(235), ra55. [DOI: 10.1126/scisignal.2002734] 5Science SignalingDimitris Kioussis and Henrique Veiga-Fernandes (31 July 2012) Andrews, Hideki Enomoto, Jeffrey Milbrandt, Vassilis Pachnis, Mark C. Coles,Alden, Jon Timmis, Katie Foster, Anna Garefalaki, Panayotis Pachnis, Paul Amisha Patel, Nicola Harker, Lara Moreira-Santos, Manuela Ferreira, KieranLymphoid and Nervous Systems

Differential RET Signaling Pathways Drive Development of the Enteric`

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D E V E L O P M E N T

Differential RET Signaling PathwaysDrive Development of the Enteric Lymphoidand Nervous SystemsAmisha Patel,1* Nicola Harker,1* Lara Moreira-Santos,2* Manuela Ferreira,2 Kieran Alden,3,4

Jon Timmis,4 Katie Foster,1 Anna Garefalaki,1 Panayotis Pachnis,1 Paul Andrews,4

Hideki Enomoto,5 Jeffrey Milbrandt,6 Vassilis Pachnis,7 Mark C. Coles,3

Dimitris Kioussis,1 Henrique Veiga-Fernandes2†

During the early development of the gastrointestinal tract, signaling through the receptor tyrosine ki-nase RET is required for initiation of lymphoid organ (Peyer’s patch) formation and for intestinal in-nervation by enteric neurons. RET signaling occurs through glial cell line–derived neurotrophic factor(GDNF) family receptor a co-receptors present in the same cell (signaling in cis). It is unclear whetherRET signaling in trans, which occurs in vitro through co-receptors from other cells, has a biologicalrole. We showed that the initial aggregation of hematopoietic cells to form lymphoid clusters occurredin a RET-dependent, chemokine-independent manner through adhesion-mediated arrest of lymphoidtissue initiator (LTin) cells. Lymphoid tissue inducer cells were not necessary for this initiation phase.LTin cells responded to all RET ligands in trans, requiring factors from other cells, whereas RET wasactivated in enteric neurons exclusively by GDNF in cis. Furthermore, genetic and molecularapproaches revealed that the versatile RET responses in LTin cells were determined by distinctpatterns of expression of the genes encoding RET and its co-receptors. Our study shows that a transRET response in LTin cells determines the initial phase of enteric lymphoid organ morphogenesis,and suggests that differential co-expression of Ret and Gfra can control the specificity of RETsignaling.

INTRODUCTION

The development of enteric (gastrointestinal) organs requires the co-ordinate growth of multiple tissues from different embryonic layers. Thisprocess is controlled in part by tissue-specific factors, but growing evi-dence suggests that the same key molecules, in particular ligands of thereceptor tyrosine kinase RET, are used in different tissues to control dis-tinct developmental end points in the gut (1). Peyer’s patches (PPs) aresecondary lymphoid organs of the gut that are important for mucosal im-mune responses. The generation of PPs involves aggregation of hemato-poietic CD4+CD3!IL-7Ra+c-Kit+CD11c! lymphoid tissue inducer (LTi)cells, CD4!CD3!c-Kit+IL-7Ra!CD11c+ cells, and mesenchymal lymph-oid tissue organizer (LTo) cells, leading to formation of primordia in themidgut of mice by embryonic day 16.5 (E16.5) (1–6). LTi cells are criticalfor this process. In the absence of LTi cells (7, 8), PPs do not form, andadoptive transfer of LTi cells into neonatal animals with impaired devel-opment of the secondary lymphoid organs rescues organogenesis of PPs

(9) and nasal-associated lymphoid tissue (10). Conversely, an increase inLTi cell numbers, induced by an increase in the abundance of cytokineinterleukin-7 (IL-7), results in increased numbers of PPs (11). Moreover,deficiencies in Ikaros (12), retinoic acid–related orphan nuclear receptor g t(RORgt) (7, 13, 14), inhibitor of DNA binding 2 (Id2) (8), tumor necrosisfactor–related activation-induced cytokine (TRANCE) and TRANCE re-ceptor (15–17), or chemokine CXCL13 and its receptor CXCR5 (18, 19)cause developmental or functional deficits in LTi cells and impairment ofPP formation, and disruption of the IL-7–IL-7 receptor a (IL-7Ra) sig-naling axis results in reduced numbers of PPs (20–22).

CD4!CD3!c-Kit+IL-7Ra!CD11c+ cells are also required for PP forma-tion. Mice partially depleted of these cells show impaired PP development,and mice deficient in RET, which is normally found on a substantial pro-portion of this cell subset, do not form PPs (2). CD4!CD3!c-Kit+IL-7Ra!CD11c+ cells are thought to participate in an early, undefined phaseof enteric lymphoid tissue formation and so are named lymphoid tissueinitiator (LTin) cells (2, 23). LTin cell behavior and the mechanism by whichthese cells operate remain obscure. LTin and LTi cells produce lymphotoxinb (LTb), which induces the maturation of LTo cells (3, 4); LTb receptor–deficient mice also lack PPs (24–26). LTo cell differentiation is accom-panied by the generation of adhesion molecules vascular cell adhesionmolecule 1 (VCAM1), intercellular adhesion molecule 1 (ICAM1), and muco-sal addressin cell adhesion molecule 1 (MAdCAM1), as well as chemokinesCXCL13, CCL19, and CCL21. LTi cells express integrins a4b7 and a4b1,which bind to MadCAM and VCAM1, respectively (27, 28), as well aschemokine receptors CXCR5 and CCR7, which respond to their respec-tive ligands CXCL13 (CXCR5) and CCL19 and CCL21 (CCR7) (29–31).Thus, the generation of these adhesion molecules and chemokines appearsto be critical for PP morphogenesis, leading to retention and attraction of

1Division of Molecular Immunology, MRC National Institute for Medical Research,The Ridgeway, Mill Hill, London NW7 1AA, UK. 2Instituto de Medicina Molecu-lar, Faculdade de Medicina de Lisboa, Av. Prof. Egas Moniz, Edifício Egas Moniz,1649-028 Lisboa, Portugal. 3Centre for Immunology and Infection, Departmentof Biology and Hull York Medical School, University of York, York YO10 5DD,UK. 4Departments of Computer Science and Electronics, University of York,York YO10 5DD, UK. 5Laboratory for Neuronal Differentiation and Regeneration,RIKEN Center for Developmental Biology, Kobe 650-0047, Japan. 6Hope Cen-tre for Neurological Disorders, Genetics Department, Washington UniversitySchool of Medicine, St. Louis, MO 63110, USA. 7Division of Molecular Neuro-biology, MRC National Institute for Medical Research, The Ridgeway, Mill Hill,London NW7 1AA, UK.*These authors contributed equally to this work.†To whom correspondence should be addressed. E-mail: jhfernandes@fm.ul.pt

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additional LTi cells within the PP primordia. Nevertheless, the earlieststeps that trigger PP initiation remain elusive.

