular organ functions as a hydrostatic skeleton to allow the larva to embryos expressing H2B-GFP to label nuclei. We recorded cell
balance in the water column, and as such it is a simple, but vital organ17. movements with respect to the anterior-posterior axis (A-P) and the
In this work, using live imaging and functional approaches, we left-right axis (L-R) (Supplementary Movie 5–8 and Supplementary
identify the intrinsic (cell migration and proliferation) and extrinsic Fig. 2a, b). With this approach, we identified two distinct phases of cell
(external cues) factors driving the first steps of tubulogenesis in this migration. In the first phase, cells made substantial movements
system. We reveal that in an early branching deuterostome, directional towards the posterior end of the embryo (Fig. 2a, b, e, g). On average,
cell migration coupled with cell division drives tubulogenesis. The cells made the most progress along the A-P axis and completed most
initial tube outgrowth, the oriented elongation, and the differentiation of their overall movements during this first phase, calculated as the
of mesodermal cells as epithelial tube cells rely of the FGF, Wnt and total migration (Fig. 2h). In the second phase, cells maintained their
Delta/Notch pathways. Our work defines a basic toolkit from where the relative positions along the A-P axis (Fig. 2c, d, e–g) and moved on the
chordate tubular organs might have evolved. L-R axis more than on the A-P axis (Fig. 2h). We thereby uncovered a
two-phase program of cell movements, in which the cells first move
Results towards the posterior axis to promote rapid tube elongation, and
The hydro-vascular epithelium polarizes in early development then along the L-R axis to promote tube expansion.
Tubulogenesis is a highly dynamic process of individualized cell Active cell migration is associated with protrusions rich in actin,
movements and tissue-level morphogenesis. To define the nature of including lamellipodia and filopodia19. To further assess active cell
sea star tube formation, we took advantage of the optical clarity of the migration we visualized actin-rich protrusions over several hours with
larvae and developed techniques to immobilize and live image mor- LifeAct and we observed the presence of dynamic filopodia projecting
phogenesis continuously from gastrulation to early larval stages from the tube epithelial cells during migration (Supplementary
(Supplementary Movie 1). To standardize the different steps of tubu- Movie 9 and Fig. 2i). These filopodia extend from the cell edge rich in
logenesis, we defined a naming scheme to summarize the critical actin that also changes dynamically, indicating active cell migration.
stages of tube development (Fig. 1b, Supplementary Fig.1). The hydro- Tubes are surrounded by a layer of laminin (Fig. 1h) that links epithelial
vascular organ derives from mesodermal progenitors18 that, from the cells to collagen in the extracellular matrix20. This layer is not uniform
tip of the growing gut, divide into two bilateral tubes which grow and there are multiple areas where laminin is missing, and where cell
towards the posterior end of the larva (Supplementary Movie 2). The protrusions expand out (Fig. 2j, k). To test whether a plastic basal
two tubes ultimately merge to form a contiguous system with one lamina is necessary for tubulogenesis, we inhibited collagen cross-
opening towards the external environment, the hydropore canal linking with beta-aminopropionitrile (BAPN), an inhibitor of the
(Fig. 1c, d–d”). This entire process happens in ~15 h, as shown in Sup- enzyme lysyl oxidase. In embryos where ECM remodeling was blocked,
plementary Movie 1. the laminin protrusion that normally forms anteriorly was missing and
Our first goal was to define the tissue morphologically. A funda- tubulogenesis did not begin (Fig. 2l 1 and 2, m). However, when the
mental feature of tubular organs is the presence of a polarized epi- drug was removed the laminin protrusion reappeared and tubes slowly
thelium, so we tested whether the cells displayed apical-basal polarity started to grow back (Fig. 2l 3 and 4, m). Altogether these results
by visualizing actin and the basal lamina component laminin. A trans- suggest that the two phases of tube extension and expansion involve
verse view through the hydropore canal showed that the tube epi- active cell migration that ECM remodeling is also required for
thelium is polarized, with the apical side of the cells rich in actin tubulogenesis.
cytoskeleton facing the lumen and a basal lamina delimiting the blas-
tocoel environment (Fig. 1d–d”). We found that the tubes were con- Mitotic growth zones contribute to tube elongation
tinuously enwrapped by a layer of laminin from where they originated We next tested whether mitosis contributes to construction of the
(Fig. 1e–j). At the onset of tubulogenesis (gastrula stage), before the hydro-vascular organ by quantifying the relative frequency of mitosis
two tubes fully separated from the gut, cells were already polarized, as along the tubes. Using EdU pulse experiments, we found that the
shown by the presence of cilia that defines the apical surface on the overall cell proliferation along the tube increased from LG until L1 (first

a Protostomes b Larva Late larva
Bilateria Echinoderms Anterior
Right
Left
Posterior
Deuterostomes Mouth
Muscles
Chordates
Hydropore
Esophagous
Canal
c (HC)
Late Gastrula Early Larva Larva
(LG) (EL) (L) Stomach
Intestine
Tubes Hydropore Canal

(Initial steps of
tubulogenesis) d d’ basal
lumen
*
Gastrula (G) Late Larva (LL)
apical transversal view

d’’
Hydro-vascular
organ
(final organ)
Laminin / Phalloidin / DAPI
Laminin DAPI (Nuclei)

e Laminin/α-tubulin/DAPI e’ e’’ g hh:mm
2
LifeAct
A H2B
1
Gastrula
gut
2
f H2B-GFP (Nuclei)
1 hh:mm
Laminin DAPI (Nuclei) Late larva

h Laminin / α-tubulin / DAPI h’ h’’
Late gastrula
mouth
i i’ i’’
Early larva
P DAPI
Laminin
α-tubulin
larval stage) and then sharply decreased until the L3 (last larval stage) cell proliferation was higher in the middle and tip cells than the stalk
(Fig. 3a). We next asked whether cell proliferation occurs stochastically cells (Fig. e). In the first larva stages (L1-L2), the middle and tip zone
along the tube, or in spatially localized growth zones. We therefore cells were dividing the most, whereas by L3 stage, proliferation had
quantified EdU positive cells within three regions of the tube: the stalk decreased throughout the tube (Fig. 3d, e).
(anterior, where cell migration starts, close to the growing gut), the The left and right tubes are morphologically distinct, with the left
middle, and tip cells (posterior-most) (Fig. 3b, d and Supplementary tube specifically branching out to create the hydropore canal, and so
Fig. 3a–c). In the initial stages of growth, late gastrula and early larva, we tested whether there were proliferative differences between them.

