Hedgehog is a lipid-linked morphogen that is carried on lipoprotein particles and that regulates both patterning and proliferation in a wide variety of vertebrate and invertebrate tissues. Hyperactivity of Hedgehog signaling causes numerous forms of cancer. Hedgehog acts by binding to its receptor Patched, relieving the suppression of Smoothened and initiating Smoothened signaling. The mechanism by which Patched represses Smoothened has been unclear, but correlates with reduced Smoothened levels on the basolateral membrane. The structural homology of Patched with the Niemann-Pick-Type C1 protein and bacterial transmembrane transporters suggests that Patched might regulate lipid trafficking to repress Smoothened. However, no endogenous lipid regulators of Smoothened have yet been identified, nor has it ever been shown that Patched actually controls lipid trafficking. This work shows that, in Drosophila melanogaster, the Sterol-Sensing Domain of Patched regulates Smoothened trafficking from Patched-positive endosomes. Furthermore, it demonstrates that Patched recruits internalized lipoproteins to Patched-positive endosomes. Thereby, Patched regulates the efflux of specific lipoprotein-derived lipids from this compartment via its Sterol-Sensing Domain and utilizes these lipids to destabilize Smoothened on the basolateral membrane. We propose that Patched normally promotes Smoothened degradation and subsequently downregulates its activity by changing the lipid composition of endosomes through which Smoothened passes. For this purpose, Patched utilizes a specific lipid – possibly a modified sterol or sphingolipid – derived from lipoproteins. Further, we suggest that the presence of Hedgehog on lipoprotein particles inhibits utilization of their lipids by Patched.

No description available.

The interplay between biochemical signals and mechanical processes during animal development is key for the formation of tissues and organs with distinct shapes and functions. An important step during the formation of many tissues is the formation of compartment boundaries which separate cells of different fates and functions. Compartment boundaries are lineage restrictions that are characterized by a straight morphology. Biochemical signaling across compartment boundaries induce the expression of morphogens in the cells along the boundaries. These morphogens then act at long-range to direct growth and patterning of the whole tissue. Compartment boundaries stabilize the position of morphogens and thereby contribute to proper tissue development. The straight morphology of compartment boundaries is challenged by cell rearrangements caused by cell division and tissue reshaping. Physical mechanisms are therefore required to maintain the straight morphology of compartment boundaries. The anteroposterior (A/P) compartment boundary in the developing Drosophila melanogaster wing is established by biochemical signals. Furthermore, mechanical processes are required to maintain the straight shape of the A/P boundary. Recent studies show that mechanical tension mediated by actomyosin motor proteins is increased along the A/P boundary. However, it was not understood how biochemical signals interact with mechanical processes to maintain the A/P boundary. Here I provide the first evidence that Hedgehog signaling regulates mechanical tension along the A/P boundary. I was able to show that differences in Hedgehog (Hh) signal transduction activity between the anterior and posterior compartments are necessary and sufficient to maintain the straight shape of the A/P boundary, which is crucial for patterning and growth of the adult wing. Moreover, differences in Hh signal transduction activity are necessary and sufficient for the increase in mechanical tension along the A/P boundary. In addition, differences in Hh signal transduction activity are sufficient to generate smooth borders and to increase mechanical tension along ectopic interfaces. Furthermore, the differential expression of the transmembrane protein Capricious is sufficient to increase mechanical tension along ectopic interfaces. It was previously suggested that mechanical tension is generated by an actomyosin-cable through which the increase in mechanical tension is transmitted between the junctions along the A/P boundary. Here I show that mechanical tension is generated locally at each cell bond and not transmitted between junctions by an actomyosin cable. My results provide new insights for our understanding of the interplay between biochemical signals and mechanical processes during animal development.

