Wnt3 Localization in the Gut Epithelium Cell Research (2016) 26:515-516

Seeing is believing: Wnt3 localization in the gut epithelium Cell Research (2016) 26:515-516. doi:10.1038/cr.2016.41; published online 25 March 2016

Wnt morphogens are notoriously the crypt and terminally differentiate conspicuously absent from the secre- elusive proteins. Thanks to a recent into the specialized cells of the villus tory granules in Paneth cells. With this study published in Nature, Clevers compartment. Proliferation in the crypts elegant genetic tool, Farin et al. then and colleagues give us a first glimpse has long been assumed to be governed examined how HA-Wnt3 is transferred of a mammalian Wnt in action in the by a gradient of Wnt activity with the between producing and receiving cells. gut epithelium. ISCs at the crypt bottom exposed to Initial experiments showed that wild- Tissue function is crucially depen- maximal levels of Wnt signaling (Figure type organoids could not rescue growth dent on the proper spatial organization 1A). Consistent with this notion, the of Wnt3 mutant organoids in co-culture of cells in three dimensions. Cellular expression levels of Wnt target genes assays and addition of neutralizing anti- architecture is established in part by is typically high in cells occupying the bodies did not block growth of Wnt3HA/ morphogens or signaling molecules crypt bottom and declines as cells move HA organoids. These results suggested expressed in concentration gradients out of the stem cell compartment higher that active Wnt3 does not function as along the forming tissues. In Lewis up the crypt. Moreover, recent evidence a diffusible factor. To examine how Wolpert’s famous French flag model, has shown that the high Wnt activity far membrane-bound HA-Wnt3 trav- distinct biological effects are induced of ISCs depends on their close contact eled from its source, Farin et al. then depending on the position of cells within with neighboring post-mitotic Paneth employed a cell re-association assay. the morphogen gradient. Several types cells. Paneth cells not only play impor- Non-labeled HA-Wnt3-producing cells of morphogens have been described tant antimicrobial functions, but also were combined with wild-type dsRED including transcription factors in the orchestrate crypt growth by releasing cells and allowed to develop into large Drosophila syncytial embryo, metabo- Wnts (e.g., Wnt3 and Wnt11) and other mosaic organoids. At the interfaces lites such as retinoic acid and of course promitotic factors from the crypt base between producing and receiving cells, secreted growth factors such as Wnt, [2]. The question posed by Farin et al. HA-Wnt3 penetrated only one or two TGFβ and Shh. was whether Paneth cell-derived Wnts cell diameters into HA-Wnt3-negative The importance of morphogens in act only locally on adjacent ISCs or domains. These data thus suggest that regulating pattern formation during em- whether long-range mechanisms exist to Wnt3 transfer is limited in range and bryonic development and tissue regen- propel Wnts beyond the stem cell zone. involves direct contact between Wnt3- eration is well described, but what is not To track Wnt proteins, Farin et al. producing Paneth cells and ISCs. so clear is how gradients of morphogen devised a strategy that bypassed the Wnts trigger their biological effects activity are established across tissues. difficult task of generating reliable by binding to Frizzled receptors. Down- In a recent issue of Nature, Clevers Wnt antibodies and instead created stream signaling is terminated at the cell and colleagues provide new insight into a HA epitope-tagged Wnt3 knockin surface by induction of transmembrane this question by visualizing the distri- mouse (Wnt3HA/HA). Since HA-Wnt3 E3 ubiquitin ligases, Rnf43 and Znrf3, bution of one particular morphogen, was functionally active both in vivo which target Frizzleds to lysosomal called Wnt3, within the intestinal crypt and in organoid cultures, the authors degradation [3]. The negative action epithelium [1]. The gut epithelium is a could then use these mice to visualize of Rn43/Znrf3 on Frizzled activity is self-renewing tissue composed of highly HA-Wnt3 in intestinal crypts by simply inhibited by the association of Lgr4/5 repetitive units called crypts and villi. performing anti-HA antibody stainings proteins with their secreted ligands R- The former are formed by invagination in combination with signal amplification spondins. This complex recruits Rnf43/ of the epithelium and are largely made steps. By staining both permeabilized Znrf3 away from Frizzleds and results in up of intensely proliferating intestinal and non-permeabilized tissues, the perdurance of Wnt stimulation. Armed stem cells (ISCs) and progenitor popula- authors concluded that HA-Wnt3 was with this knowledge, Farin et al. next tions. As part of their normal life cycle, primarily at the external surfaces of manipulated Frizzled cell surface levels most epithelial cells are