Fig. 1 | Development of the sea star larva hydro-vascular organ. a Phylogenetic side. Dotted lines indicate the polarity of a single tube cell. Scale bars 10 μm. e–e’
relationships of the main bilaterian groups. b Summary of sea star larval develop- Laminin staining showing the basal lamina protrusion (arrow) in gastrulae. Alpha
ment with a focus on the hydro-vascular organ (in magenta). The hydro-vascular tubulin marks cilia in the tube lumen (Insert 1); a cell in between the basal lamina
organ comes from mesodermal precursors located at the tip of the growing gut in (insert 2, a different z-stack of Fig. 1 E). f Single stacks from a time lapse showing
gastrula (G). Tubulogenesis starts in the late gastrula (LG), continues in the early regions of the anterior-end of the growing tubes lacking cells (arrows). g Single
larva (EL) and larval stages (L1 to L3). In larval stages, the initial tubes that form the frames from Supplementary Movie 3 showing a cell extending actin-rich protru-
organ elongate posteriorly. The left tube forms the hydropore canal, an opening sions and leaving the epithelium. h–h” Laminin protrusions still present at late
toward the outside environment. The organ is fully grown in late larva (LL), when gastrula (arrows). i, j At larval stages tubes are completely separated from the gut
the left and right tubes merge to form the closed system of the hydro-vascular and the laminin layer is continuous. All experiments were independently repeated
organ. c Summary of the main larval tissues and organs. d–d” Transversal view of at least 3 times with similar results. d: scale bar 10 μm; e, g–j: scale bars 50 μm. A
the tube trough the hydropore canal showing that the epithelium is polarized with anterior, P posterior.
actin on the apical side of the cells (facing the lumen) and laminin on the cell basal
We found that the tip cells of the left tube proliferated more than the contribution of extrinsic signaling pathways. In the sea star embryo,
right one, from the EL to L3 stages (Fig. 3f). Cell proliferation was Delta-Notch signaling is critical during gastrulation for specifying all
particularly active in the hydropore canal (Insert in Fig. 3d, transversal mesodermal cell types, including the hydro-vascular organ precursor
view). We found no difference in cell proliferation at the middle zone cells, migratory mesoderm, and muscles22,23. To test whether Delta-
between left and right tubes (Supplementary Fig. 3E). In the late larva, Notch signaling could also guide later steps of tube formation we first
cell proliferation occurred exclusively in the posterior-most region of examined the spatial expression of the Delta ligand after gastrulation
the final tube organ (Supplementary Fig. 3f). In summary, an extensive and detected Delta mRNA in different tissues, including most tube
cell division during tubulogenesis occurs within specific zones of the cells (Fig. 4a). The Notch receptor was instead expressed ubiquitously
middle and tip areas of the tubes, and especially within the left tube tip. (Supplementary Fig. 4a), as also reported in other studies22. To define
We next asked whether oriented cell division could underly the the contribution of the Delta+ tube cells to tubulogenesis we gener-
directional elongation of the tubes. We first measured the angle that ated Delta knockout embryos. In Delta KO late gastrulae, the tube was
mitotic cells make respect to the direction of growth (the anterior- not specified and there was an increase of mesodermal cells expressing
posterior axis). If orientated cell division was a driving force for tube Erg and Ets1/2 (Fig. 4b–d), markers of mesenchyme cells22,23. These
growth, we predicted a division angle close to 0° during phase 1 (A-P results are consistent with previously reported pharmacological Notch
growth, tube elongation) and close to 90° in phase 2 (L-R growth, tube inhibitions22.
expansion). We found that the orientation of cell division is non-ran- To test the contribution of Delta+ cells to later stages of tubulo-
dom, occurring with an average angle of 36.9° degrees with respect to genesis, we used the γ-secretase inhibitor DAPT to block Delta/Notch
the A-P axis (Fig. 3g–h’, Supplementary Fig. 3g), with no significant signaling with temporal control22,24. When we treated embryos at the
difference between phase 1 and phase 2 (Supplementary Fig. 3h). end of gastrulation with DAPT, after tube cells were already specified,
Despite the orientation of the division plane, after mitosis completes, Delta-Notch inhibited larvae displayed increased muscle fibers wrap-
the daughter cells do not follow the direction of the angle they divided ped around the tubes, even resulting in increased constriction
but instead continue migrating first along the A-P axis and then along (Fig. 4e–g, Supplementary Fig. 4b). These muscle fibers appeared to
the L-R axis (example in Supplementary Movie 10). These results originate from the tube itself (arrows in Fig. 4f, g, inserts 1 and 2). In
support the occurrence of oriented cell division, but it does not cor- further support of excess muscle specification in the absence of Delta/
relate with the direction of tube growth. We therefore suggest that cell Notch signaling, we detected increased expression of the muscle
movement in the tubes is distinct from cell division, and rather marker myosin heavy chain (MHC) (Fig. 4h). In addition, the treated
represents an active migration process. The observed oriented cell larvae had short and thin tubes (Fig. 4i, j). These results suggest that
division may instead function in another aspect of organ formation, continued Delta/Notch signaling is important to restrict excess muscle
such as expansion of the tube diameter. specification, and perturbations of this pathway results in
We next asked whether cell division was required for tubulo- thinner tubes.
genesis by blocking cell cycle progression in G1 using the Cdk4/6 We next asked how gene expression in the tubes was affected
inhibitor Abemaciclib (LY2835219)21. First, to test whether cell pro- under Delta/Notch inhibition. We tested a panel of known genes
liferation was necessary for initial tube outgrowth, we added the expressed in the tubes in this animal, including Six1/2, SoxE, Alx4
inhibitor from the end of gastrulation until the early larva stage. (Alx1l), and Pax618,22,25. We found that the expression of these genes was
Importantly, and consistent with a G1 arrest and failure to replicate unchanged, despite the tubes being smaller (Fig. 4h). Some Delta+ cells
DNA, the tube cells did not incorporate EdU under these conditions in the digestive system expressed the genes FoxA and GataE23, which
(Fig. 3j–l). When cell cycle progression was blocked, tube outgrowth we also found to be unaffected. As seen in previous studies22,23, Delta
was fully prevented (Fig. 3j, m). Second, to test whether ongoing cell mRNA expression was increased when Delta/Notch signaling is
division was needed after initial outgrowth to support continued blocked, indicating that the perturbation was effective (Fig. 4e). These
elongation, we instead treated embryos from late gastrula to early data suggest that although early Delta/Notch signaling is required for
larva (Fig. 3k). We found that cell cycle arrest at this stage prevented tube cell specification22,23, known markers of the tubes were not
further elongation, with the length of the tubes remaining the same as affected by later stage Notch inhibition. Instead, we found that Notch
when the drug was added (Fig. 3m). These results indicate that the signaling at later stages prevents the uncontrolled specification of
cells that make the organ are not completely segregated early in muscle-like cells from the tube cells (Fig. 4k).
embryogenesis but rather are generated by continual cell prolifera-
tion. Thus, the number of cells in the tube epithelium increases over Wnt signaling guides directional growth of the tubes
time and this cell proliferation is critical to both initiate and sustain Since the sea star tubes stereotypically extend along the anterior-
tubulogenesis. posterior axis, we asked what signals guide this polarized growth. The
Wnt pathway is a broadly conserved regulator of polarity at the orga-
Delta-Notch signaling directs cell-fate decisions nismal level and branching for many tubular organs12,13 and inhibition
Having defined that epithelial cells migrate, and that mitosis is an of Wnt signaling in the early stages of sea star development blocks the
essential intrinsic force for tube development, we next tested the formation of endomesodermal structures26. To test the later

Fig. 2 | Tube extension and expansion involve active cell migration. a–d Time phase 2, cell displacement happens along the L-R axis. Track data for n = 50 cells
lapse images of the tubes expressing H2B-GFP to track movements of single nuclei from two independent movies. i Image from Supplementary Movie 9 showing actin-
(Supplementary Movie 6). Tracked cells are labeled with unique colors/numbers to rich filopodia from tube cells (arrows), sb = 10 μm. j, k Cell protrusion marked by
visualize trajectories. In 2D projections, cell movements are recorded along the A-P tubulin extend out from a gap in the basal lamina (arrows and green dotted box);
axis (advancing direction) and the L-R axis. Note that in A and B, cell 1 has under- sb = 10 μm. Graph indicates intensity of laminin antibody staining to highlight that
gone an epithelial to mesenchymal transition (EMT) and migrates into the blas- where the cell protrusion extends out there is a drop in laminin signal intensity.
tocoel, away from the elongating tube. e Cell migration along the A-P axis of the l Pharmacological approach to block ECM remodeling inhibits tube extension;
embryo (the y axis of the movie) over time. Cell 9 from is highlighted in green to sb = 20 μm. m Tube length quantification in BAPN treatments. n = number of larvae
show the two phases of migration. Graph includes cell trajectories of both tubes for is 17 (1); 34 (2); 16 (3); 17 (4). For all graphs, statistical significance was assessed by a
2 independent movies, (Supplementary Movie 5–8; n = 30 cells). f Average cell two- sided Student’s t-test (****p < 0.0001; ns not significant). In box plots the
velocity for phase 1 and 2 for 2 independent movies. n = 19 (phase 1) and 20 (phase median is the middle line, box represents 25th and 75th percentiles, whiskers
2); ***p = 0.0008. g The trajectory of Cell 9 exemplifies cell movement along A-P indicate the minimum and maximum data range. Data are presented as mean
and L-R axes. c represents the total cell displacement and is calculated as shown. values +/− SEM. Source data are provided as a Source Data file.
h Maximum cell displacement happens in phase 1 and occurs along the A-P axis. In
contribution of Wnt signaling to tubulogenesis, we blocked secretion in controls (Fig. 5a, b). As a control, we tested the expression of a
of all Wnt ligands after gastrulation with the porcupine inhibitor ETC- known beta catenin target, Wnt826, and found it to be downregulated
15927. Importantly, treatment at this stage does not grossly affect pri- with ETC-159 treatment, while the expression of tube and muscle
mary body axis or endomesoderm specification, which are established associated genes were unchanged (Fig. 5c). Consistent with the broad
earlier in development. We found that in Wnt-inhibited larvae, the expression and role of Wnt genes26, several developmental defects
tubes elongated towards the dorsal ectoderm instead of posteriorly, as were observed, including disorganized muscle patterning and straight

a Proliferation b Late Gastrula d Larva e

50 Left Right L1 L2 L3
in tubes
n° of proliferating cells
Stalk 15
Stalk
40 10
Middle
Middle
5
Tip
Number of EdU+ cells
30 0
Tip LG EL L1 L2 L3
c Early Larva
20 Left Right
10 tv
0
Hydropore canal
LG EL L1 L2 L3 EdU / DAPI / α-tubulin
f g h g’ i Cell trajectory
TIP cells left tube
ns right tube
15 150
*** *
10
h’
A-P (µm)
200
ns b
a
*
5
R L A
250 b
a
0 90°
b 300
LG EL L1 L2 L3 a P 50 100 150
LifeAct L-R (µm)
DAPI 0°
M Abemaciclib
G1
G2
S CDK4/6-CyclinD1
Control Abemaciclib 25uM Cell
j DAPI / EdU
l proliferation m Tube length
****
G ****
150
20
LG **** 100 ****