Die Arbeit beschäftigt sich mit dem Problem der Fettlebererkrankung. In der Einleitung wird auf die aktuelle Relevanz der Gesundheitsstörung und Therapiemöglichkeiten eingegangen, insbesondere durch das, in der Therapie des Diabetes mellitus Typ 2 gebräuchliche Biguanid Metformin. Der Bezug zu molekularbiologischen Signalwegen wird hergestellt und verschiedene in vitro Modellsysteme werden vorgestellt. Anschließend wird auf die Herkunft und genetische Besonderheiten der verwendeten primären Maushepatozyten und Hepatomzellen eingegangen, bevor die angewandten Methoden vorgestellt werden. Zum Einsatz kam in dieser Arbeit vor allem die Lipidmessung mittels Fettrot, um das Ausmaß an Steatosis quantifizierbar zu machen. Im Ergebnisteil folgen zuerst Versuche zur Zytotoxizität der einzelnen Chemikalien und deren Einfluss auf intrazelluläre Energieniveaus, bevor der Einfluss auf die hepatozellulären Fetteinlagerungen im Detail untersucht wird. Unterstützt werden die Ergebnisse durch mikroskopische Bilder der Hepatozyten, welche die beschriebenen Effekte verdeutlichen. Insgesamt konnten folgende Thesen aufgestellt werden: • Zwischen primären Hepatozyten von Wildtyp- und Knockout-Mäusen, bestehen nach 24 stündiger Kultivierung Unterschiede bezüglich des intrazellulären Lipidgehaltes, welche sich nach 72 stündiger Kultivierungszeit nivellieren. • Metformin- und Fructoseinkubation senken den intrazellulären ATP-Gehalt, gleichzeitige Anwesenheit von Metformin und Glucose vermindern den Effekt. • Durch 72-stündige Inkubation der primären Hepatozyten und Behandlung mit Metformin konnte der intrazelluläre Lipidgehalt um circa 40% gesenkt werden. • Durch 72-stündige Inkubation der primären Hepatozyten mit Glucose konnte der intrazelluläre Lipidgehalt um circa 100% gesteigert werden. • Bei humanen Hepatomzellen (HuH7) konnte kein Metformin- und kein Glucoseeffekt beobachtet werden. • Der LXR-Agonist TO901317 wirkt auf den intrazellulären Lipidgehalt Metformin entgegen.

The interplay between biochemical signals and mechanical processes during animal development is key for the formation of tissues and organs with distinct shapes and functions. An important step during the formation of many tissues is the formation of compartment boundaries which separate cells of different fates and functions. Compartment boundaries are lineage restrictions that are characterized by a straight morphology. Biochemical signaling across compartment boundaries induce the expression of morphogens in the cells along the boundaries. These morphogens then act at long-range to direct growth and patterning of the whole tissue. Compartment boundaries stabilize the position of morphogens and thereby contribute to proper tissue development. The straight morphology of compartment boundaries is challenged by cell rearrangements caused by cell division and tissue reshaping. Physical mechanisms are therefore required to maintain the straight morphology of compartment boundaries. The anteroposterior (A/P) compartment boundary in the developing Drosophila melanogaster wing is established by biochemical signals. Furthermore, mechanical processes are required to maintain the straight shape of the A/P boundary. Recent studies show that mechanical tension mediated by actomyosin motor proteins is increased along the A/P boundary. However, it was not understood how biochemical signals interact with mechanical processes to maintain the A/P boundary. Here I provide the first evidence that Hedgehog signaling regulates mechanical tension along the A/P boundary. I was able to show that differences in Hedgehog (Hh) signal transduction activity between the anterior and posterior compartments are necessary and sufficient to maintain the straight shape of the A/P boundary, which is crucial for patterning and growth of the adult wing. Moreover, differences in Hh signal transduction activity are necessary and sufficient for the increase in mechanical tension along the A/P boundary. In addition, differences in Hh signal transduction activity are sufficient to generate smooth borders and to increase mechanical tension along ectopic interfaces. Furthermore, the differential expression of the transmembrane protein Capricious is sufficient to increase mechanical tension along ectopic interfaces. It was previously suggested that mechanical tension is generated by an actomyosin-cable through which the increase in mechanical tension is transmitted between the junctions along the A/P boundary. Here I show that mechanical tension is generated locally at each cell bond and not transmitted between junctions by an actomyosin cable. My results provide new insights for our understanding of the interplay between biochemical signals and mechanical processes during animal development.