Similar to the formation of the intestinal lymphoid system, formationof the enteric nervous system (ENS) depends on interactions betweenectodermally derived neural crest cells and stroma cells of the gut wall.Most of the ENS is derived from a relatively small population of vagalneural crest cells that invade the foregut and, upon receiving the appropri-ate signals from the splanchnic mesenchyme, proliferate and migrate in arostrocaudal direction to colonize the entire intestine, where they formneuronal aggregates that constitute the interconnected network of entericganglia (32). These developmental events depend on RET, which is foundon the surface of neural crest cells and is activated by its ligand glial cellline–derived neurotrophic factor (GDNF), which in turn is generated bythe stroma cells (33, 34).

RET is composed of an extracellular ligand–binding domain, whichhas four cadherin-like repeats and a cysteine-rich region; a hydrophobictransmembrane domain; and a cytoplasmic portion with a tyrosine kinasedomain (35). RET regulates several intracellular signaling cascades,including the Ras–extracellular signal–regulated kinase (ERK) and phos-phoinositide 3-kinase (PI3K)–Akt pathways, which control cell survival,differentiation, proliferation, migration, and chemotaxis (35, 36). The RETligand GDNF is the prototypical member of the GDNF family of ligands(GFL), which includes neurturin (NRTN), artemin (ARTN), and persephin(PSPN). The specificity of the interaction between each of the four GFLsand RET is controlled by one of four GDNF family receptor a (GFRa) co-receptors, GFRa1 to 4. GDNF signals preferentially through GFRa1,NRTN through GFRa2, ARTN through GFRa3, and PSPN throughGFRa4. GFLs bind to GFRa, and the GFL-GFRa complex then interactswith two RET molecules, which form a homodimer and induce signaltransduction (35).

GFLs—members of the transforming growth factor–b family—areproduced as precursors that are cleaved and activated upon secretion(35). They bind to proteoglycans of the extracellular matrix, which in-creases their local concentrations and restricts diffusion (35). The GFRaco-receptors are usually glycophosphatidylinositol (GPI)–linked cell sur-face glycoproteins, although soluble GFRa molecules can be produced byphospholipase or protease cleavage and by alternative splicing (37, 38).GFRas are more widely abundant than RET (39, 40), which suggests thatGFRa proteins may activate RET signaling in a non–cell-autonomous man-ner through soluble receptors (signaling in trans), in contrast with theconventional cell-autonomous actions of GFRa in ENS cells (signalingin cis) in which both RET and the GFRa co-receptor are expressed by thesame cells (37, 41). However, it is still not clear whether RET-GFRasignaling in trans has any physiological relevance (42). Here, we examinedthe role of RET-sensitive LTin cells in the early morphogenesis of entericlymphoid tissue. Lineage tracing and characterization of lymphoid organprogenitor cells, in combination with genetic approaches, enabled us todissect the molecular cascades that trigger PP formation.

RESULTS

LTin cells are critical for the early steps of entericlymphoid tissue morphogenesisIn human CD2–green fluorescent protein (hCD2-GFP) transgenic mice,both LTin and LTi hematopoietic cells express GFP and accumulate atsites of presumptive secondary lymphoid organ development (2, 5, 6).A subset of the LTin cells express RET and respond to the RET ligandARTN (2). To examine early events in PP induction that were controlledby RET, we studied explant cultures of intestines from E15.5 hCD2-GFP

mice incubated with ARTN-impregnated agarose beads for 24 hours. Im-munostaining of these explants revealed a pronounced accumulation ofLTin cells in the vicinity of the ARTN-soaked beads at a time whenCD4+ LTi cells were rarely present both in the control [bovine serum al-bumin (BSA)–treated] and in the ARTN-treated explants (Fig. 1, A andB). To determine whether LTin accumulation was a result of cell prolifer-ation, we incubated the explants with the thymidine analog bromodeoxy-uridine (BrdU) and found that the GFP+ hematopoietic cells were allBrdU-negative, indicating that the accumulation of LTin cells did not re-quire their proliferation (Fig. 1C). In contrast, proliferating (BrdU+) non-hematopoietic (GFP!) cells that showed a substantial increase in theabundance of the adhesion molecule VCAM1 were found in proximityto the LTin cells (Fig. 1, C and D). We concluded that the increase inVCAM1 abundance was not a direct effect of ARTN on GFP! mesenchy-mal cells because incubation of purified enteric mesenchymal cells withARTN did not result in increased VCAM1 abundance (fig. S1). Thesefindings suggested that RET-mediated initiation of PP formation is de-termined by LTin cells, whereas LTi cells are not necessary for these ini-tial steps.

To formally test this hypothesis, we analyzed explanted cultures of in-testines from RORgt-deficient (RorcGFP/GFP) mice (which are deficient inLTi cells) that were incubated with ARTN-impregnated agarose beads.Despite the failure of LTi cells to develop in these mice (7, 13, 14), LTincells efficiently accumulated near ARTN-soaked beads and were in closeproximity with cells that showed induction of VCAM1, confirming thatLTi cells were dispensable for initial events that stimulate development ofenteric lymphoid structure (Fig. 1E). This conclusion was also supportedby the finding that in intestines from E14.5 mice, LTi cells were much lessabundant than were LTin cells, a situation that changed after PP formationat E17.5 (fig. S1). Furthermore, quantitative reverse transcription–polymerasechain reaction (RT-PCR) analysis of tissue microdissected from the regionaround the beads revealed that ARTN did not alter the expression ofCcl19, Ccl21, or Cxcl13 and that the chemokines CCL19, CCL21, andCXCL13 were not required for the early phase of lymphoid tissue mor-phogenesis, as was demonstrated by chemokine-blocking experiments(Fig. 1F and fig. S1). Moreover, we found that stimulation of LTin cellswith GFLs did not increase their production of LTb and that blockage ofLTbR signaling did not impair the increase in abundance of VCAM1 onstroma cells (fig. S1). Thus, we conclude that LTin cells mediate early PPmorphogenesis in a RET-dependent, LTi-independent manner, concomi-tant with an increase in the abundance of VCAM1 in nearby mesenchymalcells that are presumably sessile.

