document s2. article plus supplemental information

Report

The Presence of a Functio
nally Tripartite Through-Gut in Ctenophora Has Implications for MetazoanCharacter Trait Evolution
Graphical Abstract

Highlights

d The Ctenophora, a non-bilaterian animal lineage, possess a

functional though-gut

d Evacuation of waste material via anal pores is controlled by

actin-rich sphincters

d We describe gut cell types involved with nutrient absorption

and distribution

d Homology of the ctenophore through-gut to the bilaterian

through-gut remains unclear

Presnell et al., 2016, Current Biology 26, 1–7October 24, 2016 ª 2016 Elsevier Ltd.http://dx.doi.org/10.1016/j.cub.2016.08.019

Authors

Jason S. Presnell, Lauren E. Vandepas,

Kaitlyn J. Warren, Billie J. Swalla,

Chris T. Amemiya, William E. Browne

[email protected]

In Brief

Ctenophores have historically been

described as having a blind, sac-like gut.

Using live imaging of ctenophore

digestion in their report, Presnell et al.

demonstrate that ctenophores possess a

functionally tripartite through-gut,

challenging the current paradigm that

assumes that the through-gut originated

within Bilateria.

mailto:[email protected]

http://dx.doi.org/10.1016/j.cub.2016.08.019

Please cite this article in press as: Presnell et al., The Presence of a Functionally Tripartite Through-Gut in Ctenophora Has Implications for MetazoanCharacter Trait Evolution, Current Biology (2016), http://dx.doi.org/10.1016/j.cub.2016.08.019

Current Biology

Report

The Presence of a Functionally TripartiteThrough-Gut in Ctenophora Has Implicationsfor Metazoan Character Trait EvolutionJason S. Presnell,1,6 Lauren E. Vandepas,2,4,6 Kaitlyn J. Warren,1 Billie J. Swalla,2,3 Chris T. Amemiya,2,4

and William E. Browne5,7,*1Department of Biology, University of Miami, Cox Science Center, 1301 Memorial Drive, Miami, FL 33146, USA2Department of Biology, University of Washington, 24 Kincaid Hall, Seattle, WA 98195, USA3Friday Harbor Laboratories, University of Washington, Friday Harbor, WA 98250, USA4Benaroya Research Institute at Virginia Mason, 1201 Ninth Avenue, Seattle, WA 98107, USA5Department of Invertebrate Zoology, Smithsonian National Museum of Natural History, MRC-163, PO Box 37012, Washington,

DC 20013-7012, USA6Co-first author7Lead Contact

*Correspondence: [email protected]


SUMMARY

The current paradigm of gut evolution assumes thatnon-bilaterian metazoan lineages either lack a gut(Porifera and Placozoa) or have a sac-like gut (Cteno-phora and Cnidaria) and that a through-gut origi-nated within Bilateria [1–8]. An important groupfor understanding earlymetazoan evolution is Cteno-phora (comb jellies), which diverged very early fromthe animal stem lineage [9–13]. The perceptionthat ctenophores possess a sac-like blind gut withonly one major opening remains a commonly heldmisconception [4, 5, 7, 14, 15]. Despite descriptionsof the ctenophore digestive system dating to Agassiz[16] that identify two openings of the digestivesystem opposite of the mouth—called ‘‘excretorypores’’ by Chun [17], referred to as an ‘‘anus’’ byMain [18], and coined ‘‘anal pores’’ by Hyman[19]—contradictory reports, particularly prominentin recent literature, posit that waste products are pri-marily expelled via themouth [4, 5, 7, 14, 19–23]. Herewe demonstrate that ctenophores possess a unidi-rectional, functionally tripartite through-gut and pro-vide an updated interpretation for the evolution of themetazoan through-gut. Our results resolve lingeringquestions regarding the functional anatomy of thectenophore gut and long-standing misconceptionsabout waste removal in ctenophores. Moreover, ourresults present an intriguing evolutionary quandarythat stands in stark contrast to the current paradigmof gut evolution: either (1) the through-gut has itsorigins very early in the metazoan stem lineage or(2) the ctenophore lineage has converged on anarrangement of organs functionally similar to the bi-laterian through-gut.

RESULTS AND DISCUSSION

The evolutionary origins of the animal through-gut are crucial

for understanding the coordinated patterning of organ systems.

The prevailing paradigm of gut evolution asserts that the

metazoan through-gut (alimentary canal) and anus originated

within Bilateria [2–5, 7, 24, 25]. The implication of this view

of gut evolution, that non-bilaterians either lack a gut altogether

or possess a blind, sac-like gut characterized by a single func-

tional opening at the oral pole, the stomodeum, is misinformed.

The idea that ctenophores possess a blind gut [4, 5, 7, 14,

21–23] appears to stem from the historical misconception

that ctenophores and other non-bilaterians are simple animals

lacking complex traits [26]. Though recent evidence supports

Ctenophora as the earliest-branching extant metazoan phylum

[9–13, 27], ctenophores possess many ‘‘complex’’ traits found

in other animals, such as definitive muscles and a nervous

system [11, 12, 28, 29]. Thus, the revised phylogenetic position

of Ctenophora has also lead to novel hypotheses reconsidering

the origin of both muscles and nerves [11, 12, 30]. Here we

provide new data for and discuss previous evidence of the

presence of a through-gut in ctenophores and the broader

implications for evolution of the metazoan through-gut and

anus.