Hedgehog is a lipid-linked morphogen that is carried on lipoprotein particles and that regulates both patterning and proliferation in a wide variety of vertebrate and invertebrate tissues. Hyperactivity of Hedgehog signaling causes numerous forms of cancer. Hedgehog acts by binding to its receptor Patched, relieving the suppression of Smoothened and initiating Smoothened signaling. The mechanism by which Patched represses Smoothened has been unclear, but correlates with reduced Smoothened levels on the basolateral membrane. The structural homology of Patched with the Niemann-Pick-Type C1 protein and bacterial transmembrane transporters suggests that Patched might regulate lipid trafficking to repress Smoothened. However, no endogenous lipid regulators of Smoothened have yet been identified, nor has it ever been shown that Patched actually controls lipid trafficking. This work shows that, in Drosophila melanogaster, the Sterol-Sensing Domain of Patched regulates Smoothened trafficking from Patched-positive endosomes. Furthermore, it demonstrates that Patched recruits internalized lipoproteins to Patched-positive endosomes. Thereby, Patched regulates the efflux of specific lipoprotein-derived lipids from this compartment via its Sterol-Sensing Domain and utilizes these lipids to destabilize Smoothened on the basolateral membrane. We propose that Patched normally promotes Smoothened degradation and subsequently downregulates its activity by changing the lipid composition of endosomes through which Smoothened passes. For this purpose, Patched utilizes a specific lipid – possibly a modified sterol or sphingolipid – derived from lipoproteins. Further, we suggest that the presence of Hedgehog on lipoprotein particles inhibits utilization of their lipids by Patched.

The liver is the central metabolic hub in organisms and a complex, intertwining regulatory network guarantees efficient liver processes. The morphogenic Hedgehog pathway was recently shown to play a role in regulating the underlying genetic program. Transgenic mouse models with hepatocyte-specific inactivation of Hedgehog signaling showed alterations in insulin-like growth factor homeostasis and in energy metabolism associated with increased lipid accumulation in the liver. In this thesis, it was possible to connect the observed infertility of female knockout mice with an unexpected activation of sex steroid synthesis in the liver. Associated with increased steroidogenic gene expression exclusively in hepatocytes, the plasma testosterone level was significantly elevated, which led to androgenization and an anovulatory phenotype. With these characteristics, the mouse model mimicked the human polycystic ovarian syndrome and suggested an influence of liver and hepatic Hedgehog signaling on reproduction under disease conditions. Further, murine liver metabolism was challenged with starvation starting at different times of day. The transcriptomic results were analyzed with a self-organizing map approach, allowing an intuitive interpretation of data and a thus far unknown diurnally different response of hepatic regulatory mechanisms due to starvation was revealed. In contrast to the manifoldly published and observed switch from energy-consuming to energy-providing processes due to starvation started in the morning, evening starvation led to a novel hepatic expression signature with decreased gluconeogenic gene expression and increased levels of lipid and steroid metabolism-related genes. These differences can be explained by the equally diurnally regulated expression of the corresponding regulatory transcription factors and hormones. Additionally, lipidome analysis confirmed the diurnal differences after starvation. Thus, this study emphasized the immense impact of circadian regulation on liver metabolism and suggests high accuracy when starvation is the focus of research to avoid varying results.:BIBLIOGRAPHISCHE DARSTELLUNG ................................................................................ II LIST OF ABBREVIATIONS .................................................................................................. III TABLE OF CONTENTS ....................................................................................................... IV SUMMARY ............................................................................................................................ 1 ZUSAMMENFASSUNG ......................................................................................................... 5 INTRODUCTION ................................................................................................................... 9 Liver architecture and metabolism ..................................................................................... 9 Diverse possibilities of liver metabolism regulation .......................................................... 