LG
µm
10
k
50
LG 0
0
)
)
EL
)
EL
)
EL
LG
LG
L)
)
)
G
G
-->
L)
-->
(E
-->
-->
(L
(L
(E
G
G
ol
(G
(G
ol
ol
(L
ol
(L
tr
tr
tr
tr
be
be
on
be
be
on
on
on
EL
A
A
A
C
A
C
C
gut (Fig. 5b). Our data suggest that Wnt signaling drives directional instead of posteriorly, which phenocopied Wnt inhibition by EC-159
growth of the tubes towards the posterior end of the larva. (Fig. 5e, f). This approach was more spatially targeted, as shown by
We next sought to define the specific Wnt signaling pathway and unaltered levels of Wnt8 (Fig. 5c). Although tubes in Fz1/2/7 KO larvae
its receptor used in tubulogenesis. A prior study identified Fz1/2/7 as looked smaller than controls, their length relative to the esophagus
initially localized broadly in the tip of the archenteron, and later (that in controls runs parallel to the tubes) was comparable, indicating
selectively in the esophagus and in the tubes that form the hydro- that in these smaller larvae, the tubes reach their maximum length
vascular organ28. We further defined the localization of Fz1/2/7 mRNA, proportionally (Fig. 5g).
and found its expression enriched in the anterior tube cells (Fig. 5d). To To assess the directionality of tube elongation we measured the
test whether tube orientation was dependent on Fz1/2/7, we generated angle that each tube formed with the larval anterior-posterior axis and
Fz1/2/7 knockout embryos and found that the tubes elongated dorsally found that this angle is similar in Fz1/2/7 KO and ETC-159 treated

Fig. 3 | Cell division during tube outgrowth. a EdU pulse experiments measure Supplementary Movie 10. Arrow indicates the mitotic event, where a and b are the
frequency of cell proliferation during tubulogenesis from tube outgrowth to larva two daughter cells. j, k EdU staining in larvae treated with Abemaciclib to arrest
stages (as defined in Supplementary Fig.1). Number of total larvae analyzed n = 60. cells in G1 from gastrula to late gastrula (during tube outgrowth) or from late
b–d EdU staining (magenta) in the three tube areas: stalk, middle and tip cells. gastrula to early larva (during tube elongation). Dotted lines indicate the tubes.
Arrowheads in (c) and (d) indicate nuclei with incorporated EdU. Insert in (d) shows Scale bar 20 μm. l EdU incorporation in presence of Abemaciclib to test inhibition
a transverse view (tv) of the hydropore canal from another specimen with nuclei of cell cycle and (m) effects on tube outgrowth and elongation. Number of larvae
stained with EdU. Scale bars 10 μm (b, c), 20 μm (d), 5 μm (insert). e Heatmap n = 16 (control LG); 32 (Abe G- > LG); 36 (control EL); 43 (Abe LG- > EL). LG (late
showing frequency of cell proliferation (white is zero, dark blue is max). gastrula); EL (early larva); L1 (larva stage 1); L2 (larva stage 2); L3 (larva stage 3).
f Frequency of cell proliferation in the tip cells for left and right tubes. n = 60 total Statistical significance was assessed by a two- sided Student’s t-test (****p < 0.0001;
number of larvae. Two-sided Student’s t-test: EL (***p-value = 0.0002), L1 (*p-value = ns not significant). In box plots the median is the middle line, box represents 25th
0.0260), L3 (*p-value = 0.0409). g, h Examples of a dividing cells from Supple- and 75th percentiles, whiskers indicate the minimum and maximum data range.
mentary Movie 10; magnification of a mitotic cell (g’) and the same cell undergoing Source data are provided as a Source Data file.
cytokinesis (h’); sb = 5 μm. i Trajectory of a cell that divides, taken from
embryos but greater than in controls (Fig. 5h). We conclude that Wnt gastrulation. We then performed differential RNA-seq on these sam-
signaling via the Fz1/2/7 receptor guides oriented growth of the left ples. In the analysis we selected candidates with an FDR SU5402/
and right tubes towards the posterior end of the larva, the site where Controls <0.05 and fold change threshold of ±1.6 log2 SU5402/Con-
the two tubes will eventually fuse to form the hydro-vascular organ of trols, as used in previous studies32. With this approach, our analysis
the late larva. identified 147 downregulated and 346 upregulated transcripts (Fig. 7a,
Supplementary Data 4). We then validated this dataset by performing
FGF initiates elongation by controlling cell proliferation fluorescent in situ hybridization to test transcript localization, and we
FGF signaling has been implicated in the morphogenesis of many kinds further investigated the function of selected genes.
of tubular structures29. Consistent with a potential role in forming the As the SU5402 treatment inhibits tube formation, we focused on
tubes, we found that the FGF receptor is expressed by scattered tube genes that were downregulated to identify candidates were uniquely
and esophageal cells (Fig. 6a). To test whether FGF signaling is required expressed in the initial tubes. Most of the downregulated genes were
for tube formation, we used the FGFR inhibitor SU54030. We found that involved in extracellular matrix homeostasis (Supplementary Fig. 6,
larvae in which FGFR was inhibited following gastrulation had partially Supplementary Data 4). For example, we identified the extracellular
missing tubes (Fig. 6b, c). As a second approach, we used the inhibitor metallo-protease matrixin as a target of FGFR (Fig. 7b, Supplementary
U0126 to block phosphorylation of the FGFR downstream effector Fig. 7). This protease is secreted and its transcripts were enriched in
kinase MEK31. This treatment also resulted in a partial lack of tubes, lateral tube cells (Fig. 7b). Our RNA-seq study also identified the
similar to the upstream FGFR inhibition (Fig. 6d). We then knocked out secreted Kazal type serine protease inhibitor FcoII as a FGFR target,
FGFR and found that also with this third approach the tubes developed which we found to be enriched in the growing tubes (Fig. 7c, d, Sup-
only partially (Fig. 6e, cartoon in f), with a significant decrease in tube plementary Fig. 8). Protease inhibitors spatially and temporarily con-
length and area compared to control early larvae (Fig. 6g–h). More- trol protease activity to tune ECM remodeling. In the first step of
over, when FGFR was knocked out or if its kinase domain was blocked, tubulogenesis a transient basal lamina protrusion appears on the
genes normally expressed in the tubes and the muscle marker MHC anterior side of the tubes and then disappears once the two tubes
were significantly downregulated (Fig. 6i). Aside from lacking the separate (Fig. 1e, f). To test its role in this remodeling, we knocked
tubes, the overall anatomy of the late larva was normal (Supplemen- down FcoII, and found that this protrusion was stabilized. Moreover,
tary Fig. 5), indicating a highly selective role of FGF signaling in tubu- the organ did not grow properly since the two bilateral tubes remained
logenesis. Consistent with inactivation of the FGF pathway, we found connected on the anterior most-end (Supplementary Fig. 9a).
that all of these manipulations resulted in a decrease in nuclear pERK, Another group of genes downregulated in FGFR blocked larvae
the target of MEK (Fig. 6j, k). After two weeks, while controls were alive, were transporters. The ATP-binding cassette sub-family C member 4
the FGFR KO larvae died, suggesting that lack of the tube organ is (MRP4 or ABCC4) is a multidrug resistance-associated protein whose
critical for larval survival. expression in the mouse brain is dependent on FGFR133. However, a
Knowing that FGF signaling promotes cell proliferation through role for ABCC4 in other morphogenetic mechanisms is unknown. We
pERK31, we tested whether FGFR influenced the cell proliferation we found that ABCC4 was a FGF target and was expressed in the initial
observed in the initial steps of tubulogenesis. We performed EdU pulse tube, particularly in the stalk cells (Fig. 7e, Supplementary Fig.10),
labeling to mark cells that have synthesized DNA in early FGFR suggesting that the link with the FGF pathway is conserved across
knockout larvae and found a significant reduction of proliferating tube species, even if the transporter is expressed in different tissues.
cells (Fig. 6m, n). This reduction in FGF-dependent cell proliferation Another class of genes downregulated when FGFR was blocked
was specific for the tube cells since cells of the esophagus that also were muscle-related genes, including troponin, myophilin, twist,
expressed FGFR (Fig. 6a) did not show pERK activity (Fig. 6j, k) and myosin light and heavy chain (Supplementary Data 4). We found that
proliferation was not reduced there when FGFR is knocked out the muscle marker myosin heavy chain (MHC) was initially expressed
(Fig. 6m). We conclude that the FGF signaling pathway initiates tube first by a few tube cells, but once the tubes elongated, they formed two
outgrowth by promoting tube cell proliferation through distinct populations of muscles (Fig. 7f, g). The first one was a muscle in
pERK (Fig. 6o). the hydropore canal (Fig. 7g, insert 1). Here, MHC transcripts marked
cells that did not have long fibers, similar to the intracellular contrac-
RNA-seq reveals FGF-induced genes during tubulogenesis tion apparatus seen in the endodermal muscles of the sea star pyloric
The FGF pathway is involved in multiple aspects of tubulogenesis, but sphincter (** in Fig. 7g) and in sea urchin larva pyloric and anal
the genes that link it to morphogenesis are still poorly understood29. sphincters34. The second muscle cells formed longitudinal fibers that
Blocking FGF signaling either genetically or pharmacologically pre- appeared in L3 stage (Fig. 7h) and later extended along the whole
cisely ablates the tube structures in sea star larvae (Fig. 6c–e). To length of the hydro-vascular organ (Fig. 7i, j). We further investigated
define the landscape of genes activated by the FGF pathway, and the function of the longitudinal muscles we identified with live imaging
potential genes that define cell types of the hydro-vascular organ, we and found that the twitching of these muscles allowed for the con-
generated tube-ablated larvae by SU5402 treatment soon after traction of the hydro-vascular organ (Supplementary Movie 11).

