Postnatal lymphatic partitioning from the bloodvasculature in the small intestine requiresfasting-induced adipose factorFredrik Backhed*, Peter A. Crawford, David O’Donnell, and Jeffrey I. Gordon†

Center for Genome Sciences, Washington University School of Medicine, St. Louis, MO 63108

Edited by Kurt J. Isselbacher, Massachusetts General Hospital Cancer Center, Charlestown, MA, and approved November 7, 2006 (received for reviewJuly 14, 2006)

Lymphatic vessels develop from specialized venous endothelialcells. Using knockout mice, we found that fasting-induced adiposefactor (Fiaf) is required for functional partitioning of postnatalintestinal lymphatic and blood vessels. In wild-type animals, levelsof intestinal Fiaf expression rise during the first postnatal day andpeak at day 2, which coincides with the onset of the lymphatico-venous partitioning abnormality in Fiaf�/� mutants on a mixed129/SvJ:C57BL/6 genetic background. Fiaf deficiency is not associ-ated with disruption of the blood vasculature or with lymphaticendothelial recruitment of smooth muscle cells. We identifiedProx1, a critical regulator of lymphangiogenesis, as a downstreamtarget for Fiaf signaling in the intestinal lymphatic endothelium.This organ-specific lymphovascular abnormality can be rescued byallowing embryonic Fiaf�/� intestinal isografts to develop inFiaf�/� recipients.

angiopoietin-like proteins � organ-specific regulator of lymphangiogenesis �postnatal gut development

Our lymphatic system performs a number of essential func-tions, ranging from the trafficking of immune cells to

draining fluids from the interstitial spaces of various organs totransporting absorbed dietary lipids to their sites of metabolicprocessing. Recent gene-targeting studies in mice support amodel of lymphatic development originally proposed by Flo-rence Sabin in 1902 (1, 2). During embryogenesis, subpopula-tions of venous endothelial cells bud to form distinct lymphaticsacs in the region of the primitive subclavian, inferior vena cava,and iliac veins; these sacs then divide extensively to createarborized lymphatic networks. A number of mediators of thecardinal stages of embryonic lymphatic development have beenidentified recently, including the homeodomain transcriptionfactor Prospero-related protein 1 (Prox1), Vegf-C, and Vegfreceptor 3 (1–5). However, information is lacking about thefactors that shape and maintain the lymphatic system during thepostnatal period.

Fasting-induced adipose factor (Fiaf), also known as angio-poietin-like protein 4 (Angptl4), is a glycosylated, secreted, andproteolytically processed protein produced by small intestinalenterocytes, white adipose tissue, liver, placenta, and muscle(6–9). Fiaf appears to have several physiological roles. It is acirculating inhibitor of lipoprotein lipase (9, 10), an enzyme thatpromotes storage of triglycerides in adipocytes. Enterocyte-specific suppression of this lipoprotein lipase inhibitor by theintestinal microbiota facilitates storage of energy, extracted fromthe diet by gut microbes, in fat cells (9). Fiaf also promotesendothelial cell survival in the gut after damage from ionizingradiation (11) and reduces Vegf-induced microvascular perme-ability in the skin (12). In this article, we identify Fiaf as anorgan-specific mediator of lymphangiogenesis that is instrumen-tal in sustaining separated blood and lymphatic circulatorysystems in the intestine, and in its supporting mesentery afterbirth.

Results and DiscussionFiaf-Deficient Animals Die During the Suckling Period with DilatedIntestinal Lymphatic Vessels. We found that newborn mice, ho-mozygous for a null allele of Fiaf (9) and with a mixed 129/SvJ:C57BL/6J (B6) genetic background, are born at a normalMendelian ratio (26.5%). Although they appear grossly normalat birth, the vast majority (41/44; 93%) dies by 3 weeks of age.In contrast, their heterozygous littermates live through adult-hood. Interestingly, Fiaf�/� mice can be rescued from the lethalphenotype by backcrossing them one generation onto a B6background: only 4.1% (5/121) of these B6-enriched Fiaf�/�animals die by postnatal day 15 (P15), with the remaindersurviving through adulthood.