10 Connection of Hedgehog signaling to hepatic metabolism ............................................... 10 Impact of feeding schemes on hepatic metabolism .......................................................... 13 Aims of the thesis ............................................................................................................ 14 References ...................................................................................................................... 15 CHAPTER 1 ........................................................................................................................ 18 CHAPTER 2 ........................................................................................................................ 39 PERSPECTIVE ................................................................................................................... 64 CURRICULUM VITAE ........................................................................................................... V PUBLICATIONS AND PRESENTATIONS ............................................................................ VI Publications ...................................................................................................................... VI Oral presentations ............................................................................................................ VI Poster presentations ........................................................................................................ VII AUTHOR CONTRIBUTION STATEMENT .......................................................................... VIII SELBSTSTÄNDIGKEITSERKLÄRUNG .............................................................................. XII DANKSAGUNG .................................................................................................................. XIII

Die Arbeit beschäftigt sich mit dem Problem der Fettlebererkrankung. In der Einleitung wird auf die aktuelle Relevanz der Gesundheitsstörung und Therapiemöglichkeiten eingegangen, insbesondere durch das, in der Therapie des Diabetes mellitus Typ 2 gebräuchliche Biguanid Metformin. Der Bezug zu molekularbiologischen Signalwegen wird hergestellt und verschiedene in vitro Modellsysteme werden vorgestellt. Anschließend wird auf die Herkunft und genetische Besonderheiten der verwendeten primären Maushepatozyten und Hepatomzellen eingegangen, bevor die angewandten Methoden vorgestellt werden. Zum Einsatz kam in dieser Arbeit vor allem die Lipidmessung mittels Fettrot, um das Ausmaß an Steatosis quantifizierbar zu machen. Im Ergebnisteil folgen zuerst Versuche zur Zytotoxizität der einzelnen Chemikalien und deren Einfluss auf intrazelluläre Energieniveaus, bevor der Einfluss auf die hepatozellulären Fetteinlagerungen im Detail untersucht wird. Unterstützt werden die Ergebnisse durch mikroskopische Bilder der Hepatozyten, welche die beschriebenen Effekte verdeutlichen. Insgesamt konnten folgende Thesen aufgestellt werden: • Zwischen primären Hepatozyten von Wildtyp- und Knockout-Mäusen, bestehen nach 24 stündiger Kultivierung Unterschiede bezüglich des intrazellulären Lipidgehaltes, welche sich nach 72 stündiger Kultivierungszeit nivellieren. • Metformin- und Fructoseinkubation senken den intrazellulären ATP-Gehalt, gleichzeitige Anwesenheit von Metformin und Glucose vermindern den Effekt. • Durch 72-stündige Inkubation der primären Hepatozyten und Behandlung mit Metformin konnte der intrazelluläre Lipidgehalt um circa 40% gesenkt werden. • Durch 72-stündige Inkubation der primären Hepatozyten mit Glucose konnte der intrazelluläre Lipidgehalt um circa 100% gesteigert werden. • Bei humanen Hepatomzellen (HuH7) konnte kein Metformin- und kein Glucoseeffekt beobachtet werden. • Der LXR-Agonist TO901317 wirkt auf den intrazellulären Lipidgehalt Metformin entgegen.

Einleitung: Das Darmmikrobiom, ein hochkomplexes Ökosystem an Mikroorganismen, geht eine lebenslange wechselseitige Beziehung mit seinem Wirt ein und beeinflusst wesentlich dessen Darmreifung post partum. Dazu interagieren Darmbakterien direkt und indirekt mit den intestinalen Stammzellen in den Krypten und regulieren Zellteilung und -differenzierung, wobei die Mechanismen nicht abschließend geklärt sind. Das Darmmikrobiom stellt eine wichtige Quelle für Mikroben-assoziierte molekulare Muster (MAMPs) dar, die von Mustererkennungsrezeptoren wie den Toll-like-Rezeptoren (TLR) auf den intestinalen Epithelzellen erkannt werden können. Somit können TLR auf den intestinalen Epithelzellen eine mögliche Verbindung zwischen Darmmikrobiom und Anpassungsreaktionen der Dünndarmmorphologie darstellen. Ziele der Untersuchung: Folgende Hypothesen sollten untersucht werden: 1. Die Anwesenheit von Darmbakterien beeinflusst die Morphologie der Dünndarmschleimhaut. 2. Diese Wirkung wird über Toll-like-Rezeptoren und den Hedgehog-Signalweg in Epithelzellen vermittelt. 