RET-dependent signals cause the arrest of enterichematopoietic cellsBecause the hematopoietic cell subsets that colonize the gut are highlymobile (2) and GFL-triggered aggregation of LTin cells did not requireproliferation (Fig. 1C), we investigated whether changes in cell motilitycontributed to the RET-mediated initial steps of PP formation. We there-fore performed time-lapse analysis of explant cultures of intestines at24 hours, a time when most aggregating cells are LTin cells (Fig. 1, A, B,D, and E). Analysis of tracks produced by hematopoietic GFP+ cells re-vealed that cells close to ARTN-loaded beads exhibited reduced tracklength, velocity, and displacement when compared to those cells at greaterdistances from the beads (Fig. 2, A and B, and movies S1 and S2). Thisresult indicated that exposure to ARTN resulted in the impaired motility ofhematopoietic cells and increased abundance of adhesion molecules onmesenchymal cells (Fig. 1, C to E). Thus, our data suggest that cell adhe-sion may control the overall motility of hematopoietic cells in the earlystages of PP morphogenesis.

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Our data could be used to provide an estimate of how cell adhesioninitiates formation of enteric lymphoid organs. Only a limited combinationof parameters yielded a result that was a simultaneous best fit with theex vivo measured track length, velocity, and displacement of cells. With astochastic, individual cell–based model, we investigated whether changesin adhesion could modulate the motility of hematopoietic cells in a waythat led to their accumulation. In silico exposure of hematopoietic cells tovariable adhesion time periods revealed that, in contrast to chemokine-related parameters (Fig. 1F and fig. S1), adhesion time profoundly influ-enced hematopoietic cell motility (Fig. 2, C to E). The theoretical valueswe obtained with best-fit parameters revealed that the simulation resulting

from an adhesion time of 60 s was an excellent fit with our experimentaldata from the ex vivo explant motility study (Fig. 2, A to E).

To further validate the hypothesis arising from our in silico PP nucle-ation model, we tested whether impairment of the adhesion activity ofVCAM1 in explant organ cultures affected RET-mediated PP formation.An antibody-mediated block of VCAM1 activity substantially reduced thecell-clustering efficiency of ARTN-soaked beads compared to that ofARTN-soaked beads incubated with an isotype control antibody (Fig.2F). The notion that these triggering alterations were irreversible andoccurred at very early stages was supported by the fact that exposure toARTN-soaked beads in explant cultures for just 24 hours stimulated an

Fig. 1. LTin cells mediate the early stages ofenteric lymphoid tissue morphogenesis. Ex-planted intestines from E15.5 hCD2-GFPmicewere incubated with agarose beads impreg-nated with BSA or ARTN and were analyzed24 hours later. (A) Representative imagesof confocal microscopic analysis of the areasurrounding the beads. Right panels showdetails of boxed areas in the images on theleft. Immunofluorescence staining: green,CD11c; red, CD4; arrows show rare CD4+

LTi cells. Scale bar, 100 mm. (B) Numbersof CD11c+ LTin cells per 105 mm2 in the vicin-ity of BSA and ARTN beads. Data are fromsix experiments for BSA and eight experi-ments for ARTN. Two-tailed t test. Error barsshow SEM. (C) Representative images ofconfocal microscopic analysis of the areasurrounding the beads. Immunofluorescencestaining: green, GFP; red, VCAM1; blue, BrdU.Data are from 10 experiments for BSA and24 experiments for ARTN. Cells positive forboth BrdU and GFP are cyan. (D) Left panel:representative images of confocal microscop-ic analysis of the area surrounding the beads.Immunofluorescence staining: green, CD11c;red, VCAM1. Scale bar, 100 mm. Right panel:intensity profiles (gray values) of VCAM1 abun-dance across the lengths of the confocalimages (excluding autofluorescence fromthe beads). Three independent profiles arerepresented. (E) Representative images ofconfocal microscopic analysis of the areasurrounding the beads. Left panel: explantedintestines from E15.5 RORgt-competent(RorcGFP/+) mice incubated with BSA- andARTN-soaked beads. Data are from eightexperiments each for BSA- and ARTN-soaked beads. Right panel: RORgt-deficient(RorcGFP/GFP ) mice incubated with BSA- andARTN-soaked beads. Data are from nineexperiments each for BSA and ARTN. Immuno-fluorescence staining: green, CD11c; red,VCAM1. Scale bar, 100 mm. (F) Intestinesfrom RorcGFP/+ and RorcGFP/GFP mice were in-cubated with agarose beads as describedearlier, and quantitative RT-PCR analysis of the expression of Ccl19, Ccl21, and Cxcl13 was performed on tissue microdissected from the regionsurrounding the beads. MC, mature mesenchymal cells obtained from formed PP primordia.

BS

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Vcam1 intensityCD11c/Vcam1

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Distance (pixels)

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BSA ARTN MCBSA ARTN BSA ARTN

BS

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BS

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CD11c/CD4 CD11c/CD4 (detail)

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increase in VCAM1 abundance on mesen-chymal cells and triggered the formation oflymphoid structures similar to those seen incultures after 96 hours of continuous expo-sure to ARTN (Fig. 2G). Thus, we conclude that exposure to the RET ligandARTN impairs the motility of LTin cells in an adhesion-dependent manner.

Hematopoietic cells respond to all GFLsBecause the specificity of RET-GFL interactions is determined by the GPI-anchored, ligand-binding co-receptors GFRa1 to GFRa4, we used geneexpression analysis to test whether the genes encoding GFRa1 to GFRa4and their ligands were expressed in the intestine during development. Thegenes encoding all four GFLs and their co-receptors (in addition to full-length Gfra4) were expressed within the developmental window fromE12.5 to E15.5 (fig. S2). We found that Gfra2, Gfra3, Nrtn, and Artn wereabundantly expressed by muscle cells, whereas Gfra2 was also highlyexpressed by enterocytes (fig. S2). These results suggested that LTin cellsmight respond to GFLs other than ARTN.