Fiaf�/� mice with the original hybrid 129/Sv:B6 backgrounddevelop shoulder girdle wasting and protuberant abdomensduring the first postnatal week of life (Fig. 1A). The smallintestine, but not the colon, assumes a bright red color beginningat P3 (Fig. 1B). By P7, small intestinal villi are filled with blood(Fig. 1C). The protuberant abdomen reflects a marked increasein the small intestine-to-body weight ratio (3.5-fold comparedwith wild-type and heterozygous littermates at P6–P10; P �0.0001; n � 11–20 animals per group) (Fig. 1D).

P7 Fiaf�/� animals are not anemic (blood hemoglobin, 16.0 �0.7 g/dl versus 15.3 � 0.6 g/dl in wild-type and Fiaf�/� litter-mates; P � 0.32, n � 6–14 mice per group). In addition, theirintestinal contents are not bloody. However, histologic analysisof P7 Fiaf�/� intestines revealed blood in dilated, thin-walledmesenchymal vessels that stain positive for lymphatic vesselendothelial hyaluronan receptor 1 (Lyve-1), a lymph-specificbiomarker that is not expressed in blood vascular endothelialcells (13) (Fig. 2 A and B). This dilatation is not accompanied bysignificant differences in levels of intestinal expression of Lyve-1and another well established lymphatic marker, podoplanin, inknockout compared with wild-type animals [see supportinginformation (SI) Figs. 6 and 7].

Defective Separation of the Intestinal Lymphatic and Blood Micro-vasculature in Fiaf�/� Animals. Blood-filled lymphatic vesselscould arise from aberrant communications between lymphatic

Author contributions: F.B. and P.A.C. contributed equally to this work; F.B., P.A.C., D.O., andJ.I.G. designed research; F.B., P.A.C., and D.O. performed research; F.B., P.A.C., and J.I.G.analyzed data; and F.B., P.A.C., and J.I.G. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS direct submission.

Freely available online through the PNAS open access option.

Abbreviations: Fiaf, fasting-induced adipose factor; qRT-PCR, quantitative RT-PCR; Pn,postnatal day n; Lyve-1, lymphatic vessel endothelial hyaluronan receptor 1; BS-1, Ban-deiraea simplicifolia 1 lectin; En, embryonic day n.

*Present address: Wallenberg Laboratory, Sahlgrenska University Hospital, SE-41345 Gote-borg, Sweden.

†To whom correspondence should be addressed. E-mail: jgordon@wustl.edu.

This article contains supporting information online at www.pnas.org/cgi/content/full/0605957104/DC1.

© 2007 by The National Academy of Sciences of the USA

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and blood vascular systems or, less likely, generalized extrava-sation from the blood vascular system and subsequent uptake ofblood cells by neighboring lymphatics. Three observations argueagainst extravasation. First, P7 Fiaf�/� mice and their �/� and�/� littermates were given a single i.v. injection of FITC-conjugated Bandeiraea simplicifolia 1 lectin (BS-1) (n � 4animals per group). BS-1 binds to �-D-galactosyl- and N-acetyl-�-D-galactosaminyl-containing glycans expressed by both bloodand lymphatic vascular endothelial cells (11, 14). In normal mice,i.v. administered FITC-BS-1 labeled endothelial cells in thesmall intestine’s subepithelial blood microvasculature, but notthe endothelium that lines its Lyve-1-positive lymphatic system,consistent with the fact that venous contents do not normallyflow into lymphatics. In contrast, FITC-BS-1 labeled bothlymphatic and blood endothelial cells in the intestines of Fiaf�/�animals (Fig. 2, compare C–E with F–H). Importantly, we did notobserve BS-1 staining outside of vessels in the Fiaf�/� intestinalmesenchyme, even though this lectin binds to epitopes com-monly found both within and outside of blood vessels. Second,transmission electron microscopic studies of blood vascularendothelial cell morphology revealed that, despite the somewhatroughened appearance of their apical surfaces, tight junctionmorphology is similar in Fiaf�/� and wild-type littermates (seeSI Fig. 8). Third, immunohistochemical assessments of theenvelopment of the intestinal blood vasculature by smoothmuscle cells (see SI Fig. 9), and TUNEL assays of endothelialapoptosis (see SI Fig. 10) in P7 Fiaf�/� and wild-type litter-mates, failed to reveal any differences.

Based on these results, we concluded that the presence of

blood within the small intestinal lymphatic microvasculature ofFiaf�/� mice was due to a functional lymphatico-venous par-titioning abnormality. However, we were not able to definitivelydetect anastamoses between Lyve-1-positive lymphatic andLyve-1-negative/CD31-positive blood vessels, despite surveying�500 sections prepared from the small intestines of P2–P17knockout mice.