3. Über diese Signalwege werden auch funktionelle Eigenschaften wie die Durchlässigkeit der Darmbarriere verändert. Tiere, Material und Methoden: Zur Untersuchung des Einflusses des Mikrobioms wurden in einem gnotobiotischen Mausmodell keimfreie Tiere mit dem minimalen mikrobiellen Konsortium Altered Schaedler Flora (ASF) besiedelt. Die ASF, welche sich aus acht definierten Bakterienarten zusammensetzt, wurde aus dem Caecum ASF-besiedelter C3H/HeNTac-Mäuse entnommen und männlichen und weiblichen keimfreien C57BL/6J-Mäusen appliziert. Der Nachweis der Bakterienspezies erfolgte aus bakterieller DNA, die aus frischen Kotpellets isoliert wurde. Der Vergleich wurde zwischen Dünndärmen ASF-kolonisierter Mäuse (n = 13), keimfreien Tieren (n = 8), konventionell gehaltenen (n = 7) und einer Gruppe Antibiotika-behandelter Mäuse (n = 8) durchgeführt. Zur Untersuchung des Einflusses der Toll-like-Rezeptoren TLR2 und TLR4 wurden epithelspezifische TLR2-defiziente (TLR2ΔIEC) bzw. TLR4-defiziente Mäuse (TLR4ΔIEC) verwendet und mit ihren jeweiligen Wildtyp-Geschwistertieren (WT) verglichen. Die morphometrische Eigenschaften des Dünndarms wurden anhand von histologischen Schnitten untersucht, die mit Hämatoxylin-Eosin oder Periodic-Acid-Schiff gefärbt waren. Hierbei wurden u.a. Mukosahöhe und Villuslänge gemessen sowie die Anzahl der Epithelzellen und Anzahl der Becherzellen pro Villus gezählt. Weiterhin wurden sowohl in Dünndarmgewebe als auch in isolierten intestinalen Epithelzellen mittels qPCR die mRNAExpression der Liganden des Hedgehog (Hh)-Signalwegs Sonic Hedgehog (Shh) und Indian Hedgehog (Ihh) sowie Hedgehog-Zielgene wie Glioma-associated oncogene transcription factor 1 (Gli-1), Patched-1 (Ptch-1) und Hedgehog Interacting Protein (Hhip) untersucht. In einem Teil der Tiere wurde der Hedgehog-Signalweg durch Applikation des Inhibitors Vismodegib gehemmt. Ein möglicher Einfluss auf die Permeabilität des Dünndarms wurde über die mRNA-Expression diverser Tight Junction-Proteine wie Occludin oder Claudin-4 sowie durch einen Permeabilitätsassay mit FITC-Dextran untersucht. Gruppenvergleiche wurden mit einer einfaktoriellen Varianzanalyse (ANOVA) und Holm-Šídák post-hoc Test bzw. zwischen den TLR2ΔIEC oder TLR4ΔIEC und WT-Mäusen mittels zweiseitigem ungepaarten t-Test durchgeführt. Unterschiede wurden bei P < 0,05 als statistisch signifikant betrachtet. Ergebnisse: Durch den Nachweis aller acht Bakterienarten in den Kotproben der behandelten Mäuse konnte die erfolgreiche Übertragung der ASF auf die Empfängertiere nachgewiesen werden. Hinsichtlich Mukosahöhe, Villuslänge, Anzahl der Epithelzellen und der Becherzellen wiesen keimfreie gegenüber konventionell gehaltenen Mäusen signifikant höhere Werte auf. Die Kolonisierung mit ASF führte zu einer signifikanten Verringerung dieser Merkmale, so dass sie sich denen der konventionell gehaltenen Tiere annäherten. Die mRNA-Expression der Hh-Liganden Shh und Ihh war sowohl im Dünndarm als auch in isolierten intestinalen Epithelzellen ASF-besiedelter Mäuse signifikant erhöht. Die Hh-Zielgene Gli-1, Ptch-1 und Hhip waren auf mRNA-Ebene im Dünndarm ASF-besiedelter Mäuse ebenfalls signifikant höher exprimiert. Mukosahöhe, Epithelzellzahl und Villuslänge waren im Jejunum von TLR2ΔIEC- und TLR4ΔIEC-Mäusen gegenüber ihren WT-Geschwistern signifikant höher. Während bei TLR2ΔIEC-Mäusen außer Shh alle untersuchten Hh-Zielgene vermehrt exprimiert waren, waren bei TLR4ΔIEC-Mäusen die gleichen Gene signifikant weniger exprimiert. Wurde hingegen der Hh-Signalweg in konventionell gehaltenen C57BL/6-Mäusen mit Vismodegib inhibiert, waren der Expression der Hh-Zielgene, des TLR2 und des TLR4 signifikant vermindert. Sowohl die Hemmung des Hh-Signalwegs mit Vismodegib als auch das Fehlen der epithelialen TLR2 oder TLR4 bedingte eine verminderte Expression von Tight Junction-Proteinen, die mit einer erhöhten Darmpermeabilität einherging. Konventionell gehaltene Mäuse hatten sowohl gegenüber keimfreien als auch gegenüber mit ASF besiedelten Tieren eine signifikant niedrigere Expression von Tight Junction-Proteinen, was mit einer signifikant höheren Darmpermeabilität verbunden war. Schlussfolgerungen: Zusammenfassend lassen die Ergebnisse dieser Studie darauf schließen, dass Darmbakterien über intestinale TLR2 und TLR4 den Hh-Signalweg regulieren und darüber die Morphologie der Dünndarmmukosa als auch deren funktionelle Eigenschaften wie die Durchlässigkeit der Darmbarriere beeinflussen. / Introduction: The intestinal microbiome, a highly complex ecosystem of microorganisms, enters into a lifelong reciprocal relationship with its host and significantly influences its intestinal maturation postpartum. To this end, intestinal bacteria interact directly and indirectly with intestinal stem cells in the crypts and regulate cell division and differentiation, although the mechanisms are not conclusively understood. The intestinal microbiome represents an important source of microbeassociated molecular patterns (MAMPs) that can be recognized by pattern recognition receptors such as Toll-like-Receptors (TLRs) on intestinal epithelial cells. Thus, TLRs on intestinal epithelial cells may represent a potential link between gut microbiome and adaptation responses of small intestinal morphology. Aims: The following hypotheses should be investigated: 1. The presence of intestinal bacteria affects the morphology of the small intestinal mucosa. 