mouse-fetal-whole-intestine-culture-system-for-ex-vivo-manipulation

Mouse Fetal Whole Intestine Culture System for <em>Ex Vivo</em> Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over ...

University of Michigan

The foxa gene is an integral component of the endoderm specification subcircuit of the endomesoderm gene regulatory network in the Strongylocentrotus purpuratus embryo. Its transcripts become confined to veg2, then veg1 endodermal territories, and, following gastrulation, throughout the gut. It is also expressed in the stomodeal ectoderm. gatae and otx genes provide input into the pregastrular regulatory system of foxa, and Foxa represses its own transcription, resulting in an oscillatory temporal expression profile. Here, we report three separate essential functions of the foxa gene: it represses mesodermal fate in the veg2 endomesoderm; it is required in postgastrular development for the expression of gut-specific genes; and it is necessary for stomodaeum formation. If its expression is reduced by a morpholino, more endomesoderm cells become pigment and other mesenchymal cell types, less gut is specified, and the larva has no mouth. Experiments in which blastomere transplantation is combined with foxa MASO treatment demonstrate that, in the normal endoderm, a crucial role of Foxa is to repress gcm expression in response to a Notch signal, and hence to repress mesodermal fate. Chimeric recombination experiments in which veg2, veg1 or ectoderm cells contained foxa MASO show which region of foxa expression controls each of the three functions. These experiments show that the foxa gene is a component of three distinct embryonic gene regulatory networks.

Repression of mesodermal fate by foxa, a key endoderm regulator of the sea urchin embryo.

The Hedgehog (Hh) and Notch signal transduction pathways control a variety of developmental processes including cell fate choice, differentiation, proliferation, patterning and boundary formation. Because many components of these pathways are conserved, it was predicted and confirmed that pathway components are largely intact in the sea urchin genome. Spatial and temporal location of these pathways in the embryo, and their function in development offer added insight into their mechanistic contributions. Accordingly, all major components of both pathways were identified and annotated in the sea urchin Strongylocentrotus purpuratus genome and the embryonic expression of key components was explored. Relationships of the pathway components, and modifiers predicted from the annotation of S. purpuratus, were compared against cnidarians, arthropods, urochordates, and vertebrates. These analyses support the prediction that the pathways are highly conserved through metazoan evolution. Further, the location of these two pathways appears to be conserved among deuterostomes, and in the case of Notch at least, display similar capacities in endomesoderm gene regulatory networks. RNA expression profiles by quantitative PCR and RNA in situ hybridization reveal that Hedgehog is produced by the endoderm beginning just prior to invagination, and signals to the secondary mesenchyme-derived tissues at least until the pluteus larva stage. RNA in situ hybridization of Notch pathway members confirms that Notch functions sequentially in the vegetal-most secondary mesenchyme cells and later in the endoderm. Functional analyses in future studies will embed these pathways into the growing knowledge of gene regulatory networks that govern early specification and morphogenesis.

Genomics and expression profiles of the Hedgehog and Notch signaling pathways in sea urchin development.

The Hedgehog (Hh) signaling pathway is essential for patterning many structures in vertebrates including the nervous system, chordamesoderm, limb and endodermal organs. In the sea urchin, a basal deuterostome, Hh signaling is shown to participate in organizing the mesoderm. At gastrulation the Hh ligand is expressed by the endoderm downstream of the Brachyury and FoxA transcription factors in the endomesoderm gene regulatory network. The co-receptors Patched (Ptc) and Smoothened (Smo) are expressed by the neighboring skeletogenic and non-skeletogenic mesoderm. Perturbations of Hh, Ptc and Smo cause embryos to develop with skeletal defects and inappropriate non-skeletogenic mesoderm patterning, although initial specification of mesoderm occurs without detectable abnormalities. Perturbations of the pathway caused late defects in skeletogenesis and in the non-skeletogenic mesoderm, including altered numbers of pigment and blastocoelar cells, randomized left-right asymmetry of coelomic pouches, and disorganized circumesophageal muscle causing an inability to swallow. Together the data support the requirement of Hh signaling in patterning each of the mesoderm subtypes in the sea urchin embryo.