delta mRNA Control Cas9 Delta KO

a b Phalloidin
DAPI
c d 4 Delta
KO
Fc (log2)
1
-1
/2
ta
g
Er
s1
73/73
el
60/60
Et
Control DAPT from LG L
e f 1 g
2
Larva
1 2
Ventral Ventral Lateral

Phalloidin / DAPI
h 4
DAPT i Tube j Tube k
length area Late Gastrula (LG)
200 **** 8000

3 ****
Fc (log2)
2 tubes gut
(µm)
(µm2)
1 100 4000
0
+ DAPT
Controls
(blocks Notch)
-1 0 0
Larva (L)
l
ol
PT
PT
ro
ta
/6
C
4
xE
x6
xA
tr
t
1/
lx
A
A
H
el
/5
on
on
Pa
So
D
Fo
D
A
M
D
a4
x
C
C
Si
at
G
Fig. 4 | Delta-Notch signaling represses a muscle phenotype in the tubes. in treated embryos the tubes are shorter than the esophagus. h The increase in
a Fluorescent in situ hybridization (FISH) shows Delta gene expression in scattered muscle-like cells is also reflected by increase in expression of the muscle marker
cells of the gut and the tubes (highlighted by dotted lines). b, c Delta knock out MHC, while other known marker genes for tubes and the digestive system do not
embryos fail to form the epithelial tubes that instead become excess mesenchymal change. Bars represent mean with standard error of the mean (SEM). n = 3 biolo-
cells. Penetrance of the Delta KO phenotype was 100% and all embryos died at the gically independent experiments. i, j Tubes are significantly smaller in DAPT treated
late gastrula stage; for genomic mutation see Supplementary Fig.12. d qPCR than control embryos. Control: n = 15 larvae; DAPT treated n = 23 larvae. k Cartoon
showing increase of mesodermal markers Erg, Ets1/2 and Pax6 in delta KO embryos. summarizing the result of Notch inhibition. All experiments are representative of 3
Bars represent mean with standard error of the mean (SEM). e–g Larvae stained biological replicates. a, b, c, f, g scale bar = 50 μm. h scale bar is 10 μm. Statistical
with phalloidin to mark muscles; Delta-Notch inhibition leads to smaller tubes significance was assessed by a two- sided Student’s t-test (****p < 0.0001). In box
constricted by muscle fibers that wrap around the tubes (arrows indicate muscles, plots the median is the middle line, box represents 25th and 75th percentiles,
magnification in inserts 1 and 2) and the foreguts. Arrows in g show that tube cells whiskers indicate the minimum and maximum data range. Source data are pro-
become muscle-like. Note that the control tubes are as long as the esophagus, while vided as a Source Data file.
The transcription factors most affected by FGFR inhibition were found that Six1/2 is expressed in a defined structure of the tube (the
Alx4, that was enriched in the tube lateral cells (Fig. 7k) and Six1/2, that opening towards the outside environment), we focused on this gene
was first enriched in the tip of the left tube during tube elongation and and found that Six1/2 knockout larvae, while otherwise morphologi-
later it marked the hydropore canal (Fig. 7l, m, arrow). Because we cally normal, did not form the hydropore canal, even 3 days after this

Control ETC159 from LG L

a Dorsal view Lateral view b Dorsal view Lateral view c
ETC159 from LG
Fzl 1/2/7 KO
d Fz1/2/7 mRNA
1
Dorsal
0
Fc (log2)
-1
-2
-3
Phalloidin
4
C
nt
lx
DAPI
H
A
M
W
Control Cas9 Fz1/2/7 KO
e f Phalloidin g Tube length h Tube angle (A-P) i
DAPI
ns Control
1.5
**** Controls Fz1/2/7 KO
Tube lemgth / esophagus length

90°
60 ****
75°
angle (deg)
60°
40
1.0
Larva
45°
Dorsal
Fz1/2/7 KO
30° or +ETC159
20 Anterior
15° (Wnt inhibition)
°0
Dorsal
Dorsal
0.5 0
Posterior
Posterior
48/56
ol
60/60
59
O
O
s
tr
K
ol
K
c1
on
tr
7
Et
2/
on
2/
C
1/
1/
C
lateral view, right side

Fz
Fz
Fig. 5 | Wnt signaling directs tube orientation through the Fzd1/2/7 receptor. Supplementary Fig.13. g Tube length measured as a ratio of tube length/esophagus
a, b Wnt inhibition with ETC159 from late gastrula to L2 caused a change in length; n = 24 larvae for controls, n = 36 larvae for Fz1/2/7 KO. h Tube orientation
orientation of the tube outgrowth from anterior-posterior to anterior-dorsal. measured as the angle that the tube makes with respect to the anterior-posterior
Confocal z-stacks are dorsal views and lateral views obtained from orthogonal axis of the larva. Number of tubes measured is n = 30 for controls, n = 30 for ETC159
projections of the same larvae. c qPCR shows Wnt8 gene expression decreases treated and n = 23 for Fzd1/2/7 KO. Same data shown as a rose plot to indicate
when ETC159 (that block secretion of Wnts) is used. Bars represent mean with difference in orientation. i Schematic summarizes the observed phenotypes. All
standard error of the mean (SEM). n = 3 biologically independent experiments. experiments are representative of 3 biological replicates. a, b, d–f scale bar is 50
d FISH showing that the Wnt receptor Fzd1/2/7 is expressed in the tube cells in early μm. a and b lateral views are 20 μm. Statistical significance was assessed by a two-
larvae. e, f In control larvae tubes grow parallel to the anterior-posterior axis, while sided Student’s t-test (****p < 0.0001; ns = not significant). In box plots the median is
in Fzd1/2/7 knock out larvae the tubes grow towards the dorsal side and recapi- the middle line, box represents 25th and 75th percentiles, whiskers indicate the
tulate the same tube orientation defects seen with ETC159 treatment. Percentage of minimum and maximum data range. Source data are provided as a Source Data file.
the larvae showing the Fz1/2/7 KO phenotype was 86%; for genomic mutation see
structure normally forms in controls (Fig. 7n, o and knock down in remodeling genes and transcription factors. These observations in the
Supplementary Fig. 9b). Since the hydropore canal forms by tissue sea star, a representative of the sister group to all other deuterostomes
bending at a defined branch point, we propose that Six1/2 activation including vertebrates, provide fundamental insight into the evolution
downstream of FGF signaling is critical for branching morphogenesis. of tubular organs.
In summary, blocking the FGFR pharmacologically we found a variety
of candidate genes that are activated by the FGF pathway and that A transition in tubulogenesis appeared in deuterostomes
defined subpopulations of tube cells during tubulogenesis. A fundamental feature of tubular organs is that the constituent epi-
thelial cells are polarized. Tubes can form either from sheets of
Discussion polarized cells or from nonpolarized cells that can acquire polarization
The tremendous diversity in strategies to form hollow tubes suggests during morphogenesis to form a lumen de novo10. We found that the
that either these structures are not homologous, or that the toolkits to hydro-vascular organ develops from pre-polarized precursor cells,
build such structures are not conserved. Here, we took advantage of therefore positioning this organ among the first category. Figure 8a
the tubular hydro-vascular organ of the sea star larva to unravel the summarizes the main steps of the sea star tubular organ development.
mechanisms of tubulogenesis in an intact, early branching deuter- Mesodermal cells from the tip of the growing gut during gastrulation
ostome. We show that tube formation and elongation require that cells evaginate to form two initial tubes formed by a simple monolayer
migrate and divide in defined zones. Cell fate depends on Notch sig- epithelium that keeps its polarity during growth. Cells are ciliated and
naling, while tube orientation requires Wnt signaling through the rich in actin at the apical side facing the lumen, while the basal side
receptor Frizzled 1/2/7. In parallel, cell proliferation downstream of faces the basal lamina (Fig.1d–g). Similarly, organs like the Drosophila
FGF signaling is critical for tube outgrowth and extension. Finally, we salivary glands and vertebrate lungs also form from polarized pre-
find that FGF reception induces the expression of genes potentially cursors that migrate to accomplish tube morphogenesis2,10. However,
involved with hydro-vascular organ function, including muscles, ECM the mechanisms of cell dynamics in tubulogenesis differ between