As early as the work of Louis Agassiz [16], it was known that

ctenophores possess digestive system openings opposite of

the mouth. Despite this, contradictory reports suggest that the

mouth alone is used to eliminate food waste, whereas other

studies have proposed that the pores are used only occasionally

to expel small waste products such as nitrogenous or intracellu-

larly digested wastes [5, 19, 20, 22–24]. Collectively, these re-

ports have misconstrued the function of the ctenophore anal

pores, resulting in support for the misconception that cteno-

phores have a simple, blind gut [4, 7, 19, 20, 23, 24].

The incongruence among these previous observations moti-

vated our experiments, whichwere explicitly designed to explore

ctenophore gut functional anatomy. Here we provide an update

to previous literature on the ctenophore digestive system using

Current Biology 26, 1–7, October 24, 2016 ª 2016 Elsevier Ltd. 1

mailto:[email protected]


Figure 1. Ctenophore Digestive System

Anatomy

(A) Schematic of the major features of the cteno-

phore digestive system. The pharyngeal axis (PA)

is to the left, and the tentacular axis (TA) is to the

right. Food enters the stomodeum and moves

aborally through the pharynx (light gray), where

digestive enzymes are secreted by the pharyngeal

folds (purple). Large particles are then mechani-

cally processed by a region of stiffened cilia, the

ciliary mill (dark gray). Small food particles then

transit into the central chamber, the infundibulum

(1), of the endodermal canal system (blue), where

isodynamic ciliary beating distributes particles

for absorption in the radial canals (2), the paired

adradial canals (3), and the paragastric canals and

tentacular canals. Both solid and fluid wastes are

eliminated from the branched endodermal canal

system by transport through the aboral canal and

anal canals (green) and evacuation through the

anal pores.

(B) Aboral schematic highlighting the major

endodermal canal system branches. The infun-

dibulum, or transverse canal (1), serves as the

central hub from which two tentacular canals (tc)

and four radial canals (2) branch. Each radial canal

bifurcates into adradial canals (3). The green circle

marks the aboral canal branch.

(C) Adult Mnemiopsis leidyi are characterized by

feeding grooves medial to oral lobes (arrows) lined

with tentacles bearing colloblasts (asterisks) that

transport prey to the stomodeum (arrowhead).

(D) In contrast, adult Pleurobrachia bachei are characterized by extensible colloblast lined tentacles (asterisks) that bring prey to the stomodeum (arrowhead).

(E) Aboral view of a juvenile cydippid stage Mnemiopsis leidyi showing the principal branches of the endodermal canal system as schematized in (B).

In all panels, the oral end is up. See also Figures S1 and S3 and Movies S4 and S5.


high-resolution in vivo time-lapse videography combined with

fluorescent labeling techniques and primary cell culture. The

main longitudinal body axis in ctenophores is the oral-aboral

axis, with the mouth at the oral pole and two anal pores at the

aboral pole [26] (Figure 1). The unidirectional ctenophore gut is

highly differentiated and organized into three functional domains

(Figure 1A). Our results resolve conflicting reports on the function

of the ctenophore anal pores, confirming their role in relation to

the overall digestive systemas secondary gut openings function-

ally equivalent to an anus. We unequivocally show that cteno-

phores possess a functional through-gut from which digestion

waste products and material distributed via the endodermal

canals are expelled to the exterior environment through terminal

anal pores that are anatomically and physiologically specialized

to control outflow from the branched endodermal canal system

(Figure 2; Movies S1, S2, and S3).

Ctenophores are highly transparent, allowing for easy in vivo

observation of internal processes [16–18, 20, 31] (Movie S1).

To visualize food passage through the ctenophore gut, we moni-

tored the digestion of zebrafish expressing fluorescent proteins

and Artemia soaked in fluorescein (Figure 2; Movies S1, S2,

S3, and S5). Digestion is spatially and temporally regulated

by coordinated activities throughout the ctenophore gut that

include characteristic cells functioning in nutrient uptake and

cells with functionally specialized cilia that control particulate

and fluid movement, as well as access to the mesoglea (Fig-

ure S1, Movies S4 and S5) [32, 33]. Food entering the stomo-

2 Current Biology 26, 1–7, October 24, 2016

deum is transported aborally in the pharynx by combined ciliary

beating and muscular contractions toward the pharyngeal folds

in the aboral medial third of the pharynx (Figure 1A). Within the

pharynx, combined mechanical and enzymatic action results in

the pre-digestion of food into large particles [20, 33] that then

enter the morphologically distinct ciliary mill (Figure 1A; Movie

S5B). The ciliary mill is uniquely characterized by dense arrays

of stiffened cilia [31] that act as a physical sieve and disrupt large

particles, allowing only small particles to pass through into the

infundibulum (transverse canal), the first chamber of the endo-

dermal canal system (Movies S5A and S5B). The filtrated mate-

rial is then supplied to four radial canals, one for each radially

symmetric quadrant of the ctenophore body plan (Figure 1).

Each radial canal bifurcates into paired adradial canals

that closely associate with several prominent organ systems,

including ctene rows, photocytes, and gonads. In the tentacular

plane, two paragastric canals extend orally and medially on

either side of the pharynx, and two tentacular canals extend

laterally through the mesoglea supplying the tentacle apparatus.