Hedgehog signaling patterns mesoderm in the sea urchin.

Hedgehog signaling is critical in gastrointestinal patterning. Mice deficient in Hedgehog signaling exhibit abnormalities that mirror deformities seen in the human VACTERL (vertebral, anal, cardiac, tracheal, esophageal, renal, limb) association. However, the direction of Hedgehog signal flow is controversial and the cellular targets of Hedgehog signaling change with time during development. We profiled cellular Hedgehog response patterns from embryonic day 10.5 (E10.5) to adult in murine antrum, pyloric region, small intestine, and colon.

Paracrine Hedgehog signaling in stomach and intestine: new roles for hedgehog in gastrointestinal patterning.

Fungiform papillae are complex taste organs that develop in a pattern on anterior tongue in rodent embryos. Several intrinsic secreted molecules are important for papilla development and patterning, including sonic hedgehog, bone morphogenetic proteins, Noggin, epidermal growth factor, and WNTs. Recent data about roles of WNTs in regulation of tongue and fungiform papilla development lead to new insights about the importance of tissue and timing contexts when studying the effects of morphogenetic proteins. WNT/beta-catenin signaling is required for formation of fungiform papillae, but not for determining tongue size and shape. In contrast, WNT5a apparently is important for tongue outgrowth, but not papilla development. Preliminary data from WNT5a mutant mice separate genetic programs for papilla number from those for tongue shape and size.

WNT5a in tongue and fungiform Papilla development.

&#x83CC;&#x72B6;&#x4E73;&#x5934;&#x662F;&#x5F00;&#x53D1;&#x6A21;&#x5F0F;&#x4E0A;&#x524D;&#x820C;&#x5728;&#x556E;&#x9F7F;&#x52A8;&#x7269;&#x80DA;&#x80CE;&#x4E2D;&#x7684;&#x590D;&#x6742;&#x5473;&#x9053;&#x673A;&#x5173;&#x3002;&#x51E0;&#x4E2A;&#x56FA;&#x6709;&#x5206;&#x6CCC;&#x7684;&#x5206;&#x5B50;&#x662F;&#x91CD;&#x8981;&#x7684;&#x4E73;&#x5934;&#x53D1;&#x5C55;&#x548C;&#x9635;&#x5217;&#xFF0C;&#x5305;&#x62EC;&#x58F0;&#x6CE2;&#x523A;&#x732C;&#x3001; &#x9AA8;&#x5F62;&#x6001;&#x53D1;&#x751F;&#x86CB;&#x767D;&#x3001; &#x8111;&#x888B;&#x3001; &#x8868;&#x76AE;&#x751F;&#x957F;&#x56E0;&#x5B50;&#x548C; WNTs&#x3002;&#x6709;&#x5173; WNTs &#x8C03;&#x63A7;&#x7684;&#x820C;&#x5934;&#x548C;&#x83CC;&#x72B6;&#x4E73;&#x5934;&#x53D1;&#x5C55;&#x89D2;&#x8272;&#x7684;&#x6700;&#x8FD1;&#x6570;&#x636E;&#x5BFC;&#x81F4;&#x7EC4;&#x7EC7;&#x548C;&#x8BA1;&#x65F6;&#x4E0A;&#x4E0B;&#x6587;&#x7684;&#x91CD;&#x8981;&#x6027;&#x6709;&#x5173;&#x7684;&#x65B0;&#x89C1;&#x89E3;&#x65F6;&#x7814;&#x7A76;&#x5F62;&#x6001;&#x53D1;&#x751F;&#x86CB;&#x767D;&#x7684;&#x5F71;&#x54CD;&#x3002;WNT &#x8FDE;&#x73AF;&#x4FE1;&#x53F7;&#x662F;&#x9700;&#x8981;&#x5F62;&#x6210;&#x7684;&#x83CC;&#x72B6;&#x4E73;&#x5934;&#xFF0C;&#x4F46;&#x4E0D;&#x662F;&#x786E;&#x5B9A;&#x7684;&#x820C;&#x5934;&#x5927;&#x5C0F;&#x548C;&#x5F62;&#x72B6;&#x3002;&#x76F8;&#x6BD4;&#x4E4B;&#x4E0B;&#xFF0C;WNT5a &#x663E;&#x7136;&#x662F;&#x91CD;&#x8981;&#x7684;&#x820C;&#x5934;&#x7684;&#x4EA7;&#x7269;&#xFF0C;&#x4F46;&#x4E73;&#x5934;&#x4E0D;&#x53D1;&#x5C55;&#x3002;&#x4ECE; WNT5a &#x57FA;&#x56E0;&#x7A81;&#x53D8;&#x5C0F;&#x9F20;&#x7684;&#x521D;&#x6B65;&#x6570;&#x636E;&#x5206;&#x5F00;&#x4E73;&#x5934;&#x6570;&#x9057;&#x4F20;&#x7684;&#x7A0B;&#x5E8F;&#x820C;&#x5F62;&#x72B6;&#x548C;&#x5927;&#x5C0F;&#x3002;