Control FGF pathway inhibition G EL: FGFR KO

FGFR mRNA
a b Phalloidin
DAPI
c SU5402 d U0126 e
SU5402 G
f g Tube length h Tube area i EL
j pERK
U0126 G EL DAPI
Gastrula (G) 6000
150 **** FGFR KO (EL)
**** **** µm2 1
****
µm)
(µ
**** 0
100 4000
-1
**** -2
area (µm2)
Fc (log2)
Early Larva (EL)
(µm)
2000 -3
50
-4
Controls + SU5402
(blocks FGFR) -5
or + U0126 0
0
(blocks MEK) -6
or FGFR KO
Tube lateral view
C
-2
1
FR l
FR l
SU rol
26
ol
O
C 6
lx
FG tro
O
FG ro
02
FR
02
H
x1
2
K
tr
M
01
t
01
t
54
54
on
on
FG
on
on
Si
U
U
SU
C
C
C
k Control SU5402 l m Control n Cell o

pERK pERK intensity proliferation
DAPI Control
**** 60
****
150000
****
n° of EdU+ cells (% of tot tube cells)

Es
Signal Intensity
FGFR KO
****
100000
40
EdU
DAPI
U0126 FGFR KO FGFR KO

pERK
50000
20
cell
proliferation
0
0
Es
O
SU ol
FG rol
02
26
K
tr
54
t
on
FR
on
01
C
Control FGFR KO
Fig. 6 | FGF signaling promotes tube outgrowth through pERK. a FISH of FGFR k pERK staining is absent when FGF signaling is blocked. l Signal intensity of pERK in
shows expression in scattered cells. b Control and early larva treated with the FGFR the above experiments. The Y axis in the graph shows the raw integrated intensity
inhibitor SU5402 (30μM) (c) or with the MEK inhibitor U0126 (10μm) (d) from the for pERK normalized by the background signal for each image. m EdU staining to
end of gastrulation to early larvae (G→EL). e Early larvae in which the FGFR is mark proliferating cells and signal quantification (n). Since the tubes of the FGFR
knocked out by Cas9 lack the tubes. Percentage of the FGFR KO larvae showing the KO larvae had fewer cells than controls, we assessed proliferation rate by counting
phenotype was 96%; for genomic mutation see Supplementary Fig.14. f Cartoon the EdU+ cells normalized to the total number of tube cells; n = 48 larvae (controls),
summarizing FGFR inhibition phenotypes. g, h Tube length and area are sig- n = 50 larvae (FGFR KO). o Schematic showing that inhibition of the FGF pathway at
nificantly decreased in larvae where the FGF pathway was blocked. n = number of different levels prevents tube growth and pERK activation. White dotted lines
tubes (controls = 26, SU5402 = 26; U0126 = 20, CAS9 control = 48; FGFR KO = 50). outline the tubes. Es esophagus. Scale bar = 50 μm (a, e); scale bar = 20 μm
i qPCR of significant genes in larvae where tube outgrowth was prevented. Bars (b–d, j, k, m). Statistical significance was assessed by a two- sided Student’s t-test
represent mean with standard error of the mean (SEM). n = 3 biologically inde- (****p < 0.0001; ns not significant). In box plots the median is the middle line, box
pendent experiments. j Immunofluorescence showing that active ERK is localized in represents 25th and 75th percentiles, whiskers indicate the minimum and max-
the nuclei of most tube cells. Arrows indicate dividing cells without pERK staining. imum data range. Source data are provided as a Source Data file.
Drosophila and mammalian tubular organs. For instance, the Droso- migration are always coupled6,9,10. However, comparisons among tub-
phila salivary gland and the tracheal precursor cells complete pro- ular organs are complex since many of these organs have diverse
liferation prior to beginning tubular morphogenesis and only in later developmental origins, like the fly trachea that is derived from the
stages do tracheal cells divide during tube remodeling3,8, while in ectoderm, vertebrate lungs from the endoderm and kidney from the
vertebrate organs like lungs and kidney cell proliferation and mesoderm3,7,15. Moreover, tubular organs can use diverse growth

a -15 Number of differentially expressed genes ECM remodelling

Controls + SU5402
FDR p-value (-log10)
vs
b c LG d L
-10 Alx4 FcoII 1
MHC
Matrixin
-5
Six1/2
ABCC4
1
0.05
Matrixin FcoII FcoII
0
-10 -5 0 5 10 Phalloidin / α -Tubulin / DAPI
Fold change (log2)
Transporters
i j
Muscles
e f g Hydropore h
Right Side
s * * Canal
1
es
m
1
t
ABCC4 MHC ** tv lv
Transcriptional factors Control Cas9 Six1/2 KO

k l left right m n o
Control
l m
t t
lateral, left side
Six1/2 KO
EL L
Alx4 Six1/2
left side 65/65 56/62
Phalloidin / α -Tubulin / DAPI
Fig. 7 | Differential RNA-seq reveals genes responsive to FGF signaling. the esophageal muscles (es in g). i, j Longitudinal muscles are localized on the tube
a Volcano plot of differentially expressed genes between controls and larvae where ventral side. k–m FISH for Alx4 and Six1/2. Six1/2 is expressed at the tip of the left
FGFR was blocked. Using the Wald test, p-values, false discovery rate (FDR) and fold tube and later in the hydropore canal (arrow in m). n, o Six1/2 KO did not form the
changes were generated. Gene/Transcripts with p value < 0.05 were called as dif- hydropore canal (arrow). Percentage of the Six1/2 KO phenotype was 90%; for
ferentially expressed genes/transcripts for the comparison. b, c FISH for genes genomic mutation see Supplementary Fig.15. All images are maximum projections
involved in extracellular matrix (ECM) remodeling, matrixin (b), and Fcoll (c, d). of confocal z-stacks. All experiments were independently repeated at least 3 times
Insert 1 in (b) shows matrixin transcripts in cells extending protrusions. e FISH for with similar results. FDR false discovery rate, s stalk, m medial, t tip, tv transverse
the transporter ABCC4 shows expression in tube stalk cells. f–h FISH showing view, lv lateral view. scale bar = 50 μm (b–g, k–o); scale bar = 20 μm (i, j); scale
myosin heavy chain (MHC) gene expression in the hydropore canal (1), the pyloric bar = 10 μm (h). Source data are provided as a Source Data file.
sphincter (** in g), the tube longitudinal muscle (h), the dorsal muscles (* in g) and
strategies at different stages. Our aim is to define the basic toolkit that angle of oriented cell division during tube growth is right in between
gave rose to the deuterostome tubular organs. Are there generalizable these two phases of elongation and expansion, an ideal angle to favor
differences between vertebrates and invertebrates? Or, does this dif- the expansion of the diameter of the tube. A question that remains
ference instead reflect the consequence of distinct life histories unanswered is whether cell migration is also necessary for tubulo-
between individual species? genesis. Cell displacement during tubulogenesis is active, since mov-
Here we show that as in vertebrates, cell migration and cell pro- ing cells have actin-rich filopodia that extend out of the basal lamina
liferation are coupled during tubulogenesis in the sea star, an early- that surround the tubes (Supplementary Movie 9). Another hint that
branched deuterostome (Fig. 8b). To establish the initial tube, cells cell migration plays a role in tubulogenesis is given by the observation
undergo a two-phase migration from the tip of the growing gut to that when collagen crosslinks are blocked, tube outgrowth is pre-
finally run parallel to the gut itself (Supplementary Movies 5–8). The vented (Fig. 2l, m), suggesting that cells migrate along the basal lamina.