Cilia lining the endodermal canal lumen sweep food particles

through the branched canal system toward the periphery during

nutrient distribution [31]. Collectively, the endodermal canals

function in both nutrient absorption and distribution (Figure S1)

[17, 18, 20, 32, 33].

Both nutrient distribution and waste elimination are aided by

peristaltic activity within the pharynx and the endodermal canals

that direct flow of material (Movie S5A). During waste removal,

Figure 2. Waste Evacuation

(A) Frame grab from Movie S1 showing waste

material being ejected from open anal pore of

Mnemiopsis leidyi.

(B) Frame grab from Movie S2 showing an aboral

view of a dilated anal pore during waste ejection

from Mnemiopsis leidyi.

(C) Frame grab from Movie S3 showing a lateral

view of a fluorescein plume emerging from an

open anal pore of Pleurobrachia bachei.

(D) Frame grab from Movie S3 showing an aboral

view of a fluorescein plume during waste material

ejection from a dilated anal pore of Pleurobrachia

bachei.

In all panels, asterisks mark the position of the

aboral apical organ, arrowheads mark the position

of the open anal pore, and arrows denote waste

plumes. (A) and (C) are lateral views, with the oral

end to the left; (B) and (D) are aboral views. See

also Movies S1, S2, and S3.


ciliary beating reverses direction, collecting waste material at the

centrally located infundibulum, prior to moving into the aboral

canals. The function of the aboral canals contrasts with the

remainder of the endodermal canal system during both nutrient

distribution and waste collection due to differentially regulated

muscle and ciliary activity. The isodynamic ciliary beat cycle

associated with nutrient distribution and waste elimination is a

regulated process that occurs with a temporally cyclic regularity

of approximately 2–2.5 hr under our continuous feeding regime.

During nutrient distribution, ciliary beating within the aboral canal

actively excludes particle entry. Additionally, muscles surround-

ing the aboral canal are relaxed, reducing the canal diameter and

further limiting particle ingress. During waste elimination, these

muscles contract, pulling on the canal walls and significantly

enlarging canal diameter (Movie S1). The muscles controlling

the relative diameter of the aboral and anal canals are stereotyp-

ically localized (Figure 3A; Movie S5C). In Mnemiopsis, muscle

bundles extend in the pharyngeal plane along the oral-aboral

axis on both sides of the pharynx. At the aboral end, they anas-

tomose with aboral and anal canals; toward the oral end, muscle

fibers separate from the bundle and anchor along the feeding

grooves. As these muscles contract, the aboral and anal canal

diameters widen, and the cilia lining the aboral canal simulta-

neously reverse beat direction, transporting material collected

in the infundibulum into the lumen of the aboral canal immedi-

ately preceding defecation. These activities were most likely

not detected in previous studies because the transparent mus-

cles are difficult to visualize, their anastomosing ends are often

C

lost during fixation, and the cyclical bi-

phasic changes associated with nutrient

distribution and waste elimination make

detection difficult without high resolution

time-lapse video.

Our results reveal that each anal canal

culminates with a sphincter, consistent

with a highly regulated system. The anal

pores are composed of actin-rich cells

in a ring configuration, consistent with a

role in contraction (Figure 3). This actin

distribution, coupled with the previous report of myosin heavy

chain expression in Pleurobrachia pilius anal pores [28], provides

molecular evidence that the anal pores function as contractile,

ring-like sphincters. During defecation, the sphincter of a single

anal canal opens and waste is forcefully expelled (Figure 2;

Movies S1, S2, and S3). After defecation, the anal pore closes

and the anal and aboral canals constrict (Movies S1 and S2).

This temporally regulated process results in the expulsion of

the entirety of material circulating in the endodermal canals (Fig-

ure 2; Movies S1 and S2). Therefore, in ctenophores, the anal

pores function as the main excretory organs for defecation and

serve as the terminal end of a functional through-gut.

Our functional anatomy study of the path of digestion in cteno-

phores confirms some of the historical observations made over

the past 150 years that the anal pores open to the external envi-

ronment [16–20]. However, many previous works have differed

significantly in their interpretations of ctenophore digestive sys-

tem function broadly, and of the role of the anal pores in partic-

ular. While some early work referred to the anal pores as an anus

that allowed for ‘‘defecation of fecal material’’ [16, 18], more

recently the ctenophore digestive tract has been interpreted as

a flow-through system, with the majority of excretion occurring

through the mouth and the minor passage of intracellular waste

products occurring through the anal pores [20]. The results pre-

sented here contradict the two prevailing ideas about waste

removal in ctenophores: (1) the relatively more recent dogma

stating that ctenophores have only one functional opening to

the gut [2, 4, 5, 7, 24] and (2) the historical perspective that

urrent Biology 26, 1–7, October 24, 2016 3

Figure 3. Aboral Region of Mnemiopsis and Pleurobrachia

(A) Differential interference contrast image ofMnemiopsis. Arrows highlight organized giant smooth muscle bundles that contact and regulate the diameter of the

aboral and anal canals.

(B) Mnemiopsis stained with phalloidin in red and DAPI in blue. The arrowhead and dashed circle mark the ring of F-actin-rich cells that contribute to the

circumference of the contractile sphincter associated with the anal pore.

(C) Pleurobrachia stained with phalloidin in red. The arrowhead and dashed circle mark the position of the ring of F-actin-rich cells contributing to the contractile

sphincter associated with the anal pore.