WNT5a &#x5728;&#x820C;&#x5934;&#x548C;&#x83CC;&#x72B6;&#x4E73;&#x5934;&#x53D1;&#x5C55;&#x3002;

Les papilles fongiformes sont les organes go&ucirc;t complexe qui se d&eacute;veloppent dans un motif sur une langue ant&eacute;rieure dans des embryons de rongeurs. Plusieurs mol&eacute;cules s&eacute;cr&eacute;t&eacute;es intrins&egrave;ques sont importants pour le d&eacute;veloppement de la papille et de structuration, notamment sonic hedgehog, prot&eacute;ines morphog&eacute;n&eacute;tiques osseuses, caboche, facteur de croissance &eacute;pidermique et WNTs. Des donn&eacute;es r&eacute;centes sur les r&ocirc;les de WNTs dans la r&eacute;gulation de la langue et le d&eacute;veloppement de papilles fongiformes conduisent &agrave; nouvelles perspectives sur l&#39;importance du tissu et des contextes de synchronisation lorsque l&#39;on &eacute;tudie les effets des prot&eacute;ines morphog&eacute;n&eacute;tiques. Signalisation WNT/b&ecirc;ta-cat&eacute;nine est requise pour la formation des papilles fongiformes, mais ne pas pour d&eacute;terminer la taille de la langue et la forme. En revanche, WNT5a est apparemment important pour l&#39;excroissance de la langue, mais pas de papille d&eacute;veloppement. Les donn&eacute;es pr&eacute;liminaires de souris mutantes WNT5a s&eacute;parent les programmes g&eacute;n&eacute;tiques pour nombre de papilles de ceux pour la taille et la forme de la langue.

WNT5a dans la langue et le d&eacute;veloppement de papilles fongiformes.

Fungiformen Papillen sind komplexe Geschmack-Organe, die in einem Muster auf vordere Zunge Nagetier Embryonen entwickeln. Mehrere systeminterne sezernierte Molek&uuml;le sind wichtig f&uuml;r die Entwicklung der Papille und Musterung, sonic Hedgehog, Bone morphogenetic Proteins, Noggin epidermalen Wachstumsfaktor und WNTs. Aktuelle Daten &uuml;ber Rollen von WNTs Verordnung der Zunge und fungiformen Papille Entwicklung f&uuml;hren zu neuen Erkenntnissen &uuml;ber die Bedeutung der Gewebe und Zeitmessung Kontexten die Auswirkungen der morphogenetic Proteins studieren. WNT/Beta-Catenin signalisieren ist erforderlich f&uuml;r die Bildung von fungiformen Papillen, aber nicht f&uuml;r die Bestimmung der Zunge Gr&ouml;&szlig;e und Form. WNT5a ist dagegen offenbar f&uuml;r Zunge Auswuchs, aber nicht Papille Entwicklung wichtig. Vorl&auml;ufige Daten aus WNT5a mutierte M&auml;use trennen genetische Programme zur Papille Anzahl von denen f&uuml;r die Zunge Form und Gr&ouml;&szlig;e.