a Gastrula Late gastrula Larva Late larva

1 2 3 4
Anterior
Right
Left
laminin (basal side) / cilia (apical side) / nuclei Posterior
b Precursor cells Tube c d left tube

Protostomes Controls Inibition of: A
A (muscles)
Bilateria
Echinoderms
D
HC
Deuterostomes
Branching
P
Vertebrates P lateral dorsal
ventral lateral
Delta/ Wnt FGFR view view
proliferating cells Notch
Fig. 8 | Summary of the intrinsic and extrinsic factors that guide tubulogenesis the relationship of the in vivo systems for the study of tubulogenesis. In proto-
of the hydro-vascular organ. a Stages of sea star hydro-vascular organ develop- stomes tubular organs form by a first step of cell proliferation followed by mor-
ment. The precursor cells of the hydro-vascular organ come from mesodermal phogenesis. In echinoderms and in vertebrates (both deuterostomes) cell
progenitors at the tip of the growing gut (1). In the first stage of tubulogenesis, pre- proliferation is continuously coupled with cell migration during morphogenesis.
polarized cells form a lumen and grow towards the posterior side of the embryo to c Cartoon summarizing the role of signaling pathways on tube formation.
form two tubes (2). During embryogenesis, left and right tubes detach from the d Cartoon showing the branch formed by the left tube, the HC (hydropore canal)
esophagus (3) and finally form a close system where the left and right tube connect that opens on the outside environment. A anterior, P posterior, D dorsal.
anteriorly and posteriorly, the hydro-vascular organ. b Phylogenetic tree showing
If cell migration is critical for tube outgrowth, inhibition of the actin- Distinct signals regulate tube elongation, cell fate and
based cell protrusions specifically in the tubes should prevent the orientation
tissue from growing. Such experiments will be possible in the future The shape of epithelial tubes is guided by signaling pathways from the
thanks to the new technologies for inducible knock outs35 or cell-type embryonic environment. However, because of the great morphologi-
specific expression of dominant negative Rho-A, an important actin cal complexity of tubular organs, our knowledge on the contribution
regulator. of the extrinsic cues to distinct steps of tubular organ establishment is
Similarly to tubulogenesis in vertebrates, subsets of cells actively still limited29. The sea star hydro-vascular organ offers a simplified
divide during these migrations in the sea star. Mitosis plays a critical system in which we defined how the Delta/Notch, Wnt and FGF path-
role in tubulogenesis, since cell number grows over time and when it is ways each uniquely contribute to distinct aspects of tubulogen-
blocked, there is a significant lack of tissue. Localized cell proliferation esis (Fig. 8c).
is an important driver of shape changes, as in many mammalian tub- Tube cells share a common origin with muscles, as both tissues
ular organs36. In the sea star tubes, we find that proliferation is derive from the mesoderm22,23,30. Here we report that when we block
downstream of FGF signaling, and it is concentrated in two main Delta/Notch signaling, an important regulator of mesodermal
proliferation zones: the middle region of both tubes and the tip of the specification39, excess muscle fibers enwrap the tubes causing con-
left tube. We propose that the extensive proliferation in the middle of striction (Fig. 4e–g). There are two possible hypotheses on the origins
the tube drives A-P elongation. Instead, proliferation at the tip of the of these fibers. First, Delta/Notch signaling acts on cells from the
left tube may drive formation of the critical branch point that forms adjacent esophageal muscles to induce migration to the tubes and
the hydropore canal (Fig. 8c). Indeed, by blocking cell cycle progres- differentiation once there. Alternatively, our data support that Delta/
sion by Cdk4/6 inhibition, or FGF perturbations at different time Notch signaling might inhibit epithelia tube cell transfating into a
points, tube initiation or elongation was disrupted. FGF signaling muscle-like phenotype. Notch inhibits skeletal muscle differentiation
regulates proliferation in the mouse Wolffian duct14, in the kidney in many contexts, including during vascular system differentiation40.
metanephric mesenchyme37, and in the mammary gland38. Similarly, Furthermore, the observation that tubes are shorter when Delta/Notch
the FGF signaling pathway is required for the formation of homo- pathway is blocked might be a consequence of cells transfating into
logous tubular pouches in the sea urchin, a related echinoderm, sug- muscles, therefore leaving the epithelium with fewer cells. From our
gesting that these mechanisms are conserved within this clade30. These data, these ectopic muscle fibers appear to originate from tube epi-
similarities with vertebrate morphogenesis suggest that the coupling thelial cells (Fig. 4f, g). Moreover, we observed a few cells from the
of proliferation with cell migration may be an ancestral deuterostome tubes undergoing EMT to become mesenchymal (cell 1 in Fig. 2b and
innovation for the formation of tubular organs. It is further possible Supplementary Movies 3, 6), suggesting that some cells retained the
that FGF signaling may be independently important for regulating cell ability to acquire a distinct mesodermal fate. This phenotypic change,
migration. However, given its required role for cell proliferation, that eventually leads to loss of epithelial function, has been observed
without which the tubes do not form, future work will be required to to be the cause of lung fibrosis41, but it is unclear what pathways guide
decouple and test these putative functions. it. Overall, our data in the sea star suggests that the Delta/Notch