To test whether other GFLs contributed to PP morphogenesis, we in-cubated explant cultures of whole embryonic guts from hCD2-GFP micewith agarose beads soaked with individual GFLs. We found that ARTN,NRTN, and PSPN were capable of inducing ectopic PP clusters to a sim-ilar extent (Fig. 3, A to C). In contrast, agarose beads soaked with GDNF,a molecule that is critical for the development of the ENS (43), did notinduce clustering of hematopoietic cells (Fig. 3, D and E). Moreover, im-munostaining of clusters induced by ARTN, NRTN, and PSPN at 72 hoursrevealed the presence of CD11c+ LTin cells and CD4+ LTi cells and showedthe increased abundance of VCAM1 on mesenchymal cells in the vicinityof the aggregates (Fig. 3F). These data showed that there is redundancy inthe effects of GFLs on RET-responsive enteric hematopoietic cells, whichis consistent with the minor decrease in the numbers of PPs observed inGfra3-deficient mice (2). Such redundancy is also consistent with our

Fig. 2. RET-mediated signals impair hema-topoietic cell motility. Intestines from E15.5hCD2-GFP mice were incubated with BSA-or ARTN-soaked beads for 24 hours. (A) Rep-resentative example of 60-min time-lapsecell tracks. Green, cells <50 mm from beads;yellow, cells >50 mm from beads. Scale bar,100 mm. (B) Summary of the time-lapse anal-ysis of cell tracks shown in (A). White bars,cells >50 mm from beads (n = 23 cells); graybars, cells <50 mm from beads (n = 17 cells).(C) Agent-based simulation of cell tracksover a 60-min analysis. Green, cells <50 mmfrom beads; yellow, cells >50 mm distanceto the beads. Scale bar, 100 mm. (D) Summa-ry of the simulations shown in (C) with a 60-sadhesion time. White bars, cells >50 mm frombeads (n = 20 cells); gray bars, cells <50 mmfrom beads (n = 19 cells). (E) “A test” scoresfor different parameter values run 500 timesfor each chemokine threshold condition. (F)Effect of blocking adhesion with an antibodyagainst VCAM1 on the percentage of ex-plants that form GFP+ cell aggregates at72 hours. Data are from 27 experiments forBSA-soaked beads, 24 experiments for ARTN-soaked beads, 23 experiments for ARTNbeads with IgG1 control antibody, and 40experiments for ARTN beads and antibodyagainst VCAM1. (G) Time course of the effectsof ARTN on GFP+ cell aggregates. Intestineswere incubated for various times with beadsimpregnated with the following compounds:BSA for 96 hours and ARTN for 12, 24, or96 hours. Two-tailed Mann-Whitney test Pvalues are indicated for (B) and (D). Two-tailedt test P values are indicated for (F) and (G).Error bars show SEM. Additional statisticalanalysis for thesedatacanbe found in theSup-plementary Materials.

Ex vivo explant culture

400

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200

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7

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P = 0.18 P = 0.004P = 0.04

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0.07

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Time (hours)

A B

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ARTNremoved at 12 h

BSA

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ARTNremoved at 24 h

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observations that mice deficient in both Artn (44)and Nrtn (45) had a normal complement of PPs(fig. S2).

Hematopoietic cells respond toGFLs in transGFRa proteins are more widely abundant thanRET (39, 40), which suggests that GFRa mightmodulate RET signaling in a non–cell-autonomousmanner (that is, they could induce signaling intrans) (37, 41). However, neurons respond mostefficiently to GFLs when RET is co-expressed withits cognate co-receptors in the same cells (whichis defined as signaling in cis) (42). These reportsand our detection of transcripts for all co-receptorsin the gut (fig. S2) raised the possibility thatGFL-dependent RET signaling in hematopoieticcells might be activated through GFRa moleculespresented in trans. To investigate this hypothesis,we incubated embryonic intestines with beads im-pregnated with ARTN, NRTN, or GDNF aloneor in combination with recombinant proteins ofeach of the three co-receptors GFRa3, GFRa2,and GFRa1. We saw the most efficient formationof ectopic lymphoid structures when ARTN wascombined with GFRa3 and when NRTN wascombined with GFRa2, although statistically sig-nificant clustering was also observed, comparedto that in control conditions, when these factorswere used alone or in combination with the other co-receptors (Fig. 4, A and B; see the SupplementaryMaterials for statistical analysis). We unexpectedlyfound that GDNF in combination with its cognateco-receptor GFRa1 [which was either impregnatedon microbeads or cleaved from the cell mem-brane of adjacent cells by phosphatidylinositol-specific phospholipase C (PIPLC) treatment,an enzyme that specifically cleaves GPI linkages(46, 47)] also induced cell clustering, whereasGDNF alone or in combination with any otherco-receptor induced no clustering above thatobserved under background control conditions(Figs. 3, D and E, and 4C and fig. S3). Our dataconfirm the specificity of each ligand for its cog-nate co-receptor. Furthermore, our data demon-strate that simultaneous exposure of hematopoieticcells to GFLs and their soluble co-receptors wassufficient to activate RET-dependent lymphoidstructure formation. We detected soluble forms ofGFRa2 and GFRa3 in E15.5 intestines (fig. S3).In addition, stochastic individual cell–basedsimulations predicted that participation of GFRain PP initiation in trans would yield numbers ofPPs similar to those induced by GFRa usage incis (Fig. 4, D and E, and movies S3 and S4).Therefore, our data suggest that hematopoieticcells respond in an unconventional, but efficient,manner to all RET ligands by responding toGFRa co-receptors in a non–cell-autonomousmanner.

BSA ARTN

24 h

48 h

72 h

24 h

BSA PSPN

48 h

72 h

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Fig. 3. All GFLs can initiate enteric lymphoid organ formation. Explant cultures of intestines fromE15.5hCD2-GFP mice were incubated for 72 hours with agarose beads impregnated with ARTN, NRTN, orPSPN. (A to D) Representative examples of stereo microscopic analysis of intestine surfaces aftertreatment with (A) BSA or ARTN, (B) BSA or NRTN, (C) BSA or PSPN, or (D) BSA or GDNF. BSA wasusedasanegativecontrol.Darkcircles, agarosebeads;whitedots,GFP+cells; arrows,GFP+cell clusters.Similar results were obtained in five independent experiments. (E) Percentages of individual explantsthat formedcell aggregates in the vicinity ofGFL-soakedagarosebeads at 72 hours of culture. For (A),BSA beads, n = 35 experiments; ARTN beads, n = 25 experiments; two-tailed t test P = 0.0008. For(B), BSA beads, n = 20 experiments; NRTN beads, n = 25 experiments; two-tailed t test P = 0.0300.For (C), BSA beads, n = 15 experiments; PSPN beads, n = 15 experiments; two-tailed t test P =0.0029. For (D), BSA beads, n = 45 experiments; GDNF beads, n = 40 experiments; two-tailed t testP=0.3329. Error bars showSEM. (F) Confocalmicroscopic analysis of ARTN-,NRTN-, andPSPN-inducedGFP+ cell aggregates at 72 hours. Left panels in each pair: green, CD4; red, CD11c. Right panels ineach pair: green, GFP; red, VCAM1. Similar results were obtained in five independent experiments.