(D)Mnemiopsis aboral view frommerged brightfield and fluorescencemicroscopy. The arrowhead and dashed circle mark the position of a fully dilated anal pore.

Digested fluorescent fish particles appear as bright spots in the anal canals.

(E) Mnemiopsis stained with phalloidin in red and DAPI in blue. The arrowhead and dashed circle mark the position of F-actin associated with one of the two

contractile anal pores.

(F) Pleurobrachia stained with phalloidin in red. Arrowheads and dashed circles mark F-actin-rich cells contributing to the contractile sphincters associated with

the anal pores.

In all panels, arrowheads indicate the position of anal pores. Lateral views of the pharyngeal plane are shown in (A)–(C), and aboral views are shown in (D)–(F). See

also Movie S5.


both themouth and anal pores are used to remove digested food

waste [19, 20, 31]. Notably, authors have also disagreed as to

whether the aboral pores constitute an anus (e.g., [19]).

In contrast to prior studies, we did not observe food waste

expulsion or regurgitation through the mouth during our feeding

experiments with either Mnemiopsis or Pleurobrachia. Cteno-

phore regurgitation is associated with three events: (1) over-

feeding [20, 34, 35], (2) ingestion of material that cannot be

predigested and/or preprocessed in the pharynx for distribution

through the endodermal canals [34, 35], and (3) ingestion of

unpalatable material [34]. Unsurprisingly, most metazoans will

regurgitate material through the mouth under these conditions.

When captive ctenophores are over-handled or fed excessively,

they will regurgitate material from the pharynx. The ctenophore

pharynx can also reach a maximum concentration threshold, af-

ter which material contained within the pharynx is regurgitated

[35]. In their natural environment, the likelihood of reaching crit-

ical prey threshold within the pharynx is low. We argue that in

previous studies supporting the mouth as a major site of waste

removal, feeding regimes surpassed this threshold and the re-

sulting regurgitation behavior was thus interpreted as a normal

mode of waste removal. In addition, it is clear that prey with sig-

nificant cuticle and/or chitinous exoskeletons can reside in the

pharynx for as long as 7 hr during digestion [36]. In our long-

term lab-cultured animals, we observe extended residence

time in the pharynx for rotifer and crustacean exoskeletons


during digestion. As long as the pharynx is not overwhelmed,

exoskeletons are typically broken down, passed into the endo-

dermal canals, and expelled from the anal pores (Movie S3A).

Our long-term, multigenerational ctenophore cultures coupled

with our functional anatomical results and previous descriptions

of ctenophore digestion clearly demonstrate that ctenophores

possess a functionally tripartite through-gut. This directly refutes

the historical characterization of ctenophores as possessing a

sac-like gut with a single opening. The results presented here

show that ingested food processed by the pharynx and distrib-

uted through the endodermal canal system is subsequently

expelled en masse at regular intervals through the anal pores

(Movies S1 and S2).

Comparatively, the functional anatomy of theMnemiopsis and

Pleurobrachia digestive systems is typical of Ctenophora,

though some ctenophore body plans have modifications to

endodermal canal peripheral branching patterns. We have

observed the same digestive system function across a range

of ctenophore body plans, including representatives from the

genera Beroe, Bolinopsis, Ocyropsis, Cestum, Coeloplana, and

Vallicula. Among other non-bilaterians, pores connecting the

gastric cavity to the external environment have been reported

in Cnidaria, including both hydrozoan medusa and some sessile

corals exhibiting true fluid flow-through systems [21, 37, 38].

However, cnidarians are still considered to possess a blind gut

[2, 4, 19, 20, 22, 39, 40]. Thus, our results represent an additional

Figure 4. Evolutionary Scenarios for the Origins of the Animal

Through-Gut

(A) Scenario for the convergent acquisition of a functional through-gut in

Ctenophora. After the divergence of Ctenophora, the through-gut organ sys-

tem evolved independently in both the ctenophore lineage and the bilaterian

lineage after the divergence of Acoelomorpha [3, 4].

(B) Scenario for a single origin of the through-gut prior to the divergence of the

ctenophore lineage. Under this scenario, several non-bilaterian lineages and

the Acoelomorpha would be inferred to have subsequently lost a functional

through-gut, rejecting the initial origins of a functional through-gut within Bi-

lateria. Although phylogenetic relationships between early branching animals

remain contentious, our animal phylogeny is based on recent phylogenomic

inferences [9–13].

Organism silhouettes by Noah Schottman, Mali’o Kodis, Oliver Voigt, and

Joseph Ryan are available from the PhyloPic database (http://phylopic.org)

and are used here under the Creative Commons Attribution 3.0 license. See

also Figure S2.


significant morphological challenge to the historical grouping of

ctenophores with cnidarians as the Coelenterata [7, 41, 42].

The recognition of a through-gut in ctenophores has broad im-

plications for our understanding of the evolution of the metazoan

anus and the unidirectional alimentary canal (Figures 4 and S2).