The Small Intestinal Lymphatico-Venous Partitioning AbnormalityDoes Not Develop Until After Birth and Extends to the Mesentery. Wedid not observe blood in lymphatics until P2–P3 in Fiaf�/� pups:embryonic day 18 (E18) and P0 Fiaf�/� animals exhibitedidentical histology as wild type (n � 5 per group) (see Fig. 3),indicating that embryonic lymphatico-venous partitioning isintact at birth. Consistent with these findings, we documented amarked increase of intestinal Fiaf expression on the first post-natal day in wild-type mice, peaking at P2 (17.9 � 1.5-foldrelative to E18) (Fig. 4A). These results indicate that functionalseparation of the lymphatic and blood vasculature is activelypreserved during postnatal life through a process that requiresFiaf.

The ‘‘conjoined’’ lymphatico-venous abnormality extended tothe small intestinal mesentery. Lyve-1 staining of paraffin-embedded sections of mesentery revealed blood-filled and di-lated lymphatics in P7–P12 Fiaf�/� animals but not in �/� or�/� littermates (n � 5 per group) (SI Fig. 11). Gastric gavagewith Nile red, a neutral lipid-binding fluorescent dye that isabsorbed into the lymphatic system from the gut lumen, dis-closed ectatic lymphatic vessels that resembled ‘‘strings of sau-sages’’ as well as microscopic leakage of the dye, but noterythrocytes, into the surrounding mesentery. Neither of thesemesenteric phenotypes was observed in age-matched wild-typelittermates (see SI Fig. 11). We interpret the Nile red results asindicating that there are low levels of leakiness in the lymphaticsof Fiaf-deficient mice: this leakiness is not of sufficient magni-tude to produce chylous ascites but is sufficient to be detectedwith this sensitive fluorescent dye.

Intravenous injection of FITC-BS-1, intradermal injection ofEvans blue dye (a commonly used lymphatic tracer that bindsalbumin), plus Lyve-1 immunohistochemical studies of E18–P12Fiaf�/� mice and their age-matched wild-type and Fiaf�/�littermates disclosed that the lymphatic vascular defects associ-ated with Fiaf deficiency did not extend to involve centrallymphatics (see SI Fig. 12) or the lymphatic microvasculature ofthe skin (see SI Fig. 13). The liver, which also expresses highlevels of Fiaf, developed normal vascular features (data notshown). We did note dilated blood-filled lymphatic vessels in thecolons of the small subset of Fiaf�/� mice that survived untilP17 (see SI Fig. 14), emphasizing the evolving nature of thisdefect within the gut. Fiaf-deficient mice do not have thoraciceffusions or edematous and hemorrhagic skin (n � 41), provid-ing additional evidence that the abnormality is gut-specific.

Development of the Lymphatico-Venous Partitioning AbnormalityOccurs Independent of the Gut Microbiota. We have shown previ-ously that the intestinal microbiota is important for postnataldevelopment of the intestine’s blood vascular system (15) andthat Fiaf expression is higher in the villus epithelium of germ-free compared with conventionally raised wild-type mice (9, 11).Therefore, we rederived 129/Sv:B6 Fiaf�/� mice as germ-free toexamine whether expression of this abnormal lymphatic pheno-type was elicited by the microbial colonization of the postnatalgut. We found that the lymphatico-venous abnormality andpostnatal lethality were fully penetrant in these germ-free ani-mals and indistinguishable from what was noted in their synge-neic, conventionally raised Fiaf�/� counterparts. Thus, theimpact of Fiaf deficiency on postnatal development of the

Fig. 1. Fiaf-deficient mice have blood-filled small intestinal villi. (A) A P12Fiaf�/� mouse (mixed 129/SvJ:B6 genetic background) exhibits cachexia anda protuberant abdomen compared with its Fiaf�/� littermate. (B) Smallintestine of the P12 Fiaf�/� animal shown in A appears bright red, thickened,and foreshortened. These gross abnormalities terminate abruptly at the ileal–cecal junction (arrows). S, stomach. (C) Whole-mount preparation of smallintestines from the mice in A, demonstrating blood-filled Fiaf�/� villi. (Scalebars: 1 mm.) (D) Increased small intestine-to-body weight ratio in P6–P10Fiaf�/� mice (n � 11) compared with Fiaf�/� and Fiaf�/� littermates (n � 20)(P � 0.001). Mean values � SE are plotted.