WNT5a in der Zunge und fungiformen Papille-Entwicklung.

Papille fungiformi sono organi di gusto complesso che si sviluppano in un modello sulla linguetta anteriore in embrioni di roditori. Diverse molecole secrete intrinseci sono importanti per lo sviluppo della papilla e patterning, tra cui sonic hedgehog, bone morphogenetic proteins, Noggin, fattore di crescita epidermico e WNTs. Dati recenti sui ruoli di WNTs nella regolamentazione della lingua e sviluppo fungiformi portano a nuove intuizioni circa l&#39;importanza del tessuto e contesti di temporizzazione quando studiando gli effetti delle proteine morfogenetiche. Segnalazione WNT/beta-catenina &egrave; richiesto per la formazione di papille fungiformi, ma non per determinare la lingua dimensione e forma. Al contrario, a quanto pare WNT5a &egrave; importante per escrescenza lingua, ma non lo sviluppo papilla. Dati preliminari da topi mutanti WNT5a separano programmi genetici per numero di papilla da quelli per la lingua forma e dimensione.

WNT5a in lingua e lo sviluppo di papille fungiformi.

&#x8338;&#x72B6;&#x4E73;&#x982D;&#x9F67;&#x6B6F;&#x52D5;&#x7269;&#x306E;&#x80DA;&#x306B;&#x304A;&#x3051;&#x308B;&#x820C;&#x306E;&#x524D;&#x65B9;&#x306E;&#x30D1;&#x30BF;&#x30FC;&#x30F3;&#x3092;&#x958B;&#x767A;&#x3059;&#x308B;&#x8907;&#x96D1;&#x306A;&#x5473;&#x306E;&#x5668;&#x5B98;&#x3067;&#x3042;&#x308A;&#x307E;&#x3059;&#x3002;&#x3044;&#x304F;&#x3064;&#x304B;&#x306E;&#x7D44;&#x307F;&#x8FBC;&#x307F;&#x306E;&#x5206;&#x6CCC;&#x306E;&#x5206;&#x5B50;&#x306F;&#x4E73;&#x982D;&#x958B;&#x767A;&#x3068;&#x30BD;&#x30CB;&#x30C3;&#x30AF; &#x30D8;&#x30C3;&#x30B8;&#x30DB;&#x30C3;&#x30B0;&#x9AA8;&#x5F62;&#x6210;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x3001;&#x9152;&#x3092;&#x98F2;&#x3080;&#x3053;&#x3068;&#x3001;&#x8868;&#x76AE;&#x306E;&#x6210;&#x9577;&#x56E0;&#x5B50;&#x3001;WNTs &#x3092;&#x542B;&#x3080;&#x30D1;&#x30BF;&#x30FC;&#x30CB;&#x30F3;&#x30B0;&#x306E;&#x305F;&#x3081;&#x91CD;&#x8981;&#x3067;&#x3059;&#x3002;&#x820C;&#x3068;&#x8338;&#x72B6;&#x4E73;&#x982D;&#x958B;&#x767A;&#x306E;&#x898F;&#x5236;&#x306B;&#x304A;&#x3051;&#x308B; WNTs &#x306E;&#x5F79;&#x5272;&#x306B;&#x3064;&#x3044;&#x3066;&#x306E;&#x6700;&#x8FD1;&#x306E;&#x30C7;&#x30FC;&#x30BF;&#x306F;&#x5F62;&#x6210;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x306E;&#x52B9;&#x679C;&#x3092;&#x52C9;&#x5F37;&#x3059;&#x308B;&#x3068;&#x304D;&#x7D44;&#x7E54;&#x3068;&#x30BF;&#x30A4;&#x30DF;&#x30F3;&#x30B0;&#x306E;&#x30B3;&#x30F3;&#x30C6;&#x30AD;&#x30B9;&#x30C8;&#x306E;&#x91CD;&#x8981;&#x6027;&#x306B;&#x3064;&#x3044;&#x3066;&#x306E;&#x65B0;&#x3057;&#x3044;&#x6D1E;&#x5BDF;&#x306B; &#x3057;&#x307E;&#x3059;&#x3002;WNT/&beta;-&#x30AB;&#x30C6;&#x30CB;&#x30F3; &#x30B7;&#x30B0;&#x30CA;&#x30EB;&#x4F1D;&#x9054;&#x306E;&#x8338;&#x72B6;&#x4E73;&#x982D;&#x5F62;&#x6210;&#x304C;&#x820C;&#x306E;&#x5927;&#x304D;&#x3055;&#x3068;&#x5F62;&#x72B6;&#x3092;&#x6C7A;&#x5B9A;&#x3059;&#x308B;&#x306E;&#x3067;&#x306F;&#x306A;&#x3044;&#x304C;&#x5FC5;&#x8981;&#x3067;&#x3059;&#x3002;&#x5BFE;&#x7167;&#x7684;&#x306B;&#x3001;WNT5a &#x3069;&#x3046;&#x3084;&#x3089;&#x820C;&#x4F38;&#x9577;&#x304C;&#x306A;&#x3044;&#x4E73;&#x982D;&#x306E;&#x958B;&#x767A;&#x304C;&#x91CD;&#x8981;&#x3067;&#x3059;&#x3002;WNT5a &#x5909;&#x7570;&#x30DE;&#x30A6;&#x30B9;&#x304B;&#x3089;&#x306E;&#x4E88;&#x5099;&#x7684;&#x30C7;&#x30FC;&#x30BF;&#x306F;&#x820C;&#x306E;&#x5F62;&#x72B6;&#x3068;&#x30B5;&#x30A4;&#x30BA;&#x306E;&#x3082;&#x306E;&#x4E73;&#x982D;&#x6570;&#x306E;&#x907A;&#x4F1D;&#x7684;&#x30D7;&#x30ED;&#x30B0;&#x30E9;&#x30E0;&#x3092;&#x533A;&#x5207;&#x308A;&#x307E;&#x3059;&#x3002;