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Enteric neurons respond to GFLs only in cisAlthough formation of the PPs and the ENS depends on RET activation(2, 34), previous genetic studies suggest that there are distinct GFL re-quirements for hematopoietic and enteric neural crest cells (2, 34, 42).To test whether in trans RET signaling was used in cells other than LTincells, we studied the responses of enteric neurons in explanted entericorgan cultures to various GFLs alone or in combination with differ-ent co-receptors. Enteric neurons responded efficiently to GDNF, as re-vealed by the attraction of enteric axonal projections toward the beads,but failed to respond to beads soaked with ARTN, NRTN, or PSPN(4 mM), and provision of soluble GFRa2 or GFRa3 failed to induce neu-ral responses to NRTN or ARTN, respectively (Fig. 5, A to D, and fig.S4), even at high concentrations of GFLs (40 mM) (fig. S4). These datademonstrate that, although hematopoietic cells responded to all RET lig-ands by using GFRa co-receptors in trans, enteric neurons respondedonly to GDNF.

Expression of GFRa1 in cis reprograms RET responsesin hematopoietic cellsEnteric neural crest cells and neurons co-express RET and GFRa1 (in thesame cells, that is, in cis) (42), but RET+ hematopoietic cells lackGFRa co-receptors (2). In light of this, our data raised the possibility that the absenceofGFRa co-receptorsmight renderRET+hematopoietic cells promiscuouslyresponsive to different combinations of GFLs and their soluble co-receptors.To investigate this hypothesis, we introduced the hCD2-GFP transgene (2)into RetGfra1 knock-in mice (42) to generate mice in which all GFP+RET+

hematopoietic cells were reprogrammed to co-express GFRa1 in cis. In ex-periments with these mice, which developed a normal complement of PPs(fig. S5), we examined the kinetics of ectopic lymphoid structure formationin explant organ cultures in the presence of different GFLs. As predicted,GDNF failed to induce aggregation of GFP+ hematopoietic cells in the in-testines of E15.5 Ret+/+ (wild-type) mice but triggered efficient aggregationof GFP+ cells in explant cultures from hCD2-GFP RetGfra1mice (Fig. 6A).

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Fig. 4. Hematopoietic cells respond to GFLs in trans. Explanted organcultures of intestines from E15.5 hCD2-GFP mice were incubated with agar-ose beads impregnated with different combinations of GFLs and GFRaco-receptors. (A) Clustering efficiency in the vicinity of beads incubatedwith ARTN alone or with different GFRa co-receptors. Two-tailed t testP value at 96 hours comparing ARTN alone with ARTN and GFRa3. Errorbars show SEM. (B) Clustering efficiency as described for (A) but withbeads impregnated with NRTN alone or with different GFRa co-receptors.Two-tailed t test P value at 96 hours comparing NRTN alone with NRTN andGFRa2. Error bars show SEM. (C) Clustering efficiency as described for (A)but with beads impregnated with GDNF alone or with different GFRas. Two-

tailed t test P value at 96 hours comparing GDNF alone with GDNF andGFRa1. Error bars show SEM. (D) Image from the simulation of PP forma-tion by RET signaling in trans and in cis in 10% of the midgut length. Simu-lations were started at E14.5 in silico and run for 72 hours. Results showsnapshots of PP formation at 72 hours under conditions in which 0.1 or0.2% of total gut cells express GFLs. Red, LTin cells; green, LTi cells.(E) Summary of results from the simulation of PP formation per intestineby RET signaling in trans (white bars, n = 20 experiments) and in cis (graybars, n = 20 experiments). Two-tailed Mann-Whitney test P values. Errorbars show SEM. Additional statistical analysis of the data in this figurecan be found in the Supplementary Materials.

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We also noted that, although the combination of ARTN and soluble GFRa3induced efficient clustering of Ret+/+ GFP+ hematopoietic cells, RetGfra1

GFP+ hematopoietic cells responded poorly to this combination of mole-cules (Fig. 6B). These findings indicate that expression of GFRa1 in cisrestricts the response of the hematopoietic lineage to the available combina-tions of GFLs and GFRa molecules.

To further test the hypothesis that GFRa co-receptor expression patternsdetermine the response of RET-expressing cells to GFLs, we examined howneural crest–derived cells depleted of GFRa1 responded to GDNF in em-bryonic gut explant cultures. To achieve this, we dissected intestines fromE12.5Wnt1-Cre; Rosa26eYFPmice, in which enhanced yellow fluorescentprotein (eYFP) is found in all neural crest cells and their progeny (48, 49),and cultured them for 12 hours in three-dimensional (3D) collagenmatricesafter the cells had undergone a short treatment with either BSA or PIPLC.As expected, cleavage of GPI-linked GFRa1 caused a marked reduction inthe ability of neural crest cells to invade the collagen matrix in response toGDNF, although neurons could still respond more efficiently to treatmentwith the combination of GDNF and soluble GFRa1 (Fig. 6, C and D). Inaddition, medium supplemented with ARTN and GFRa3 induced PIPLC-treated cells to invade the collagenmatrix efficiently,whereas eYFP+neuronsfrom control guts, which expressed GFRa1 in cis, failed to respond to theARTN and its preferred co-receptor GFRa3 (Fig. 6, C and D).

DISCUSSIONOur data indicate that the initiation of PP formation is mediated by un-conventional RET signaling in which receptor stimulation is provided in

a non–cell-autonomous manner by GFLs and soluble GFRa proteins.These soluble GFRa molecules may capture diffusible GFLs, thus creat-ing enteric microenvironments that are privileged sites for GFL-inducedLTin modulation, although the identity of the cells that produce GFLs andGFRa remains elusive. We further showed that engagement of RET onenteric LTin cells led to their accumulation by adhesion-mediated arrest.Chemokines were not required for this initial phase; rather, the adhesionmolecule VCAM1 was necessary, and blockage of VCAM1 functioncaused a large reduction in the extent of cell-clustering efficiency. A smallnumber of hematopoietic cell aggregates were still observed when VCAM1was blocked, suggesting that other adhesion molecules may also contrib-ute to RET-dependent arrest of LTin cells. Our results are consistent withprevious findings that indicated impaired PP development upon VCAM1blockage in vivo (9) and the detection of minor enteric VCAM1-expressingcell aggregates before the accumulation of IL-7Ra+ cells in vivo (50). Al-though it seemed reasonable to speculate that an increase in VCAM1abundance might depend on LTb!LTbR signaling (2, 23), we found thatstimulation of LTin cells with GFLs did not increase their production ofLTb and that blockage of LTbR signaling did not impair the increase inabundance of VCAM1 on stroma cells (fig. S1). These data suggest thatthe initial triggering of enteric lymphoid tissue does not require LTb, whichis in agreement with previous studies of the initial phase of lymph nodedevelopment (51).