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intestinal lymphovascular system occurs independent of the gutmicrobiota.

Prox1, a Homeobox Gene Required for Normal Lymphangiogenesis, IsRegulated by Fiaf. To identify additional molecular correlates ofthe lymphatico-venous abnormality, we performed quantitativeRT-PCR (qRT-PCR) studies of total small intestinal RNAisolated from P7 Fiaf�/� mice and their wild-type littermates(n � 4 animals per group, each analyzed individually). Therewere modest (�2-fold) reductions in expression of known bi-omarkers of lymphatic vascular development (Vegf-C and Neu-ropilin 2, P � 0.05) whereas, as noted above, levels of mRNAsencoding other lymphatic markers (Lyve-1, podoplanin, andVegr 3) did not change (see SI Fig. 6). These results, togetherwith our immuohistochemical analyses, indicate that the ob-served lymphatico-venous malformations are manifestations ofa specific rather than a global change in the nature of lymphaticvessels.

Prox1 is a homeobox gene essential for normal embryonicdevelopment (16–18): Prox1�/� mice die at approximatelyE14.5 without any lymphatics (1, 16). Heterozygotes do notsurvive unless they are on an NMRI-enriched genetic back-ground (16), and only a small proportion of these animals livepast the postnatal period (19). Surviving Prox1�/� mice havedilated and leaky intestinal lymphatics but no aberrant lym-phatico-venous connections. This lymphatic phenotype is mostpronounced in the small bowel (19). These observationsprompted us to assess the relationship between Fiaf and Prox1expression. qRT-PCR studies revealed that the perinatal induc-tion of Prox1 parallels that of Fiaf in wild-type mice (Fig. 4B).Fiaf deficiency is associated with a 3-fold diminution in smallintestinal Prox1 expression at P7 (P � 0.05) but no significant

change in its expression before that time or in the skin at P7 (P �0.92) (Fig. 4B and SI Fig. 13). Follow-up immunohistochemicalstudies disclosed that, in contrast to wild-type mice, the Lyve-1-positive lymphatic endothelium of P7 Fiaf�/� animals rarelycontain Prox1-positive nuclei (Fig. 4 C–F).

We cannot yet provide a definitive mechanistic explanationabout how Fiaf regulates Prox1, or why a very small subset ofLyve-1-positive cells retains the ability to express normal levelsof Prox1 in P7 Fiaf�/� animals. Any effects of Fiaf on lymphaticendothelial Prox1 expression are likely to occur via paracrinesignaling. Fiaf, which encodes a secreted protein, is prominentlyexpressed in the epithelium but not in the underlying mesen-chymal compartment of the small intestine where lymphaticendothelial cells reside, whether judged by qRT-PCR of lasercapture microdissected cell populations or �-galactosidase im-munohistochemistry of small intestinal sections prepared fromFiaf�/� mice where the disrupted locus contains an insertedlacZ reporter (ref. 9 and data not shown).

Although these findings demonstrate that Prox1 expression inthe postnatal intestinal lymphatic endothelium is modulated byFiaf, the difference in phenotypes between Prox1-deficient andFiaf-deficient mice suggests that Fiaf can also effect maintenanceof lymphatico-venous partitioning through Prox1-independentpathways. The Prox1-deficient phenotype consists of absent orseverely abnormal vessels that are leaky: hence the chylousascites. The phenotype in Fiaf�/� mice is qualitatively different:the aberrant postnatal lymphatico-venous communication inthese animals does not lead to chylous ascites; however, suchcommunication could produce retrograde flow within lymphat-ics causing the small bowel wall edema that we observe (see Fig.1 and SI Fig. 8B Inset).

Fig. 2. Functionally conjoined lymphatic and blood vasculature in the small intestines of Fiaf�/� mice. (A and B) Bright-field immunohistochemistry showingLyve-1 (red) in hematoxylin-stained sections of small intestine from P7 Fiaf�/� (A) and Fiaf�/� (B) littermates. Cross-sections of villi from Fiaf�/� and Fiaf�/�small intestine are displayed in A Right and B Right, respectively. The dilated lymphatic vasculature in the villus core mesenchyme of the Fiaf-deficient mouseis lined by Lyve-1-positive endothelial cells (e.g., arrow) and filled with blood cells (asterisks). (C–H) Immunostaining for Lyve-1 (red) in the small intestines of P7Fiaf�/� and Fiaf�/� mice that received an i.v. injection of FITC-labeled BS-1 (blood and lymphatic vascular endothelial cell-binding lectin; green) 30 min beforethey were killed. Note the presence of dilated FITC-BS-1/Lyve-1-positive lymphatic vessels (orange) at the base of Fiaf�/� villi. Dashed lines in E and H outlinethe margins of villi. (Scale bars: 50 �m in A and B and 100 �m in C–H.)