WNT5a &#x820C;&#x3068;&#x8338;&#x72B6;&#x4E73;&#x982D;&#x306E;&#x958B;&#x767A;&#x3067;&#x306F;&#x3002;

Fungiform papillae&#xB294; &#xC124;&#xCE58;&#xB958; &#xD0DC;&#xC544;&#xC5D0; &#xC55E;&#xCABD;&#xC5D0; &#xD600;&#xC5D0; &#xD328;&#xD134;&#xC744; &#xAC1C;&#xBC1C; &#xD558;&#xB294; &#xBCF5;&#xC7A1; &#xD55C; &#xB9DB; &#xAE30;&#xAD00;. &#xC5EC;&#xB7EC; &#xB0B4;&#xC7A5; &#xBE44; &#xBD84;&#xC790;&#xB294; &#xC2DC; &#xAC1C;&#xBC1C; &#xD328;&#xD134;&#xD654;, &#xC18C;&#xB2C9; &#xD5E4; &#xC9C0; &#xD638; &#xADF8;, &#xBF08; &#xC804;&#xCCB4;&#xC801; &#xB2E8;&#xBC31;&#xC9C8;, &#xBA38;&#xB9AC;, &#xD45C; &#xD53C; &#xC131;&#xC7A5; &#xC778;&#xC790; &#xBC0F; Wnts&#xB97C; &#xD3EC;&#xD568; &#xD558; &#xC5EC; &#xC911;&#xC694; &#xD569;&#xB2C8;&#xB2E4;. &#xD600;&#xC640; fungiform &#xC2DC; &#xAC1C;&#xBC1C; &#xADDC;&#xC81C;&#xC5D0; Wnts&#xC758; &#xC5ED;&#xD560;&#xC5D0; &#xB300; &#xD55C; &#xCD5C;&#xADFC; &#xB370;&#xC774;&#xD130; &#xC804;&#xCCB4;&#xC801; &#xB2E8;&#xBC31;&#xC9C8;&#xC758; &#xD6A8;&#xACFC; &#xC5F0;&#xAD6C; &#xD558;&#xB294; &#xACBD;&#xC6B0; &#xC870;&#xC9C1;&#xACFC; &#xD0C0;&#xC774;&#xBC0D; &#xC0C1;&#xD669;&#xC758; &#xC911;&#xC694;&#xC131;&#xC5D0; &#xB300; &#xD55C; &#xC0C8;&#xB85C;&#xC6B4; &#xD1B5;&#xCC30;&#xB825;&#xC73C;&#xB85C; &#xC774;&#xC5B4;&#xC9C8;. WNT/&#xBCA0;&#xD0C0;-catenin &#xC2E0;&#xD638; &#xD558;&#xB294; &#xAC83;&#xC740; fungiform papillae&#xC758; &#xD615;&#xC131;&#xC5D0; &#xB300; &#xD55C; &#xD558;&#xC9C0;&#xB9CC; &#xD558;&#xC9C0; &#xD600; &#xD06C;&#xAE30;&#xC640; &#xBAA8;&#xC591;&#xC744; &#xACB0;&#xC815; &#xD558;&#xB294; &#xB370; &#xD544;&#xC694; &#xD569;&#xB2C8;&#xB2E4;. &#xBC18;&#xBA74;, WNT5a &#xBD84;&#xBA85;&#xD788; &#xD600; &#xD30C;&#xC0DD;&#xBB3C;, &#xD558;&#xC9C0;&#xB9CC; &#xD558;&#xC9C0; &#xC2DC; &#xAC1C;&#xBC1C;&#xC5D0; &#xB300; &#xD55C; &#xC911;&#xC694; &#xD558;&#xB2E4;. &#xC608;&#xBE44; &#xB370;&#xC774;&#xD130; WNT5a &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; &#xC0DD;&#xC950;&#xB97C; &#xD600; &#xBAA8;&#xC591; &#xBC0F; &#xD06C;&#xAE30;&#xC5D0;&#xC11C; &#xC720;&#xC804; &#xD504;&#xB85C;&#xADF8;&#xB7A8; &#xC2DC; &#xC218;&#xC5D0; &#xB300; &#xD55C; &#xAD6C;&#xBD84;&#xD569;&#xB2C8;&#xB2E4;.

&#xD600;&#xC640; &#xC816;&#xAF2D;&#xC9C0; &#xAC1C;&#xBC1C; fungiform wnt5a&#xC785;&#xB2C8;&#xB2E4;.

Las papilas fungiformes son &oacute;rganos de sabor complejo que se desarrollan en un patr&oacute;n en lengua anterior en embriones de roedores. Varias mol&eacute;culas secretadas intr&iacute;nsecas son importantes para el desarrollo de la papila y patrones, incluyendo sonic hedgehog, prote&iacute;nas morfogen&eacute;ticas &oacute;seas, Noggin, factor de crecimiento epid&eacute;rmico y WNTs. Datos recientes sobre roles de WNTs en regulaci&oacute;n de la lengua y el desarrollo de las papilas fungiformes llevan nuevas ideas sobre la importancia del tejido y contextos de sincronizaci&oacute;n al estudiar los efectos de las prote&iacute;nas morfogen&eacute;ticas. Es necesaria para la formaci&oacute;n de las papilas fungiformes, pero no para determinar el tama&ntilde;o de la lengua y la forma la se&ntilde;alizaci&oacute;n de WNT/beta-catenina. Por el contrario, WNT5a aparentemente es importante consecuencia de la lengua, pero no desarrollo de papila. Los datos preliminares de ratones mutantes WNT5a separan programas gen&eacute;ticos para el n&uacute;mero de la papila de los de tama&ntilde;o y forma de la lengua.

WNT5a en lengua y desarrollo de papilas fungiformes.