forced out of basolateral membranes of crypt cell and indirectly by modulating R-spondin1 npg 516 extracellular matrix. How this relates to Wnt3 transport in the crypt epithelium or other tissue types is unknown. The only available clue is that retromer transport appears not to be necessary for Wnt signaling in the gut [9]. Hopefully, purification of HA-Wnt3 from the gut epithelium combined with the use of mass spectrometry can shed light on these issues. Finally, given that other sources of Wnts can sustain crypt homeostasis in Figure 1 Schematic view of the intestinal crypt and Wnt transfer from Paneth cells vivo in the absence of Paneth cells [10], to intestinal stem cells (ISCs). (A) A gradient of Wnt activity subdivides the crypt into a stem cell zone composed of ISCs and Paneth cells at the crypt bottom and it will also be interesting to see how a transit-amplifying zone made up of progenitor cells. (B) Wnt secretion in Paneth these Wnts get to their target cells. For cells depends partly on Porcupine and once released Wnts are captured by Friz- example, an important source of Wnts, zled receptors on ISCs. Frizzled turnover is mediated by Rnf43/Znrf3. The molecu- absent from the organoid system are lar mechanisms underlying Wnt secretion and transfer from Paneth cells to ISCs remain unclear. surrounding stromal cells. How do these stromally derived Wnts traverse the basement membrane and which epithelial cells do they target? Obviously, these and Rnf43 expression in organoids. Porcupine inhibitor to release newly fundamental questions will continue to These experiments showed that Friz- produced HA-Wnt3. After 48 h, crypts challenge researchers for several years. zleds are required to anchor HA-Wnt3 regained a normal gradient of HA-Wnt3 However, having now found Wnt3, we at the cell surface. Wnt ligands are expression. However, in the presence of are no longer in the dark. lipid modified, which is required for mitotic inhibitors blocking Egfr, Mek or its biological activity, but also presents Cdk4/6, HA-Wnt3 remained localized Alex Gregorieff1, somewhat of a conundrum for under- near producing cells. Therefore these Jeffrey L Wrana1, 2 standing how it acts at a distance [4]. data suggested that Wnt3 once bound Lipidation is mediated by, Porcupine, to ISCs travels up the crypt by piggy- 1Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Sinai Health System, 600 Univer- an ER-resident protein and o-aceyl backing on proliferating ISCs and their sity Avenue, Toronto, Ontario M5G 1X5, Canada; transferase that palmitoylates Wnts. The progeny rather than diffusion. 2Department of Molecular Genetics, University authors therefore explored HA-Wnt3 Now that we have a clear view of of Toronto, Toronto, Ontario M5S 1A8, Canada processing in Paneth cells using an in- at least one mammalian Wnt in its Correspondence: Jeffrey L Wrana E-mail: [email protected] hibitor of Porcupine. Indeed, treatment native environment, the next question of organoids with Porcupine inhibitor becomes one of mechanism. More References led to a loss of HA-Wnt3 at cell mem- specifically, how is Wnt3 transferred branes and corresponding cytoplasmic from producing cell to receiving cell. 1 Farin HF, Jordens I, Mosa MH, et al. Nature accumulation in Paneth cells within 24 Here numerous options exist. Follow- 2016; 530:340-343. 2 Sato T, van Es JH, Snippert HJ, et al. Nature h, consistent with Porcupine’s essential ing post-translational modifications in 2011; 469:415-418. role in secretion and lipid modification the ER and Golgi, Wnts are known to 3 de Lau W, Peng WC, Gros P, et al. Genes of Wnts. associate with the sorting receptor, Wls, Dev 2014; 28:305-316. Evidence so far suggested that HA- and are released into the extracellular 4 Bartscherer K, Boutros M. EMBO Rep 2008; 9:977-982. Wnt3 is secreted by Paneth cells via space [5]. Endosomes and the retromer 5 Willert K, Nusse R. Cold Spring Harb Per- Porcupine and captured by Frizzleds in complex recycle Wls, allowing for spect Biol 2012; 4:a007864. surrounding ISCs (Figure 1B). Because multiple rounds of Wnt secretion. Once 6 Stanganello E, Scholpp S. J Cell Sci 2016; ISCs are continuously undergoing released, Wnts can utilize several modes 129:665-672. 7 Steinhauer J, Treisman JE. Curr Opin Genet cell division, Farin et al. then asked of transport including lipoprotein parti- Dev 2009; 19:308-314. whether further propagation of HA- cles, exosomes and cytonemes [6-8]. To 8 Zhang L, Wrana JL. Curr Opin Genet Dev Wnt3 beyond the stem cell compartment complicate matters further, spreading of 2014; 27:14-19. depends on ISC cell division. Pulse- Wnts also depends on interactions with 9 de Groot RE, Farin HF, Macůrková M, et al. PLoS One 2013; 8:e76971. chase experiments were conducted by various heparan sulfate proteoglycans 10 Kabiri Z, Greicius G, Madan B, et al. Devel- washing out organoids treated with that reside in membranes and in the opment 2014; 141:2206-2215.

Cell Research | Vol 26 No 5 | May 2016