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Implantation of Fiaf�/� Isografts into Fiaf�/� Recipients Rescues theLymphatico-Venous Partitioning Abnormality. To further confirm acentral role for Fiaf in postnatal intestinal lymphovasculardevelopment, we harvested small intestines from E15 or E18Fiaf�/� mice and their �/� littermates (all on a mixed 129/Sv:B6 background) and implanted them into the dorsal s.c. tissueof syngeneic young adult Fiaf�/� recipients. The intestinalisografts, which had been harvested from donors and placed inrecipients under sterile conditions, were recovered 14 days afterimplantation and characterized by using histochemical andimmunohistochemical methods. Our previous studies using wild-type donors and recipients had shown that an ‘‘extrinsic’’ vas-culature from the adult recipient animal rapidly envelops theisograft and supports subsequent growth, villus morphogenesis,and epithelial cytodifferentiation of the intestinal implant in itss.c. habitat (20, 21).

Remarkably, intestinal isografts obtained from embryonicwild-type and Fiaf-deficient donors both exhibited normal lym-phovascular development after implantation into Fiaf-expressing wild-type recipients, as judged by Lyve-1 and Prox1staining of serially sectioned, well oriented, nascent crypt villusunits (four isografts per group; n � 200 villi per genotype)(Fig. 5).

These experiments indicate that development of the aberrantintestinal lymphatic phenotype in Fiaf�/� intestinal isograftscan be prevented if the late gestation/perinatal gut is exposed toan environment where there is circulating Fiaf. Although therescued Fiaf�/� isografts do not contain Fiaf locally produced

from their lining epithelium, they are exposed to a milieu inwhich circulating host intestine-derived Fiaf is present (9).Similarly, maternal Fiaf may cross the placental barrier, whichwould explain why Fiaf�/� pups are normal at birth but developa severe phenotype soon thereafter. Given the rapid neonatalinduction of Fiaf that occurs in the intact normal small intestine,our findings are consistent with the notion that sustainedlymphovascular partitioning requires locally produced Fiaf tosignal through an as yet undefined intestinal receptor.

Prospectus. The molecular mechanisms that regulate normalblood vasculature development have been the subject of inten-sive investigation, and a detailed picture is emerging about theunderlying signaling pathways (22). Despite its importance tohealth and disease, considerably less is known about the mech-anisms that support proper lymphatic development. In thisarticle we present evidence that separation of the intestinalmucosal lymphatic and blood microvasculature is a processwhose regulation continues well beyond fetal life and demon-strate the essential, organ-specific role of Fiaf in postnatallymphatic development.

The phenotype exhibited by Fiaf�/� mice is unique. Micehomozygous for a null allele of Slp-76, an SH2 domain-harboringadapter molecule, or the tyrosine kinase Syk, exhibit aberrantcommunications between their blood and lymphatic vessels (23).qRT-PCR assays of total small intestinal RNA disclose that Fiafdeficiency is associated with a 2.8 � 0.3 increase in Syk expres-sion (P � 0.03) (SI Fig. 6), although this may simply reflect thefact that Syk is expressed in blood cells that accumulate in theectatic lymphatic vessels of these Fiaf knockout animals (23).Unlike Fiaf�/� mice, the defects in Syk�/� and Slp76�/�animals appear during embryogenesis and are not gut-specific.Mice deficient in Angpt2, a member of the same superfamily asFiaf, also show postnatal intestinal lymphatic dilatation (24), butlymphatico-venous malformations are not present, and the phe-notype is not intestine-specific. Fiaf is the first described tissue-specific regulator of postnatal lymphangiogenesis: as such, thisobservation raises the question of whether there are additionalselective modulators of this process that operate in other organs.