It was suggested that the initiation of lymph node formation may bemediated by retinoic acid signals that are provided by adjacent neurons(52). It remains unclear whether this signaling axis operates in the intestine.

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Fig. 5. Response of neural crest–derived cells to GFLs and soluble GFRaco-receptors. Explanted organ cultures of intestines from E15.5 hCD2-GFPmice were incubated for 96 hours with agarose beads impregnated withdifferent GFLs and GFRa co-receptors. (A) Gut cultures were incubatedwith agarose beads impregnated with ARTN, NRTN, or GDNF (4 mM),immunostained for GFP (green) and neuronal marker TUJ1 (red), and ana-lyzed by confocal microscopy. Similar results were obtained in five in-dependent experiments. Beads are indicated with a white “B.” Thedashed white line indicates the gut wall. (B) Explant organ cultures were

incubated with BSA-impregnated beads as a negative control and wereanalyzed as described in (A). (C) Explant organ cultures were incubatedfor 96 hours with agarose beads impregnated with combinations of ARTNand GFRa3, NRTN and GFRa2, or GDNF and GFRa1 and immunostainedfor GFP (green) and TUJ1 (red). (D) Explant organs were cultured withGDNF-impregnated beads. Arrows show the location of two beads en-circled with a dotted line. Ninety-six hours after incubation, samples werefixed and the cell tracker CM-Dil was applied in the wall of the intestine at adistance from the beads. Samples were analyzed by confocal microscopy.

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Gdnf or Gfra1 null embryos that fail to de-velop a myenteric nervous plexus havenormal PP development, arguing againstsuch a hypothesis (2, 43, 53). Nevertheless,parasympathetic axons, sympathetic axons,or both, which are still present in the gutsof these mutants, may provide such retinoicacid cues for PP initiation. Retinoic acidmodulates RET abundance during kidneydevelopment (54), suggesting that both sig-naling pathways may exhibit cross talk dur-ing enteric lymphoid morphogenesis. Thus,we propose a two-stepmodel of enteric lymph-oid organ morphogenesis in which RET sig-nals initiate lymphoid tissue formation throughthe adhesion-dependent arrest and accumula-tion of LTin cells and the consequent prim-ing of mesenchymal cells into LTo cells.These LTo cells would then ensure adhesion-dependent interactions with LTi cells thatultimately provide cues for the productionof chemokines by the LTo cells and theconsequent chemoattraction of large num-bers of LTi cells.

Our data showed that hematopoieticRET+GFRa! cells in the gut depended onexogenous GFRas for a successful responseto RET ligands. In contrast, because theyexpress the co-receptor GFRa1 in cis, RET+

neural crest–derived cells failed to respondto soluble combinations of GFLs and GFRamolecules in gut explant cultures. Therefore,in addition to their well-established cell-autonomous activities acting as co-receptorsfor RET, we showed that GFRas also actedas soluble co-receptors, which exerted phys-iological effects by modulating RET signal-ing in a non–cell-autonomous manner.

It remains unclear why the co-expressionof GFRa1 by RET-responsive cells pre-cludes them from responding to other GFL-GFRa combinations presented in trans. Itis possible that GPI-linked GFRa presentedin cis may enable more efficient RET-GFLinteractions than those that are formed bysoluble GFRa molecules presented in trans.Development of the ENS depends specifi-cally on a RET-GFRa1-GDNFaxis in differ-ent vertebrate species (55); however, organizedenteric lymphoid organs only arose in mam-mals. Thus, we suggest that evolutionarypressure may have been selected for enterichematopoietic RET+GFRa! cells that, incontrast to neurons, could use soluble GFLand GFRa guiding cues in a redundant andextremely versatile manner. The existence ofgut cells that produce GFLs and GFRa mol-ecules, but do not express RET, may haveenabled the evolution of hematopoietic cellsthat use RET signaling in trans, which could

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Fig. 6. The responses of hematopoietic and neural crest–derived cells to GFLs can be reprogrammed. (A)Intestines from E15.5 hCD2-GFPmice were cultured with beads impregnated with GDNF or BSA, and clusteringefficiencywas determined.Wild type (WT), n=8 experiments; GFRa1 cis, n=8 experiments. Two-tailed t testP values at 96 hours were as follows: P = 0.4070 for BSA versus WT, P = 0.0024 for BSA versus GFRa1 cis,andP=0.0015 forWTversusGFRa1cis. Error bars showSEM. (B) Beads impregnatedwithARTNandGFRa3or with BSA were cultured with intestines, and cell clustering was determined as described in (A). WT, n = 19experiments; GFRa1 cis, n=20 experiments. Two-tailed t testP values at 96 hourswere as follows:P=0.0026for BSA versusWT,P=0.2410 for BSA versusGFRa1 cis, andP=0.0192 forWT versusGFRa1 cis. Error barsshowSEM. (C) Representative intestines fromE12.5Wnt1-Cre;Rosa26eYFPmice treatedwithBSAasacontrol(CON) or with PIPLC were then cultured for 14 hours with GDNF, GDNF and GFRa1, or ARTN and GFRa3.Arrows indicate neurons or axons that exited thewall of the gut. (D) Quantification of the numbers of eYFP+ cellbodies andaxonal projections over the zplane in one field of viewat!24magnification. (Top)GDNF:CON,n=4 experiments; PIPLC, n=4 experiments; two-tailed t test P= 0.0241. (Middle) GDNF andGFRa1: CON, n= 5experiments; PIPLC, n = 6 experiments; two-tailed t test P = 0.0273. (Bottom) medium containing ARTN andGFRa3:CON,n=9experiments; PIPLC,n=9experiments; two-tailed t testP=0.00003. Error bars showSEM.

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have advantages compared to neurons that had used the same signaling sys-tem exclusively in cis.