One scenario for the evolution of the extant ctenophore through-

gut is that an anus formed independently in the ctenophore stem

lineage (Figure 4A). This scenario does not explicitly reject

current hypotheses regarding bilaterian through-gut evolution

[3–5]. A second scenario is that the sophisticated morphological

and cellular organization of the through-gut has very early origins

in the metazoan stem lineage, suggesting the loss of a functional

through-gut in several non-bilaterian lineages (Figure 4B). Sec-

ondary loss of organ systems is not atypical. Loss of the

through-gut and anus has occurred multiple times within Bilate-

ria, for example within the deuterostomes among the echi-

noderms and among members of the Platyzoa [4]. The latter

scenario would reject current hypotheses that posit that the

anus first evolved after the divergence of the Acoelomorpha

[3–5]. Though functionally analogous, the homology of the cteno-

phore anal pore to the bilaterian anus remains unclear. For the

two evolutionary scenarios to be resolved, further research will

be needed to distinguish between the functional and structural

similarities of the ctenophore and bilaterian anus [43].

Our findings draw attention to the evolutionary importance of

the anus. The mouth is currently considered to be homologous

across most of Metazoa [5, 44]. In ctenophores, as well as

many protostomes, the position of the mouth corresponds with

the blastopore [45]. Moreover, the ctenophore blastopore ex-

hibits the same conserved gene expression patterns seen in

bilaterians and cnidarians [44, 46, 47]. However, the anal open-

ing among bilaterians has a varied phylogenetic distribution,

suggesting independent evolution of the through-gut and anus

in many lineages [3, 4]. Genes with clear orthology to those

expressed in the bilaterian hindgut and anus (e.g., NK2.1, fox

genes, and FGF8/17/18) [4] are not present in either the Mne-

miopsis or Pleurobrachia genomes [11, 12], whereas a number

of genes expressed in the bilaterian gut have clear orthologs

that are expressed in ctenophores (Figure S3). Additional inves-

tigation of gene expression in ctenophore anal pores and more

detailed functional and structural morphology analyses in these

and other non-bilaterian animals will certainly provide more

insight into the evolution of the anus and the metazoan alimen-

tary canal [4, 43]. An improved understanding of the through-

gut organ system is crucial for improving our understanding of

body plan evolution and diversification in the metazoans.

EXPERIMENTAL PROCEDURES

For additional details, see the Supplemental Experimental Procedures.

Feeding Experiments

Lab-reared Mnemiopsis leidyi were kept at room temperature and starved for

�24 hr prior to feeding experiments. Time-lapse video was captured with a

RED EPIC (RED). For aboral views, at 4 days post-hatching, juveniles were

fed Brainbow fluorescent zebrafish larvae [48] and filmed on a Zeiss SteREO

Discovery.V8 with Zeiss AxioCam MRm rev3.

Wild-caught Pleurobrachia bachei adults were kept at 11�C and starved for

�24 hr prior to feeding experiments.Marine rotifers andArtemiawere added to

sterile sea water with 0.1 mg/mL fluorescein (Sigma Aldrich). Pleurobrachia

were added and fed in the dark for 90 min. Imaging was performed on a Leica

M205FA stereoscope with DFC360FX CCD and DFC425C CCD.

Mnemiopsis Ex Vivo Tissue and Cell Culture

A thin explant of lobe material containing endodermal canals was excised with

a scalpel and covered with a nutrient-rich ctenophore mesogleal serum (CMS)

overlay and incubated at 16�C to make tissue envelopes. Small explants con-

taining endodermal canal tissue were isolated, covered in CMS, and incubated

at 16�C for �48 hr to generate primary cell cultures. Time-lapse video and

images were acquired using a Zeiss SteREO Discovery.V8 or Zeiss Axio

Imager.Z2, with a RED EPIC or Zeiss AxioCam MRm rev3.

Current Biology 26, 1–7, October 24, 2016 5

http://phylopic.org


Immunohistochemical Staining

Whole Pleurobrachia adults were stained for phalloidin as previously

described [11] and imaged on a Leica M205FA stereoscope with DFC360FX

CCD. Whole Mnemiopsis juveniles and excised lobe tissue were relaxed in a

6.5% MgCl2 solution mixed with filtered sea water (FSW) at 1:1 volumetric ra-

tio, fixed in 4% paraformaldehyde, washed in PBS Tween 20 (PTw), incubated

with phalloidin (Molecular Probes) in PBS (1:100), andmounted with SlowFade

Diamond Antifade Mountant with DAPI (Molecular Probes). Fixed excised

endodermal canals were permeabilized with 0.2% Triton X-100 in PBS, incu-

bated in blocking solution, incubated with E7 antibody (Developmental Studies

Hybridoma Bank) (1:20) in blocking solution at 4�C, and incubated at 4�C with

secondary antibody (Jackson ImmunoResearch Laboratories) at 1:250 in

blocking solution and phalloidin-488 (Cytoskeleton) at 100 nM in PBS.

Samples were mounted in SlowFade Diamond Antifade Mountant with DAPI.

Images were acquired with a Zeiss Axio Imager.Z2 with Zeiss AxioCam

MRm rev3 or with a Leica SP5 confocal microscope.

Vital Dye Staining

Mnemiopsis juveniles were incubated 2 days post-hatching in FSW contain-

ing 5 mg/mL Hoechst (Molecular Probes) and 100 nM Lysotracker Red

DND-99 (Molecular Probes). Tissue envelopes and primary cell cultures

were incubated with 5 mg/mL Hoechst and 66 nM Lysotracker Red

DND-99. Images were acquired using a Zeiss Axio Imager.Z2 with Zeiss

AxioCam MRm rev3.

SUPPLEMENTAL INFORMATION

Supplemental Information includes Supplemental Experimental Procedures,

three figures, and five movies and can be found with this article online at

http://dx.doi.org/10.1016/j.cub.2016.08.019.