signaling might be responsible for such phenotypic change in complex not previously described in the sea star larva: muscles in the hydropore
organs. canal and longitudinal muscle fibers along the tubes (Fig. 7f–j). The
Although correct orientation is critical for tube function, very longitudinal muscle allows for the tubular organ to contract (Supple-
little is known about how tubular organs orient within the growing mentary Movie 11), while the movements of cilia inside the lumen helps
embryo29. Taking advantage of the simple morphology of the sea star moving fluids or particles in the tubes (Supplementary Movie 4). Stu-
tubes, we found that the Wnt signaling through the Frizzled 1/2/7 dies in other echinoderms showed that larvae have smooth muscles30,
receptor regulates tube orientation. Frizzled 2 receptor controls suggesting this is also the case of the longitudinal muscles around the
branch formation and shape of the epithelial tube of the highly bran- hydro-vascular system. Our findings show that the hydro-vascular
ched mouse lungs42, but a clear link with the overall orientation of this organ also includes muscles that surround this organ in a way that
structure was not established. A question raised by these findings is the resembles the vasculature or the intestine, specialized tubes that can
mechanism through which Fz1/2/7 affects orientation of tube growth. autonomously contract to move their contents forward.
We hypothesize that Wnt signaling may be acting through the planar
cell polarity pathway rather than the canonical pathway to guide shape A conserved regulatory network for branching morphogenesis
changes and tube directionality. In support of this premise, Wnt sig- In our RNAseq analysis of FGF targets, we identified the transcription
naling has been found to coordinate cell polarity in groups of cells, for factor Six1/2, which is selectively expressed in the hydropore canal
instance by controlled cell division orientation in the mouse stratified (Fig. 7l, m). By Cas9 knockout, we found it to be essential and
epithelium43. Another hypothesis to consider is that activation of Fz1/ selective for forming the hydropore canal branch point (Fig. 7n, o). In
2/7 drives expression of membrane proteins that bind to ECM com- earlier stages, Six1/2 was found to be expressed in the mesodermal
ponents to properly orient the tube outgrowth directionality. Last, the tube precursor cells in both sea stars and sea urchins18,22,30. In an
fact that the esophagus and the tubes, both expressing Fz1/2/7, were example of potentially homologous function, in the mouse, Six1
shorter than controls but kept the same length ratio raises the possi- plays a critical role in a subpopulation of collecting tubule epithelial
bility that Wnt signaling also stimulates cell proliferation in these tis- cells in the developing kidney50. Six1/2 is also expressed by distal
sues by stimulating cyclin genes, as has been shown in the pancreas44. epithelial tips of branching tubules in embryonic lungs where it
These findings all highlight the conserved importance of Wnt coordinates FGF signaling51. These important parallels between for-
signaling in defining the shape and orientation of complex organs mation of the branch point in the sea star hydro-vascular organ and
across evolution. Importantly, our study adds that Wnt signaling spe- different examples in mice suggest that Six1/2 activation by FGF may
cifically guides the 3D orientation of the growing tube in be a conserved node in the regulation of branching morphogenesis.
embryogenesis. An advantage of the sea star system is that while mammalian organs
have multiple branches, the sea star tubes offer a simple system with
FGF target genes reveal a breadth of mechanistic insight a single defined branched point observable in vivo (Fig. 8d). This
Unraveling the downstream targets of the FGF signaling pathway, and system will enable future studies to unravel what aspects of
understanding the conservation of molecular pathways in different branching are specifically regulated by Six1/2 and its interactions
models, are critical objectives that need to be addressed in with FGF signaling.
tubulogenesis29. A unique aspect of our study is that we found FGF
perturbation to selectively ablate the tubes, allowing us to use RNAseq The hydro-vascular organ offers insight into organ evolution
to discover putative transcriptional targets of the FGF pathway. In the Our results support the idea that the basal toolkit to make vertebrate
brittle star Ophiura filiformi, another echinoderm, a similar RNAseq tubular organs was already established at the root of the deuterostome
study of embryos without functional FGFR showed a lack of skeleton clade, the group that includes vertebrates. We revealed that, as in
formation32. As in the sea star hydrovascular organ, Alx4, Six1/2 and vertebrate organs, tubulogenesis involves cell migration coupled with
MHC were downregulated when FGFR was blocked, though other tar- proliferation in the sea star hydro-vascular organ. Like in vertebrate
gets like the transporters and the ECM remodeling enzymes were not organs, proliferation happens in growth zones, especially at the tube
affected. Indeed, the most abundant candidates we found in our ana- tips, and it is regulated by FGF signaling. Six1/2 is a conserved target of
lysis were metalloproteases and tissue inhibitors of metalloproteases FGF signaling that is critical for establishing branch points. Given the
(expression examples for matrixin and FcoII, Fig. 7b–d). Similarly, a simplified morphology of the sea star tubes compared to highly
recent study found that tube cell precursors are enriched in genes branched vertebrate organs, and the technical advantages that the
related to the extracellular matrix and adhesion45. Dynamic remodel- system offers for in vivo studies, we were able to discover additional
ing of the extracellular matrix components (ECM) at the basal and roles of signaling pathways in tubulogenesis. We propose that the
apical compartments of tubes is a fundamental factor in tube shape development of the sea star hydro-vascular organ provides important
and growth46. For instance, the basal lamina controls the size of the insight into the fundamental evolutionary steps that preceded the
Drosophila tracheal tubes47, and remodeling of the ECM through appearance of the more complex vertebrate organs.
metalloproteases is essential for the elongation of the sea urchin larva
skeleton, a syncytial tube48. We showed that the metalloprotease Methods
inhibitor FcoII contributes to remodeling of the basal lamina at the Embryo and larva cultures
anterior end of the growing tubes (Supplementary Fig. 9a), and both Patiria miniata adult animals were obtained from Pete Halmay
matrixin and FcoII proteins have a signal peptide, suggesting they are (peterhalmay@gmail.com) and Marinus Scientific (info@mar-
secreted (Supplementary Figs. 7 and 8). However, further studies inusscientific.com) and kept at 15 °C in a sea water aquaria. Gonads
should address the mechanism used by the metalloprotease and its were surgically obtained by small cuts on the oral side. Embryos and
inhibitors to contribute to ECM remodeling around the growing tube. larvae were cultured at 16 °C. Oocytes were microinjected, then trea-
In many systems, the balance of these factors is regulated by the FGF ted with 1-methyladenine (Acros Organics) at a final concentration of
pathway. 10 μM to induce meiotic resumption. Adult sea stars do not show any
FGF is essential for myotube guidance in fly muscles, and for phenotypic trait that can differentiate among individuals; therefore,
muscle specification in sea urchin larva30,49. Also in the sea star system animals were randomly chosen. The sex of the larvae is always
another group of FGF downstream candidates were muscle genes, and unknown and individual larvae with the same genotype were identical,
we focused on the expression of the terminal differentiation gene MHC therefore allocation in experimental groups was not relevant in our
since it is highly abundant. We discovered two populations of muscles study and sex of the larvae was not considered. Animals are collected

from the ocean and it is not possible to assess age. Sea stars are Live imaging, single nucleus tracking and statistical analyses
invertebrate and do not require prior ethical approval. For time-lapse recordings, larvae were embedded in 1% low melting
point agarose (Sigma), covered with sea water, mounted on a MatTek
Plasmid constructs and RNA in vitro transcription 35 mm glass bottom dish, and imaged at room temperature (22 °C).
The H2B-GFP construct was amplified from first strand cDNA reverse For tube elongation movies, larvae of two independent experiments
transcribed from total ovary mRNA and then cloned into pCS2 + 8 as expressing H2B-GFP were imaged for 2 days capturing z-stacks with 3
C-terminal GFP fusions52. LifeAct-mcherry construct was a gift from μm step size every 5 min. For each experiment, both the left and the
Cynthia Bradham. Both plasmids were linearized with NotI to obtain a right tubes were imaged, and nuclei were tracked. Movies and images
linear template DNA. mRNA was transcribed in vitro with the mMes- of fix samples were taken on a Nikon Yokogawa W1 spinning disk
sage mMachine SP6 followed by the polyadenylation kit (Life Tech- microscope (NIS Elements version 5.21.03) with 40× silicon immersion
nologies, #AM1340), then purified using lithium chloride. In vitro oil objective. Images were processed and analyzed in Fiji (https://fiji.sc/
transcribed mRNA was injected at a final concentration of 500 ng/ul lmageJ 1.53c) using the manual tracking plugin (Fabrice Cordelières,
for each marker. Prophase arrested oocytes were injected with ~20 Institut Curie, Orsay). To track the displacement of individual nuclei,
picoliters of H2B mRNA. we first normalized the movies by defining the starting point of tube
elongation (t = 0) as the tubes were 50 ± 5 μm long, and the final point
Perturbation experiments with CRISPR and Cas9 MASO of tracking (t = 40–45 h) as the tubes were 80 ± 10 μm long. Only nuclei
injection of the tube epithelial cells that were in focus and moving along the x
Guide RNAs (gRNAs) were designed with the online software www. and y axes were tracked. Bright field movies were taken with a Zeiss
crisprscan.org and purchased from Synthego. gRNAs stock con- Axio Vert.A1 (Zen 2.5 Blue Edition version 2.5.75.0) in continuous
centration was100pmol/μl each. The Cas9 plasmid used was pCS2- mode. DIC Supplementary Movie of cilia moving in the hydro-vascular
3xFLAG-NLS-SpCas9-NLS (Yonglong Chen, Addgene plasmid #51307). organ (Supplementary Movie 4) was taken on an Olympus FV3000
The plasmid was linearized with NotI, mRNA was transcribed with SP6 confocal microscope (Olympus Fluoview V4.2, Brown University Leduc
polymerase and stored in nuclease-free dH2O. For each knock out Facility). Student’s t-test was performed to assess statistical sig-
experiment, injection solutions had Cas9 mRNA at a final concentra- nificance, values of p < 0.001 are represented as ****. Graphs and sta-
tion of 750 ng/μl and a mixture of 2–3 gRNAs to target genomic DNA at tistical analyses were performed using Prism (GraphPad Software,
a final concentration of 150 ng/μl each. Control larvae were injected V.7.0.0). Rose plots were made with GeoRose (https://www.
with Cas9 mRNA only. Embryos were kept at 18 °C. Guide RNAs yongtechnology.com V.0.5.1.1). Figures were made with Adobe Illus-
sequences are in Supplementary Data 1. Genomic DNA of individual trator (V.27.2).
larvae (n = 3–6) was sequenced to test for CRISPR Cas9 induced
mutations as previously described53. Genomic sequences used to find EdU incorporation and pharmacological perturbations
Patiria miniata genes and to design gRNAs were analyzed with the 5-ethynyl-2-deoxyuridine (EdU) pulse labeling was performed with the
resources at echinobase.org54. The frequency of mutations in each Click-IT EdU imaging kit (Life Technologies; Cat.#:C10340) as descri-
larva was 100%, every single larva sequenced had mutations. bed in57. A final concentration of 10 mM EdU in sea water for 30 min
A MASO (GeneTools, Philomath, OR) complementary to Pm-Six1/2 was used. 25 to 50 tubes were analyzed for each stage. The Cdk4/6
transcript 5′ UTR (5’-CGTGAAGCCAAACGACGGCAACATG-3’) and a inhibitor Abemaciclib (LY2835219, Selleckchem Cat.#S5716)21 was
MASO complementary to Pm-FcoII (5’-GTTAATTCACGTCCT-3’) were added to larval cultures at 16 °C at a final concentration of 10μM. The γ-
used at a final concentration of 800 μM each to block protein trans- secretase inhibitor DAPT (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-
lation. A scrambled MASO sequence was used as control. Prophase phenylglycine t-butyl ester, FisherScientific, Cat.#J65864.MA)39 was
arrested oocytes were injected with 20 picoliters of morpholino anti- added to a final concentration of 10 μM. To perturb the Wnt pathway,
sense oligonucleotide (MASO) or Cas9+gRNAs solutions in nuclease the small molecule porcupine inhibitor ETC-15927 was used at a final
free water. concentration of 5 μM (Selleckchem, Cat.#S6616). The FGFR inhibitor
SU5402 was used at 40 μM final concentration (Sigma, Cat.#
Individual Larva genotyping and TIDE Spectral Decay Analyses SML0443) and the MEK inhibitor U0126 at 10 μM (Selleckchem,
Individual larvae were genotyped using PCR primers and the resulting Cat.#S1102). Beta-aminopropionitrile (BAPN, Sigma Cat.#A3134) was
amplicons specific to each targeted region (Supplementary Data 2). All used at a final concentration of 300 μM in sea water. All other drugs
individual mutant embryo PCRs were sequenced. For mutagenesis were dissolved in DMSO and the optimal drug concentration was
analysis: sequence trace files (.ab1) for CRISPR-Cas9 edited and Cas9 chosen after testing concentration ranges from 0.1 to 100 μM. All
injected control larvae were retrieved following sequencing per- drugs were added at the end of gastrulation, prior to the development
formed through Azenta Genewiz (https://www.genewiz.com/). For all of the hydro-vascular organ tubes. A corresponding volume of DMSO
genotypes relevant, Cas9/sgRNA mutation penetrance and distribu- was added to sea water in controls. Experiments were performed in
tion of mutation types were analyzed using differential sequence-trace three independent biological replicates.
chromatograph analysis with two different analytical packages. The
first package: TIDE (Tracking of Indels by Decomposition (V. 3.3.0);55 is Fluorescent in situ hybridization (FISH) and
used for the Six1/2 and Delta mutants. For larger indels, SynthegoICE immunofluorescence
(V.3) was used (Inference of CRISPR Editing; ice.synthego.com56. Total For fluorescence in situ hybridization (FISH), larvae previously fixed in
Efficiency, corresponding to the fraction of mutant sequences detec- 4% paraformaldehyde were hybridized for 1 week with each
ted is displayed on each spectra for CRISPR mutants (Supplementary digoxigenin-labeled riboprobe that were transcribed from linearized
Fig.11). Each of the results show complete penetrance of the Cas9 DNA (list of primers in Supplementary Data 3) with the DIG-RNA
mutations, that is, each larvae showing the mutant phenotype had labeling kit (Sigma-Aldrich, Cat:#11175025910). Signal was detected
corresponding genomic mutations, within the target site, and each with an anti-DIG antibody (Roche Cat:#11207733) diluted 1:2000 fol-
larva with a genomic mutation shown a mutant phenotype. The lowing the protocol of the tyramide signal amplification kit (Perki-
mutations also showed broad size and distribution of mutations as nElmer, Cat:#NEL760) as shown in ref. 58. For immunofluorescence
expected in an F0 mutagenesis. An estimated percentage of mosaicism experiments, larvae were fixed in 2% PFA in PBS for 20 min at RT fol-
is quantified from spectral data and is shown in the supplement lowed by 10 min in 100% methanol (this step was skipped only for
(Supplementary Fig.11). experiments where phalloidin was added). To image laminin together