The Hedgehog (Hh) pathway plays multiple patterning roles during development of the mammalian gastrointestinal tract, but its role in adult gut function has not been extensively examined. Here we show that chronic reduction in the combined epithelial Indian (Ihh) and Sonic (Shh) hedgehog signal leads to mislocalization of intestinal subepithelial myofibroblasts, loss of smooth muscle in villus cores and muscularis mucosa as well as crypt hyperplasia. In contrast, chronic over-expression of Ihh in the intestinal epithelium leads to progressive expansion of villus smooth muscle, but does not result in reduced epithelial proliferation. Together, these mouse models show that smooth muscle populations in the adult intestinal lamina propria are highly sensitive to the level of Hh ligand. We demonstrate further that Hh ligand drives smooth muscle differentiation in primary intestinal mesenchyme cultures and that cell-autonomous Hh signal transduction in C3H10T1/2 cells activates the smooth muscle master regulator Myocardin (Myocd) and induces smooth muscle differentiation. The rapid kinetics of Myocd activation by Hh ligands as well as the presence of an unusual concentration of Gli sties in this gene suggest that regulation of Myocd by Hh might be direct. Thus, these data indicate that Hh is a critical regulator of adult intestinal smooth muscle homeostasis and suggest an important link between Hh signaling and Myocd activation. Moreover, the data support the idea that lowered Hh signals promote crypt expansion and increased epithelial cell proliferation, but indicate that chronically increased Hh ligand levels do not dampen crypt proliferation as previously proposed.

Hedgehog signaling controls homeostasis of adult intestinal smooth muscle.

In the adult intestine, an organized array of finger-like projections, called villi, provide an enormous epithelial surface area for absorptive function. Villi first emerge at embryonic day (E) 14.5 from a previously flat luminal surface. Here, we analyze the cell biology of villus formation and examine the role of paracrine epithelial Hedgehog (Hh) signals in this process. We find that, before villus emergence, tight clusters of Hh-responsive mesenchymal cells form just beneath the epithelium. Cluster formation is dynamic; clusters first form dorsally and anteriorly and spread circumferentially and posteriorly. Statistical analysis of cluster distribution reveals a patterned array; with time, new clusters form in spaces between existing clusters, promoting approximately four rounds of villus emergence by E18.5. Cells within mesenchymal clusters express Patched1 and Gli1, as well as Pdgfrα, a receptor previously shown to participate in villus development. BrdU-labeling experiments show that clusters form by migration and aggregation of Hh-responsive cells. Inhibition of Hh signaling prevents cluster formation and villus development, but does not prevent emergence of villi in areas where clusters have already formed. Conversely, increasing Hh signaling increases the size of villus clusters and results in exceptionally wide villi. We conclude that Hh signals dictate the initial aspects of the formation of each villus by controlling mesenchymal cluster aggregation and regulating cluster size.

Hedgehog-responsive mesenchymal clusters direct patterning and emergence of intestinal villi.

First identified in Drosophila, the Crumbs (Crb) proteins are important in epithelial polarity, apical membrane formation, and tight junction (TJ) assembly. The conserved Crb intracellular region includes a FERM (band 4.1/ezrin/radixin/moesin) binding domain (FBD) whose mammalian binding partners are not well understood and a PDZ binding motif that interacts with mammalian Pals1 (protein associated with lin seven) (also known as MPP5). Pals1 binds Patj (Pals1-associated tight-junction protein), a multi-PDZ-domain protein that associates with many tight junction proteins. The Crb complex also binds the conserved Par3/Par6/atypical protein kinase C (aPKC) polarity cassette that restricts migration of basolateral proteins through phosphorylation. Here, we describe a Crb3 knockout mouse that demonstrates extensive defects in epithelial morphogenesis. The mice die shortly after birth, with cystic kidneys and proteinaceous debris throughout the lungs. The intestines display villus fusion, apical membrane blebs, and disrupted microvilli. These intestinal defects phenocopy those of Ezrin knockout mice, and we demonstrate an interaction between Crumbs3 and ezrin. Taken together, our data indicate that Crumbs3 is crucial for epithelial morphogenesis and plays a role in linking the apical membrane to the underlying ezrin-containing cytoskeleton.

Katherine D. Walton

Cell and Developmental Biology

University of Michigan

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development