MethodsAnimals. Specified pathogen-free Fiaf�/�, �/�, and �/� litter-mates on a mixed C57BL/6J:129/Sv background were housed inmicroisolater cages in a barrier facility and maintained under astrict 12-h light cycle. To test the effects of genetic backgroundon phenotypes associated with Fiaf deficiency, male Fiaf�/�mice with this hybrid B6:129/Sv background were bred towild-type C57BL/6J females (The Jackson Laboratory, BarHarbor, ME). The resulting Fiaf�/� progeny were intercrossedto generate Fiaf�/� animals (defined in this article as ‘‘back-crossed one generation onto a C57BL/6J background’’). Fiaf�/�animals with both genetic backgrounds were rederived as germ-free according to previously described protocols and reared ingnotobiotic isolators (9).

All animals were fed a standard chow diet (Lab Diet 5058;Purina Mills) (autoclaved in the case of germ-free mice). Allexperiments involving mice were conducted by using protocolsapproved by the Animal Studies Committee of WashingtonUniversity.

Histochemistry and Immunohistochemistry. Small intestines wereremoved en bloc and fixed overnight at 4°C in freshly prepared4% paraformaldehyde (in PBS). Fixed specimens used forhistochemistry were washed in 70% ethanol, mounted in 2%agar, and embedded in paraffin. Sections (7 �m thick) were cutfor subsequent staining with hematoxylin and eosin. For immu-nohistochemical studies, sections were deparaffinized in xylene,treated with isopropanol, blocked in 1% BSA/0.1% Triton

Fig. 3. Development of small intestinal lymphatics in wild-type and Fiaf�/�mice. (A–D) Close approximation of anatomically distinct Lyve-1-positive lym-phovasculature (L) and blood vasculature (b) in E18 and P0 wild-type (A and C)and Fiaf�/� (B and D) littermates. At these stages of development, noerythrocytes are seen in the lymphovasculature of knockout (or wild-type)littermates. (E and F) By P3, blood cells are present within the lymphaticvasculature (L*), but not in the extravascular space, of Fiaf�/� animals (com-pare Insets). (Scale bars: 50 �m in A, B, E, and F and 25 �m in C and D.)

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X-100/PBS, and incubated with rabbit anti-mouse Lyve-1(1:1,000; Upstate Cell Signaling Solutions). Antigen–antibodycomplexes were visualized by using Vectastain Elite and Nova

Red substrate (Vector Laboratories). Sections were counter-stained with hematoxylin.

For immunofluorescence studies, fixed tissue was cryopro-tected in 10% sucrose in PBS (3 h at 4°C), immersed in 20%sucrose/10% glycerol in PBS (overnight at 4°C), and then frozenin OCT TissueTek compound (VWR Scientific) before prepar-ing 8-�m-thick cryosections. A rat monoclonal antibody tomouse Lyve-1 (R & D Systems), rabbit anti-mouse/human Prox1(Abcam), Syrian hamster anti-mouse podoplanin (AngioBio),and mouse anti-mouse/human smooth muscle actin conjugatedto Cy3 (Sigma, St. Louis, MO) were diluted 1:100 in blockingbuffer before use. Secondary Alexa Fluor 594- or Alexa Fluor647-conjugated antibodies to rat, rabbit, or hamster Igs (1:100;Molecular Probes) were used to visualize antigen–antibodycomplexes. Nuclei were stained with DAPI (40 ng/ml blockingbuffer). An Axiomat inverted microscope and AxioVision 4software (Zeiss) were used to acquire and process immunoflu-orescent images. To visualize the microvasculature underlyingthe small intestinal epithelium, FITC-labeled BS-1 (Sigma) wasinjected retroorbitally (25 mg/kg of body weight) 30 min beforeanimals were killed (11).

Assessment of Programmed Cell Death. TUNEL assays were per-formed on fresh-frozen intestinal cryosections by using theTUNEL kit from Roche and the manufacturer’s suggestedprotocols. Sections were incubated with rat anti-Lyve-1 andAlexa Fluor 647-conjugated goat anti-rat Ig before the TUNELassay (11).