Here, we describe an initiation phase of lymphoid organ developmentin the gut that relies on cell adhesion–mediated arrest of LTin cells, whichis conveyed through a versatile, yet unconventional, RET response systemin which the RET ligand and the co-receptor are produced in soluble formin trans by other nearby cells. Our data provide one illustration of howstimulation of onemolecule, RET, can cause different responses in differenttargets, lymphoid cells, and neurons. During enteric organogenesis, by usingdifferent co-expression patterns of RET and GFRa co-receptors, lymphoidand neural cells can both discriminate and respond to RETand yet maintaintheir identity and ensure distinct biological outcomes and functions.

MATERIALS AND METHODS

MicehCD2-GFP (2), RorcGFP/GFP (13), Wnt1 Cre (48), ROSA 26 eYFP (49),RetGfra1/Gfra1 (42), Artn–/– (44), and Nrtn!/! (45) mice have been describedpreviously. All mice were bred at the National Institute for Medical Re-search and the Instituto de Medicina Molecular (IMM) animal facilities.Animal use and procedures were in accordance with national and institu-tional guidelines.

Explant culturesIntestines were removed from E15.5 mice and were cultured in vitro at37°C in 5% CO2. To study the responses of hematopoietic cells to com-binations of soluble GFLs and GFRa molecules, we impregnated agarosemicrobeads overnight with solutions of ARTN (PeproTech), GDNF (R&DSystems), NRTN (PeproTech), PSPN (PeproTech), GFRa1 (R&D Sys-tems), GFRa2 (R&D Systems), GFRa3 (R&D Systems), or BSA (Sigma)(all at 100 ng/ml). These microbeads were then incubated with the intestinecultures. Pictures were taken with a Zeiss M2Bio (Carl Zeiss Ltd.) micro-scope. Cluster efficiency was determined by the percentage of samples inwhich at least one GFP+ cell aggregate (larger than 50 mm in diameter)was observed in the vicinity of impregnated beads (at a distance of lessthan 500 mm from the bead). Blocking antibody against VCAM1 (6C7.1,Hycult Biotech) (56) and antibodies against CCL19 (AF880, R&D Sys-tems), CCL21 (AF457, R&D Systems), and CXCL13 (AF470, R&D Sys-tems), as well as rat immunoglobulin G1 (IgG1) isotype control (RTK2071,BioLegend) and goat IgG isotype control antibodies (ab37388, Abcam),were used at a concentration of 5 mg/ml. LTbR:Fc (10 mg/ml; Alexis Bio-chemicals) was used. Microdissection of enteric areas surrounding agarosebeads was performed with a Zeiss SteREO Lumar.V12 (Carl Zeiss Ltd.)microscope and Dumont 0.01-mm forceps. Intestines from E15.5 micewere incubated at 37°C for 8 hours with PIPLC (0.2 U; Sigma) or BSA(10 ng/ml). Intestines from E12.5 Wnt1-Cre; Rosa26eYFP mice (48, 49)were incubated at 37°C for 6 hours with PIPLC (0.2 U) or BSA (10 ng/ml).Treated intestines were placed in a rat tail collagen matrix (BD Biosciences)and incubated with a medium containing recombinant GDNF (10 ng/ml;R&D Systems) or ARTN (10 ng/ml; PeproTech) and GFRa3 (10 ng/ml;R&D Systems). Numbers of cell bodies and axonal projections weremeasured over the z plane in one field of view with !24 magnificationwith a Zeiss M2Bio microscope and OpenLab software (Improvision).

Stereo and time-lapse microscopyStereomicroscopywas performedwith a ZeissM2Biomicroscope. Pictureswere captured with an ORCA-ER camera (Hamamatsu) and OpenLabsoftware (Improvision). Time-lapse images were acquired with the OpenLabsoftware. Images were taken every 60 s for 60 min. Time-lapse images were

taken after 24 hours of culture of intestine explants. Movie sequencing andcell tracking were performed with Volocity 4.0 (Improvision).

In silico simulationComputer simulations were developed with the MASON agent-basedsimulation platform (57), Java version 1.6. There are four cell types in thesimulation: LTin, LTo, LTi, and a non-LTo cell type that produces RETligand. We constructed an agent-based simulation in which each cell typewas represented explicitly as an agent, of which a number was introducedto an environment, and the interactions between agents were monitoredand analyzed. A description of the cell types and steps used to create thein cis and in trans RET signaling model is depicted in the activity dia-grams (figs. S6 and S12). The current understanding of the behavior ofeach of these cell types was initially modeled with a notation similar tounified modeling language (UML). These diagrams document the statesin which a cell may exist and the interactions that can occur for the cellstates to change and form our domain model (figs. S7 to S10 and S13to S16). The parameters identified in the creation of the model werederived from experimental data, and where data were not obtainable, suit-able assumptions were made and documented (fig. S11). These modelswere then used to create the platform model, again with a modified ver-sion of the UML (figs. S7 to S10 and S13 to S16). The platform modelcaptures how the biological understanding documented in the domainmodel translates to the encoding of cell state and interactions in the sim-ulation. One iteration of the simulation equals 60 s of development time.Initially, LTo cells were randomly distributed along a simulated tissue rep-resenting 10% of the entire length of the intestine. This measurement wasgained from taking 10% of the average length of the intestines of six miceat E15.5. The number of LTin and LTi cells in the simulation was cal-culated on the basis of cell counts obtained by flow cytometric analysisat E15.5 (LTin, 0.3 to 0.6%; LTi, 0.25 to 0.5%) and confocal analysis. Bytaking the midpoints of the measured percentage ranges, we calculated thenumber of cells that would be present at E15.5 and used this to generate arate of input that, if accumulation started at E14.5, would lead to this pop-ulation target being met at E15.5. LTin and LTi cells were assigned aspeed with a Gaussian distribution, with the limits taken from experimen-tal results (minimum rate, 3.8 mm/min; maximum rate, 8.8 mm/min). Thesimulation was initialized with 20% of the tract surface populated by LTocells, a percentage obtained from flow cytometric analysis (CD45!GP38+

cells). An arbitrary percentage of these cells was then set as being producersof RET ligand. When non-LTo RET ligand producer cells were introducedinto the simulation, and these were set to populate 1% of the stroma, againrandomly distributed along the intestine length. Each simulation was per-formed 20 times, and median values were taken and reported at 12 and72 hours of simulated time intervals. Data were logged into a comma-separated file at each iteration of the simulation and thenwere analyzedwithscripts written in Java, with statistical analysis performed in R. The simu-lation is accessible online at http://www.cs.york.ac.uk/immunesims/ppatch.