AUTHOR CONTRIBUTIONS

Conceptualization, W.E.B.; Methodology, J.S.P., L.E.V., and W.E.B.; Investi-

gation, J.S.P., L.E.V., K.J.W., and W.E.B.; Writing – Original Draft, J.S.P.,

L.E.V., and W.E.B.; Writing – Review & Editing, J.S.P., L.E.V., C.T.A., and

W.E.B.; Visualization, J.S.P., L.E.V., and W.E.B.; Resources, B.J.S., C.T.A.,

and W.E.B.

ACKNOWLEDGMENTS

J.S.P. was supported by the University of Miami College of Arts and Sci-

ences. L.E.V. was supported by a Graduate Research Fellowship from the

National Science Foundation and the University of Washington Department

of Biology Edmondson Award. We thank Kathryn Tosney, James Baker,

and Julia Dallman for comments on early versions of this manuscript. We

thank Ricardo Cepeda, Joshua Swore, and Michael Rego for additional an-

imal support.

Received: July 20, 2016

Revised: August 3, 2016

Accepted: August 4, 2016

Published: August 25, 2016

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Current Biology, Volume 26

Supplemental Information

The Presence of a Functionally Tripartite

Through-Gut in Ctenophora Has Implications

for Metazoan Character Trait Evolution

Jason S. Presnell, Lauren E. Vandepas, Kaitlyn J. Warren, Billie J. Swalla, Chris T.Amemiya, and William E. Browne

Figure S1 | Specialized cell types associated with the ctenophore gut. Related to Figure 1 and Movies S4-S5. A and B are live Mnemiopsis leidyi 2 days post hatching. Oral end is oriented up and arrows highlight the pharynx and gut lumen. (A) DIC image. (B) Fluorescence image, nuclei are stained with Hoechst in blue and lysosomes are stained with Lysotracker in yellow. Lysosome signal strongly marks cells lining the oral region of the gut as well as cells scattered throughout the pharynx. (C) Bright-field image of endodermal canal in ex vivo tissue envelope isolated from an adult Mnemiopsis. Arrows highlight the asymmetrically distributed digestive cells that contain darkly pigmented lysosome vacuoles. (D) Adult Mnemiopsis endodermal canal with Hoechst stained nuclei in blue. (E) Adult Mnemiopsis endodermal canal with Lysotracker stained lysosomes in yellow. (F) Merged image of D and E showing the distinct asymmetric localization of digestive cells containing lysosomes in the endodermal canal wall. (G) Adult Mnemiopsis endodermal canal with Hoechst stained nuclei in blue. (H) Adult Mnemiopsis endodermal canal with RFP signal derived from digested Brainbow zebrafish. (I) Merged image of G and H showing the differential retention of RFP in digestive cells along the endodermal canal wall. (J) Bright-field image of in vitro cell culture derived from endodermal canal tissue. Arrows highlight darkly pigmented lysosome vacuoles in mature digestive cells. (K) Bright-field image of an isolated endodermal cell with a large centrally located lysosome vacuole. (L) Fluorescence image of K with Hoechst stained nuclei in blue and the central lysosome stained with Lysotracker in yellow. Mature digestive cells are typically bi-nucleated and contain one or more large lysosome vacuoles. Images M and N are of asymmetrically distributed cell types in the ctenophore gut associated with ultrafiltration. (M) DIC image of cells in the endodermal canal wall organized into a ciliated rosette. The mesogleal space is at the top and the endodermal canal lumen is at the bottom. Each ciliated rosette contains a central pore that provides access between the lumen of the gut and the mesoglea. The asterisk marks specialized stiffened cilia that project into the gut lumen, the arrowhead marks long cilia that project into the mesoglea. (N) Merged fluorescent confocal image of a ciliated rosette. Nuclei are marked by DAPI in blue. F-actin is marked by phalloidin in yellow. β-tubulin is marked by E7 in purple. The ciliated rosette is formed from two concentric rings of eight cells, each having a β-tubulin rich region facing the central pore. The lower ring of cells project cilia into the gut lumen, indicated by an asterisk. The upper ring of cells project cilia into the mesoglea, indicated by an arrowhead. The two groups of specialized cilia function as ultrafilters preventing transit of, and/or blockage by, large particles between the two compartments.

Figure S2 | Alternative scenarios of animal through-gut evolution. Related to Figure 4. Panels represent alternative phylogenetic hypotheses for the position of Ctenophora relative to other major lineages of metazoans based on recent phylogenomic analyses. Blue squares represent the presence of a through-gut while a red X represents an inferred loss of the through-gut. Panels (A) and (B) depict Ctenophora as the earliest branching extant metazoan lineage based on data from [S1, S2]. Panels (C) and (D) depict the divergence of Porifera at the base of the Metazoa based on analyses from [S3]. Panels (E), (F), and (G) depict the ‘Coelenterata’ clade inferred by [S4, S5]. All three scenarios allow for the independent, convergent acquisition of a through-gut within the Ctenophora lineage as presented in panels A, C, and E. Explicit scenarios for the ancestral evolution of a through-gut in the metazoan stem lineage with subsequent losses among Porifera, Placozoa, Cnidaria, and/or Acoelomorpha are presented in panels B, D, and G. Panel F represents a scenario in which the independent, convergent evolution of a through-gut occurred in the ‘Coelenterata’ stem lineage and was subsequently lost in Cnidarians. The phylogenetic placement of Acoelomorpha along with the absence of a through-gut is based on [S6].