2. This effect is mediated via Toll-like-Receptors and the Hedgehog signaling pathway in epithelial cells. 3. Functional properties such as the permeability of the intestinal barrier are also altered via these signaling pathways. Animals, materials and methods: To investigate the influence of the microbiome, germ-free animals were colonized with the minimal microbial consortium Altered Schaedler Flora (ASF) in a gnotobiotic mouse model. The ASF, which is composed of eight defined bacterial species, was harvested from the caecum of ASF-colonized C3H/HeNTac mice and applied to male and female germ-free C57BL/6J mice. Bacterial species were detected from bacterial DNA isolated from fresh fecal pellets. Comparison was made between small intestines of ASF-colonized mice (n = 13), germ-free animals (n = 8), conventionally housed (n = 7), and a group of antibiotic-treated mice (n = 8). To investigate the influence of Toll-like-Receptors TLR2 and TLR4, epithelial-specific TLR2-deficient (TLR2ΔIEC) or TLR4-deficient mice (TLR4ΔIEC) were used and compared with their respective wild-type (WT) littermates. Morphometric characteristics of the small intestine were examined using histological sections stained with hematoxylin-eosin or periodic-acid-Schiff. Here, mucosa height and villus length were measured, and the number of epithelial cells and number of goblet cells per villus were counted, among other measurements. Furthermore, mRNA expression of the ligands of the Hedgehog (Hh) signaling pathway Sonic Hedgehog (Shh) and Indian Hedgehog (Ihh) as well as Hedgehog target genes such as Gliomaassociated oncogene transcription factor 1 (Gli-1), Patched-1 (Ptch-1) and Hedgehog Interacting Protein (Hhip) were examined in small intestinal tissue as well as in isolated intestinal epithelial cells by qPCR. In a subset of animals, Hedgehog signaling was inhibited by application of the inhibitor Vismodegib. A possible influence on small intestinal permeability was investigated by mRNA expression of various tight junction proteins such as Occludin or Claudin-4 and by permeability assay with FITC-dextran. Group comparisons were performed with a one-way analysis of variance (ANOVA) and Holm-Šídák post-hoc test or between the TLR2ΔIEC or TLR4ΔIEC and WT mice by two-tailed unpaired t test. Differences were considered statistically significant at P < 0.05. Results: Detection of all eight bacterial species in the fecal samples of treated mice demonstrated successful transfer of ASF to recipient animals. In terms of mucosa height, villus length, number of epithelial cells and goblet cells, germ-free mice had significantly higher values compared to conventionally housed mice. Colonization with ASF significantly decreased these characteristics to approach those of conventionally housed animals. The mRNA expression of the Hh ligands Shh and Ihh was significantly increased in the small intestine as well as in isolated intestinal epithelial cells of ASF colonized mice. The Hh target genes Gli-1, Ptch-1, and Hhip were also significantly higher expressed at the mRNA level in the small intestine of ASF-colonized mice. Mucosa height, epithelial cell number, and villus length were significantly higher in the jejunum of TLR2ΔIEC and TLR4ΔIEC mice compared with their WT littermates. Whereas in TLR2ΔIEC mice, except for Shh, all Hh target genes examined were increased in expression, the same genes were significantly less expressed in TLR4ΔIEC mice. In contrast, when the Hh pathway was inhibited with Vismodegib in conventionally maintained C57BL/6J mice, the expression of Hh target genes, TLR2, and TLR4 were significantly decreased. Both inhibition of the Hh pathway with Vismodegib and absence of epithelial TLR2 or TLR4 caused decreased expression of tight junction proteins, which was associated with increased intestinal permeability. Conventionally housed mice had significantly lower expression of tight junction proteins compared with both germ-free and ASF-populated animals, which was associated with significantly higher intestinal permeability. Conclusions: In summary, the results of this study suggest that intestinal bacteria regulate the Hh signaling pathway via intestinal TLR2 and TLR4 and thereby influence the morphology of the small intestinal mucosa as well as its functional properties such as intestinal barrier permeability.