with phalloidin the methanol step was skipped, resulting in a less sharp processed RNA-seq data and the GO term analysis results are provided
laminin staining. Larvae were then washed in PBST (0.1% Tween20) and as Supplementary Data 4. The Supplementary Movies in Figs. 2 and 3
incubated overnight at 4 °C with 1 mg/ml BSA and 4% sheep serum in analyzed in this study are provided as Supplementary Movies 5, 6, 7, 8.
PBST and one or more of the following: Alexa-fluor 488 Phalloidin The Supplementary Movie used for images in Fig. 1g is provided in
1:300 (Molecular Probes, Cat.#A12379), anti-beta tubulin antibody Supplementary Movie 4; the Supplementary Movie used for Fig. 2i is
1:100 (Hybridoma bank, Cat.#E7), anti-laminin antibody 1:300 (Abcam, provided in Supplementary Movie 9; the video used to make Fig. 3g, h
Cat.#AB11575), monoclonal Anti-MAP Kinase, Activated (Dipho- is provided in Supplementary Movie 10. Source data are provided with
sphorylated ERK-1&2) pERK 1:100 (Millipore Sigma, Cat.#M8159). this paper.
Samples were then washed three times with PBST and incubated with
the corresponding AlexaFluo secondary antibodies (Invitrogen anti- References
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essential for normal mammary gland development and stem cell Acknowledgements
function. Stem Cells 31, 178–189 (2013). The authors are grateful for the support for this work from the Eunice
39. Materna, S. C., Swartz, S. Z. & Smith, J. Notch and Nodal control Kennedy Shriver National Institute of Child Health and Human Devel-
forkhead factor expression in the specification of multipotent pro- opment (K99HD099315) to S.Z.S. and the National Institutes of Health
genitors in sea urchin. Development 140, 1796–1806 (2013). 1R35GM140897 to G.M.W. The authors thank Mary Jane Tsang and Iain

Cheeseman for sharing reagents, Cynthia Bradham’s lab for sharing the Peer review information Nature Communications thanks the anon-
LifeAct construct, PRS for technical assistance and Dave Matus from ymous reviewers for their contribution to the peer review of this work.
Arcadia Science for insightful comments on the manuscript.
Reprints and permissions information is available at
Author contributions http://www.nature.com/reprints
M.P. and G.M.W. conceived and designed the study. M.P. performed all
the experiments and analyzed the data; C.P. performed GO Term ana- Publisher’s note Springer Nature remains neutral with regard to jur-
lysis on the RNAseq and submitted the transcriptomic data to NCBI; isdictional claims in published maps and institutional affiliations.
G.M.W. performed CRISPR CAS9 sequencing and TIDE analyses. M.P.,
S.Z.S., C.P. and G.M.W. wrote the paper. All authors read and approved Open Access This article is licensed under a Creative Commons
the final manuscript. Correspondence to M.P. and G.M.W. Attribution 4.0 International License, which permits use, sharing,
adaptation, distribution and reproduction in any medium or format, as
Competing interests long as you give appropriate credit to the original author(s) and the
Synthego provided free samples of FGFR gRNAs. Other gRNAs and all source, provide a link to the Creative Commons license, and indicate if
other products used in this study were purchased at market value. The changes were made. The images or other third party material in this
authors declare no other competing interests. article are included in the article’s Creative Commons license, unless
indicated otherwise in a credit line to the material. If material is not
Additional information included in the article’s Creative Commons license and your intended
Supplementary information The online version contains use is not permitted by statutory regulation or exceeds the permitted
supplementary material available at use, you will need to obtain permission directly from the copyright
https://doi.org/10.1038/s41467-023-37947-2. holder. To view a copy of this license, visit http://creativecommons.org/
licenses/by/4.0/.
Correspondence and requests for materials should be addressed to
Margherita Perillo or Gary M. Wessel. © The Author(s) 2023

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Article https://doi.org/10.

Molecular mechanisms of tubulogenesis

A fundamental goal in the organogenesis ﬁeld is to understand how cells

Nature Communications | (2023)14:2402 1

Nature Communications | (2023)14:2402 2

a Protostomes b Larva Late larva

Bilateria Echinoderms Anterior

Tubes Hydropore Canal

apical transversal view

Laminin / Phalloidin / DAPI

Laminin DAPI (Nuclei)

Laminin DAPI (Nuclei) Late larva

Nature Communications | (2023)14:2402 3

Nature Communications | (2023)14:2402 4

Nature Communications | (2023)14:2402 5

a Proliferation b Late Gastrula d Larva e

LG **** 100 ****

Nature Communications | (2023)14:2402 6

Nature Communications | (2023)14:2402 7

delta mRNA Control Cas9 Delta KO

Ventral Ventral Lateral

200 **** 8000

Nature Communications | (2023)14:2402 8

Control ETC159 from LG L

Tube lemgth / esophagus length

lateral view, right side

Nature Communications | (2023)14:2402 9

Control FGF pathway inhibition G EL: FGFR KO

k Control SU5402 l m Control n Cell o

n° of EdU+ cells (% of tot tube cells)

U0126 FGFR KO FGFR KO

Nature Communications | (2023)14:2402 10

a -15 Number of differentially expressed genes ECM remodelling

-10 Alx4 FcoII 1

Transcriptional factors Control Cas9 Six1/2 KO

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a Gastrula Late gastrula Larva Late larva

b Precursor cells Tube c d left tube

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