Figure S3 | Gene expression associated with the ctenophore gut. Related to Figure 1. (A) Schematic of the ctenophore tripartite digestive system. The oral end is oriented to the left and major features are labeled as in Figure 1. The digestive system has been divided into four regions of gene expression based on prior gene expression studies highlighted in panel B. (B) Table of known gene expression in the ctenophore digestive system by region, including relevant citations [S7-S15].

Supplemental Experimental Procedures: Feeding experiments

Lab-reared Mnemiopsis leidyi were kept at room temperature and starved for ~24 hours prior to feeding experiments. For lateral views, adult animals were pinned in 8-inch silicon-coated dishes (SYLGARD–184, Dow Corning, Inc.) and fed zebrafish larvae. Time-lapse video was captured with a RED EPIC camera (RED, Inc.). For aboral views, 4-day post hatching juveniles were fed Brainbow fluorescent zebrafish larvae [S16], and filmed on a Zeiss SteREO Discovery.V8 with a Zeiss AxioCam MRm rev3 using Zeiss AxioVision software (Release 4.8.2). Annotations, images and video were processed with REDCINEX-PRO (RED, Inc.), Adobe Photoshop, and QuickTime Pro (Apple, Inc).

Pleurobrachia bachei adults, collected from Puget Sound WA, were kept at 11C and starved for ~24 hours prior to feeding experiments. Marine rotifers and Artemia were added to sterile seawater with 0.1mg/mL fluorescein (Sigma Aldrich, USA). Pleurobrachia were added and fed in the dark for 90 minutes. Imaging was performed on a Leica M205FA stereoscope equipped with a DFC360FX monochrome CCD camera and a DFC425C color CCD camera. Annotations, images and video were processed with Adobe Photoshop and QuickTime Pro.

Mnemiopsis ex vivo tissue and cell culture Lobe tissue was removed from adult animals with a scalpel and washed several times in filtered

seawater (FSW) containing 1x penicillin/streptomycin. A nutrient rich Ctenophore Mesogleal Serum (CMS) overlay for cell culture was prepared by homogenizing excised tissue with a mortar and pestle followed by heat inactivation at 56°C for 30 minutes, centrifugation, and mixed at 1:1 volumetric ratio with FSW containing 1x pen/strep. A thin explant of lobe material containing endodermal canals was excised with a scalpel to make tissue envelopes, covered with CMS overlay, and incubated at 16°C. To generate primary cell cultures, small explants containing endodermal canal tissue were isolated, covered in CMS and incubated at 16°C for ~48 hours. Explants were then removed from the cell culture. A pipette was used to transfer identified endodermal cells to a new slide. Time-lapse video and images were acquired using a Zeiss SteREO Discovery.V8 or Zeiss Axio Imager.Z2, with a RED EPIC Camera (RED, Inc.) or Zeiss AxioCam MRm rev3 and Zeiss AxioVision software (Release 4.8.2). Images were processed with REDCINEX-PRO, QuickTime Pro, or Adobe Photoshop.

Immunohistochemical staining Whole Pleurobrachia adults were stained for phalloidin as previously described [S17] and imaged

on a Leica M205FA stereoscope with a DFC360FX monochrome CCD camera. Whole Mnemiopsis juveniles and excised lobe tissue were relaxed in a 6.5% MgCl2 solution mixed with FSW in a 1:1 volumetric ratio and fixed in 4% paraformaldehyde at room temperature. Fixed samples were washed with PTw (0.1% Tween-20 in PBS) and incubated with phalloidin (Alexa Fluor 568 phalloidin, Molecular Probes) in PBS (1:100) for 1 hour at room temperature, washed with PBS, and incubated in SlowFade Diamond Antifade Mountant with DAPI (Molecular Probes) at room temperature. Fixed excised endodermal canals were permeablized with 0.2% Triton X-100 in PBS, incubated in blocking solution (10% Goat serum, 0.2% Triton X-100, in PBS), and incubated with E7 antibody (Developmental Studies Hybridoma Bank) diluted 1:20 in blocking solution overnight at 4°C. Samples were incubated overnight at 4°C with secondary antibody at 1:250 in blocking solution and phalloidin-488 (Cytoskeleton, Inc.) at 100 nM in PBS. Either Alexa Fluor Goat anti-Mouse 594 or Alexa Fluor Chicken anti-Mouse 647 was used (Jackson ImmunoResearch Laboratories, Inc.). Samples were mounted in SlowFade Diamond Antifade Mountant with DAPI (Molecular Probes). Images were acquired using a Zeiss Axio Imager.Z2 with a Zeiss AxioCam MRm rev3 and Zeiss AxioVision software (Release 4.8.2), or with a Leica SP5 laser scanning microscope. Images were processed with either Adobe Photoshop or FIJI [S18].

Vital dye staining Mnemiopsis juveniles 2 days post-hatching were incubated in FSW containing 5µg/mL Hoechst

(Molecular Probes) and 100nM Lysotracker® Red DND-99 (Molecular Probes). Tissue envelopes and cell cultures were incubated with 5µg/mL Hoechst and 66nM Lysotracker® Red DND-99 (Molecular Probes). Images were acquired using a Zeiss Axio Imager.Z2 with a Zeiss AxioCam MRm rev3 and Zeiss AxioVision software (Release 4.8.2) and edited using Adobe Photoshop or FIJI [S18].