Nile Red Staining. Nile red fluoresces intensely in the presence ofboth neutral and polar lipids (25). P12 pups were gavaged with400 �l of a Nile red solution (12.5 �g/ml PBS; Molecular Probes)60 min before they were killed. The gut was removed en bloc, and

Fig. 4. Prox1 expression is regulated in the small intestine by Fiaf. (A) qRT-PCR analysis of the relative expression of Fiaf in the small intestine of wild-type E18–P24mice (n � 4 per group, each intestine analyzed individually; mean values � SE are plotted relative to E18). *, P � 0.05; **, P � 0.01. Control experiments showed thatFiaf expression remains constant in the liver during this period (data not shown). (B) Expression of Prox1 in the small intestines of Fiaf�/� and Fiaf�/� littermates (n �4 per group, each intestine analyzed individually; mean values � SE are plotted relative to E18; note that Fiaf�/� mice do not survive to P24, so no data are shown forthis time point). ***, P � 0.001 compared with wild type. (C–F) Multilabel immunofluorescence study demonstrating Prox1-positive nuclei (yellow) in Lyve-1-positivesmall intestinal lymphovascular endothelium (red). In wild-type mice (C and D), nearly all lymphatic endothelial cell nuclei are Prox1-positive, whereas in Fiaf�/� mice(E and F), �90% are Prox1-negative (arrowheads). The arrows in E and F point to a rare Prox1-positive lymphatic endothelial cell nucleus within the Fiaf�/� smallintestine. (Scale bars: 50 �m.)

Fig. 5. Circulating Fiaf rescues the conjoined lymphatico-venous abnormal-ity in Fiaf�/� isografts. Small intestinal isografts were harvested from E15Fiaf�/� or Fiaf�/� donors and implanted for 14 days into the dorsal s.c. tissueof adult Fiaf�/� recipients. Serial sections of harvested isografts were stainedwith antibodies to Lyve-1 (red) or Prox1 (yellow) and counterstained withhematoxylin. Representative nascent crypt villus units are shown. Lymphovas-cular development is indistinguishable in wild-type (A) and Fiaf�/� (B)isografts. Black-bordered insets show enlarged views of the black boxed areas:note the absence of dilated, blood-filled lymphatic vessels in the Fiaf�/�isograft. White-bordered insets show Lyve-1-positive lymphatic endotheliumwith Prox1-positive nuclei (arrows). (Scale bars: 25 �m.)

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lipids in the mesentery were visualized with an Olympus SZX12stereomicroscope.

Evans Blue Staining. P7 pups were injected with Evans blue dye(Sigma) in the hind paws 15 min before they were killed. Centrallymphatics were visualized with an Olympus SZX12 microscope.

qRT-PCR. RNA was isolated from the small intestines of E18, P2,P7, and P24 wild-type and E18, PO, P2, and P7 Fiaf�/� mice(n � 4 per group; RNeasy; Qiagen). Each RNA preparationfrom each individual animal was reverse-transcribed by usingSuperScript II and dT15 primers (Invitrogen). qRT-PCR assayswere performed in triplicate with gene-specific primers (SI Table1) and SYBR green (Abgene), as described previously (9, 11).Data were normalized to L32 mRNA (��CT analysis).

Isografts. Pregnant mice were killed on day 15 or day 18 ofgestation. The gastrointestinal tracts of their fetuses were re-moved en bloc after transection at the gastroesophageal junctionand the rectum and placed into RPMI medium 1640 (Gibco/BRL), maintained at 4°C. The stomach was removed by severingthe pyloric–duodenal junction, and the small intestine was

isolated by cutting the ileal–cecal junction. The small intestinalspecimen was then implanted into a young adult isogenic recip-ient mouse (20, 21). To do so, each recipient animal wasanesthetized with rodent anesthesia mixture [0.15 ml of asolution of ketamine (100 mg/ml) and xylazine (20 mg/ml)], anda midline incision was made in their paravertebral region. Apocket was created in the s.c. fascia with a blunt-ended probe,and the fetal isograft was placed into this space. The ends of theisograft were fastened and sealed with 7-0 proline sutures, andthe incision was closed with surgical clips. The isograft wasremoved by careful dissection 14 days after implantation andfixed in 4% paraformaldehyde/PBS as described above.

Statistical Analysis. Data were analyzed by using Student’s t testor ANOVA with Tukey’s post hoc analysis.

We thank Maria Karlsson, Sabrina Wagoner, and Howard Wynder forsuperb technical assistance and John Rawls, Justin Sonnenburg, EricMartens, Jeffrey Milbrandt, and Clay Semenkovich for helpful sug-gestions. This work was supported in part by National Institutes ofHealth Grants DK70977 (to J.I.G.) and DK073282 (to P.A.C.). F.B.was the recipient of a postdoctoral fellowship from the Wenner-GrenFoundation.

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