Confocal microscopyIntestines were fixed in 4% paraformaldehyde at room temperature for20 min and then stained with Alexa Fluor 647–conjugated antibodyagainst CD4 (Serotec), Alexa Fluor 488–conjugated antibody againstGFP (Invitrogen), purified rat antibody against VCAM1 (BD Pharmingen),PE-conjugated antibody against CD11c (eBioscience), Alexa Fluor 488–conjugated antibody CD11c (BioLegend), antibody against TUJ1 (Covance),antibody against BrdU (PRB-1) (Invitrogen), Alexa Fluor 647–conjugatedgoat antibody against rat antibody (Invitrogen), and Alexa Fluor 647–conjugated goat antibody against rabbit antibody (Molecular Probes).CM-Dil (Invitrogen) was used according to the manufacturer’s instructions.

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Samples were optically cleared in benzyl alcohol and benzyl benzoate(Sigma) and acquired on a Leica SP2 microscope (Leica Microsystems)and Zeiss LSM 710 microscope (Carl Zeiss Ltd.) with a !10/0.3– to0.4–numerical aperture objective lens. Images were processed with Vo-locity (Improvision) and Zeiss LSM Image Browser 4.2 (Carl Zeiss Ltd.).Snapshot pictures were obtained from 3D images. Depending on the an-tibody combinations used for immunostaining, it was not always possibleto use the same color channel for a specific molecule. Agarose beads areautofluorescent at greater than 560 nm. Thus, depending on the number ofexcitation wavelengths greater than this value, beads can exhibit differentcolors because of overlap between different wavelengths.

Western blotting analysisIntestines from E15.5 mice were cultured in serum-free medium for48 hours, after which the conditioned medium was collected, clarified,and concentrated with Vivaspin columns (Sartorius Stedim Biotech). SDS–polyacrylamide gel electrophoresis and Western blotting analysis were per-formed as previously described (58), with antibodies against GFRa2 (AF429,R&D Systems) and GFRa3 (ab8028, Abcam), as well as horseradish perox-idase (HRP)–conjugated goat antibody against rabbit IgG (sc-2004, SantaCruz Biotechnology) and HRP-conjugated donkey antibody against goatIgG (sc-2020, Santa Cruz Biotechnology) as secondary reagents.

RT-PCR analysisRT-PCR analysis was performed as previously described (2). Primer se-quences can be found in the Supplementary Materials. Primers for theamplification of full-length Gfra4 were previously described (59).

Statistical analysisStatistical analysis was performed with Microsoft Excel and Prism 4.1.Variance was analyzed with the F test. The Student’s t test was performedon homocedastic populations, and the Student’s t test with Welch correc-tion was applied to samples with different variances. Equality of severalmeans was determined by analysis of variance (ANOVA) test. The Mann-Whitney test was used to analyze non–Gaussian-distributed populations.The A test score is between 0 and 1. A score of 0.5 indicates “no difference”between two sets of results that are being compared. The difference in twodistributions becomes more significant as the results approach 0 or 1.

SUPPLEMENTARY MATERIALSwww.sciencesignaling.org/cgi/content/full/5/235/ra55/DC1MethodsFig. S1. Stroma cell activation, LTi and LTin cell frequencies, chemokine blocking, lympho-toxin modulation, and A test results for chemokine simulations.Fig. S2. RET co-receptors in the gut.Fig. S3. GDNF and GFRa1 signaling in lymphoid organ formation.Fig. S4. Neural projections induced by GFLs.Fig. S5. PP development in GFRa1 cis mice.Fig. S6. Activity diagram of cellular events leading to PP triggering.Fig. S7. State diagram of LTin cells.Fig. S8. State diagram of LTi cells.Fig. S9. State diagram of LTo cells.Fig. S10. State diagram of non-LTo RET ligand producer cells.Fig. S11. Parameter descriptions.Fig. S12. Activity diagram of trans RET signaling events leading to PP formation.Fig. S13. State diagram of LTin cells when RET signaling in trans is used.Fig. S14. State diagram of LTi cells when RET signaling in trans is used.Fig. S15. State diagram of LTo cells when RET signaling in trans is used.Fig. S16. State diagram of non-LTo RET ligand producer cells for in trans RET signaling.MovieS1.Motility of hematopoieticGFP+cells in explantedgut cultureswithARTN-soakedbeads.MovieS2.Motility of hematopoieticGFP+ cells in explantedgut cultureswithBSA-soakedbeads.Movie S3. Motility of hematopoietic cells in silico using RET signaling in cis.Movie S4. Motility of hematopoietic cells in silico using RET signaling in trans.Simulation code

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Acknowledgments: We thank V. Sasselli, K. Roderick, T. Norton, K. Williams, and theIMM imaging department for technical assistance. Funding: L.M.-S. and M.F. weresupported by scholarships from Fundação para a Ciência e Tecnologia, Portugal. Workin this paper was funded by the Medical Research Council, UK (MRC U117512796 andMRC G0601156); the Fundação para a Ciência e Tecnologia, Portugal (PTDC/SAU-MII/100016/2008); the European Molecular Biology Organization (project 1648); and the Eu-ropean Research Council (project 207057). Author contributions: A.P., N.H., L.M.-S., M.F.,K.A., K.F., A.G., P.P., and H.V.-F. performed the experiments and the data analysis; K.A.,J.T., P.A., and M.C.C. designed the in silico experiments; H.E. and J.M. provided RetGfra1/Gfra1

mice; V.P., D.K., and H.V.-F. designed the in vivo and ex vivo experiments; and H.V.-F.directed the study and wrote the manuscript. Competing interests: The authors declarethat they have no competing interests. Data and materials availability: The sequencesfor the following can be found in GenBank with the following accession numbers: Gdnf,107430; Artn, 1333791; Nrtn, 108417; Pspn, 1201684; Gfra1, 1100842; Gfra2, 1195462;Gfra3, 1201403; Gfra4, 1341873; Hprt1, 96217; Ccl19, 1346316; Ccl21, 1891386; Cxcl13,1888499; Ltb, 104796.

Submitted 28 November 2011Accepted 13 July 2012Final Publication 31 July 201210.1126/scisignal.2002734Citation: A. Patel, N. Harker, L. Moreira-Santos, M. Ferreira, K. Alden, J. Timmis,K. Foster, A. Garefalaki, P. Pachnis, P. Andrews, H. Enomoto, J. Milbrandt, V. Pachnis,M. C. Coles, D. Kioussis, H. Veiga-Fernandes, Differential RET signaling pathways drivedevelopment of the enteric lymphoid and nervous systems. Sci. Signal. 5, ra55 (2012).

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