Supplemental References: S1 Dunn, C.W., Hejnol, A., Matus, D.Q., Pang, K., Browne, W.E., Smith, S.A., Seaver, E., Rouse, G.W., Obst,

M., Edgecombe, G.D., et al. (2008). Broad phylogenomic sampling improves resolution of the animal tree of life. Nature 452, 745-749.

S2 Hejnol, A., Obst, M., Stamatakis, A., Ott, M., Rouse, G.W., Edgecombe, G.D., Martinez, P., Baguñà, J., Bailly, X., Jondelius, U., et al. (2009). Assessing the root of bilaterian animals with scalable phylogenomic methods. Proc. R. Soc. Lond. B 276, 4261-4270.

S3 Pick, K.S., Philippe, H., Schreiber, F., Erpenbeck, D., Jackson, D.J., Wrede, P., Wiens, M., Alié, A., Morgenstern, B., Manuel, M., et al. (2010). Improved phylogenomic taxon sampling noticeably affects nonbilaterian relationships. Mol. Biol. Evol. 27, 1983-1987.

S4 Philippe, H., Derelle, R., Lopez, P., Pick, K., Borchiellini, C., Boury-Esnault, N., Vacelet, J., Renard, E., Houliston, E., Quéinnec, E., et al. (2013). Phylogenomics revives traditional views on deep animal relationships. Curr. Biol. 19, 706-712.

S5 Nosenko, T., Schreiber, F., Adamska, M., Adamski, M., Eitel, M., Hammel, J., Maldonado, M., Müller, W.E.G., Nickel, M., Schierwater, B., et al. (2013). Deep metazoan phylogeny: When different genes tell different stories. Mol. Phylogenet. Evol. 67, 223-233.

S6 Hejnol, A., and Martindale, M.Q. (2008). Acoel development indicates the independent evolution of the bilaterian mouth and anus. Nature 456, 382-386.

S7 Yamada, A., Pang, K., Martindale, M.Q., and Tochinai, S. (2007). Surprisingly complex T-box gene complement in diploblastic metazoans. Evol. Dev. 9, 220-230.

S8 Pang, K., and Martindale, M.Q. (2008). Developmental expression of homeobox genes in the ctenophore Mnemiopsis leidyi. Dev. Genes Evol. 218, 307-319.

S9 Pang, K., Ryan, J.F., Mullikin, J.C., Baxevanis, A.D., Martindale, M.Q., and Program, N.C.S. (2010). Genomic insights into Wnt signaling in an early diverging metazoan, the ctenophore Mnemiopsis leidyi. EvoDevo 1, 10.

S10 Pang, K., Ryan, J.F., Baxevanis, A.D., and Martindale, M.Q. (2011). Evolution of the TGF-beta signaling pathway and its potential role in the ctenophore, Mnemiopsis leidyi. PLoS One 6, e24152.

S11 Reitzel, A.M., Pang, K., Ryan, J.F., Mullikin, J.C., Martindale, M.Q., Baxevanis, A.D., and Tarrant, A.M. (2011). Nuclear receptors from the ctenophore Mnemiopsis leidyi lack a zinc-finger DNA-binding domain: Lineage-specific loss or ancestral condition in the emergence of the nuclear receptor superfamily? EvoDevo 2, 3.

S12 Simmons, D.K., Pang, K., and Martindale, M.Q. (2012). Lim homeobox genes in the ctenophore Mnemiopsis leidyi: The evolution of neural cell type specification. EvoDevo 3, 2.

S13 Schnitzler, C.E., Simmons, D.K., Pang, K., Martindale, M.Q., and Baxevanis, A.D. (2014). Expression of multiple Sox genes through embryonic development in the ctenophore Mnemiopsis leidyi is spatially restricted to zones of cell proliferation. EvoDevo 5, 15.

S14 Dayraud, C., Alie, A., Jager, M., Chang, P., Le Guyader, H., Manuel, M., and Queinnec, E. (2012). Independent specialisation of myosin II paralogues in muscle vs. non-muscle functions during early animal evolution: A ctenophore perspective. BMC Evol. Biol. 12, 107.

S15 Jager, M., Dayraud, C., Mialot, A., Queinnec, E., le Guyader, H., and Manuel, M. (2013). Evidence for involvement of Wnt signalling in body polarities, cell proliferation, and the neuro-sensory system in an adult ctenophore. PLoS One 8, e84363.

S16 Pan, Y.A., Livet, J., Sanes, J.R., Lichtman, J.W., and Schier, A.F. (2011). Multicolor brainbow imaging in zebrafish. Cold Spring Harb. Protoc. 2011, pdb.prot5546.

S17. Moroz, L.L., Kocot, K.M., Citarella, M.R., Dosung, S., Norekian, T.P., Povolotskaya, I.S., Grigorenko, A.P., Dailey, C., Berezikov, E., Buckley, K.M., et al. (2014). The ctenophore genome and the evolutionary origins of neural systems. Nature 510, 109-114.

S18 Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., Preibisch, S., Rueden, C., Saalfeld, S., Schmid, B., et al. (2012). Fiji: An open-source platform for biological-image analysis. Nat. Methods 9, 676-682.

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