Mesenchymal Compartment Research Articles

Isolation, Expansion and Characterization of BM-Mesenchimal Cells (MSCs) in Patients Affected By Chronic Myeloproliferative neoplasm (MPN)

Introduction MSCs constitute a pivotal cell type capable of shaping both the architecture of the microenvironment and modulating communication between the various cell types through effects on the extracellular matrix (ECM) and by secretion of various growth factors and cytokines. MSCs and hematopoietic stem cells are thought to share the same mesenchymal origin. Some data confirm that MSCs express a functional erythropoietin receptor and JAK2-transduction pathway, but their role in the development and evolution of MPN is still not well known so our aim was the isolation, expansion and characterization of MSCs in patients affected by MPN. Some data indicates that BM-MSCs of patients affected by MPN do not carry the JAK2-V617F mutation. We studied 20 patients affected by MPN with the following characteristics: M/F 12/8, median age 53years, 8 affected by PV, 8 by ET and 4 by PMF. 15 patients were positive for JAK2 V617F mutation, 1 pts for cMPL and 2 were CARL mutations carriers.Methods: MSC were isolated by bone marrow fraction by gradient separation on Lympholyte cell separation media and expanded in culture with a specific medium (MesenCult) in plastic-adherent cultures up to the second passage. DNA was extracted from MSC using QIAmp DNA Mini kit and the study of recurrent alterations (JAK2, MPL and CARL gene mutations was performed).Results: MCS were expanded in 14 on 21 patients. Flow citometry analysis confirmed the standard MSC phenotype (CD45 negative, CD73 positive, CD90 positive and CD105 positive). The molecular analysis of JAKV617F, cMPL and CARL mutations resulted negative in all analyzed samples both in patients carriers of mutations and in wild type ones.Conclusions: We conclude that common mutations markers of MPN neoplasm are absent in the mesenchymal compartment of bone marrows of patients affected by MPN and are restricted to the neoplastic clone.This research project was supported by a grant from Associazione Italiana per la Ricerca sul Cancro (A.I.R.C.) DisclosuresNo relevant conflicts of interest to declare.

HAND2 Targets Define a Network of Transcriptional Regulators that Compartmentalize the Early Limb Bud Mesenchyme

The genetic networks that govern vertebrate development are well studied, but how the interactions of trans-acting factors with cis-regulatory modules (CRMs) are integrated into spatiotemporal regulation of gene expression is not clear. The transcriptional regulator HAND2 is required during limb, heart, and branchial arch development. Here, we identify the genomic regions enriched in HAND2 chromatin complexes from mouse embryos and limb buds. Then we analyze the HAND2 target CRMs in the genomic landscapes encoding transcriptional regulators required in early limb buds. HAND2 controls the expression of genes functioning in the proximal limb bud and orchestrates the establishment of anterior and posterior polarity of the nascent limb bud mesenchyme by impacting Gli3 and Tbx3 expression. TBX3 is required downstream of HAND2 to refine the posterior Gli3 expression boundary. Our analysis uncovers the transcriptional circuits that function in establishing distinct mesenchymal compartments downstream of HAND2 and upstream of SHH signaling.

Abstract LB-34: 3-Dimensional air-liquid interface organoid culture of primary human tumor biopsies

Abstract The application of primary organoid cultures to cancer modeling holds promise for combining the accurate multilineage differentiation and physiology of in vivo systems with the facile in vitro manipulation of transformed cell lines. We have previously described a single air-liquid interface culture method that can be used to engineer oncogenic mutations into primary epithelial/mesenchymal organoids from mouse colon, stomach, and pancreas (Ootani et al., Nature Medicine 15(6):701-6 (2009); Li et al., in press). We have recently modified this methodology to allow the robust long-term 3-dimensional culture of a wide variety of primary neoplasms from human biopsy samples. These primary tumor organoids accurately recapitulate the architecture and histopathology of the tumors from which they were derived, including epithelial and mesenchymal compartments. These tumor organoids can be further passaged into 96-well format to enable a variety of genetic and pharmacologic manipulations with the overall goal of enabling mechanistic studies of tumorigenesis and obtaining clinically actionable information regarding chemosensitivity and resistance to therapy. Citation Format: James T. Neal, Michael Cantrell, Paul Rack, Calvin J. Kuo. 3-Dimensional air-liquid interface organoid culture of primary human tumor biopsies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-34. doi:10.1158/1538-7445.AM2014-LB-34

Crosstalk between Epithelial and Mesenchymal Tissues in Tumorigenesis and Imaginal Disc Development

Cancers develop in a complex mutational landscape. Interaction of genetically abnormal cancer cells with normal stromal cells can modify the local microenvironment to promote disease progression for some tumor types. Genetic models of tumorigenesis provide the opportunity to explore how combinations of cancer driver mutations confer distinct properties on tumors. Previous Drosophila models of EGFR-driven cancer have focused on epithelial neoplasia. Here, we report a Drosophila genetic model of EGFR-driven tumorigenesis in which the neoplastic transformation depends on interaction between epithelial and mesenchymal cells. We provide evidence that the secreted proteoglycan Perlecan can act as a context-dependent oncogene cooperating with EGFR to promote tumorigenesis. Coexpression of Perlecan in the EGFR-expressing epithelial cells potentiates endogenous Wg/Wnt and Dpp/BMP signals from the epithelial cells to support expansion of a mesenchymal compartment. Wg activity is required in the epithelial compartment, whereas Dpp activity is required in the mesenchymal compartment. This genetically normal mesenchymal compartment is required to support growth and neoplastic transformation of the genetically modified epithelial population. We report a genetic model of tumor formation that depends on crosstalk between a genetically modified epithelial cell population and normal host mesenchymal cells. Tumorigenesis in this model co-opts a regulatory mechanism that is normally involved in controlling growth of the imaginal disc during development.

Mechanisms that mediate the development of fibrosis in patients with Crohn's disease.

Crohn's disease is complicated by the development of fibrosis and stricture in approximately 30% to 50% of patients over time. The pathogenesis of fibrostenotic disease is multifactorial involving the activation of mesenchymal cells by cytokines, growth factors, and other mediators released by immune cells, epithelial cells, and mesenchymal cells. Transforming growth factor β, a key activator of mesenchymal cells, is central to the process of fibrosis and regulates numerous genes involved in the disordered wound healing including collagens, and other extracellular matrix proteins, connective tissue growth factor, and insulin-like growth factors. The activated mesenchymal compartment is expanded by recruitment of new mesenchymal cells through epithelial to mesenchymal transition, endothelial to mesenchymal transition, and invasion of circulating fibrocytes. Cellular hyperplasia and increased extracellular matrix production, particularly collagens, from fibroblasts, myofibroblasts, and smooth muscle cells add to the disturbed architecture and scarring on the intestine. Extracellular matrix homeostasis is further disrupted by alterations in the expression of matrix metalloproteinases and tissue inhibitors of metalloproteinase in the gut. Among the 163 susceptibility genes identified that contribute to susceptibility in inflammatory bowel disease mutations in NOD2/CARD15, innate immune system components and autophagy jointly contribute to the activation of mesenchymal cells and pathogenesis of fibrosis in this polygenic disorder. Numerous growth factors cytokines and other mediators also contribute to development of fibrosis in the susceptible patient. This review focuses on the molecular mechanisms that regulate mesenchymal cell function, particularly smooth muscle cells, the largest compartment of mesenchyme in the intestine, that lead to fibrosis in Crohn's disease.

An Essential Mesenchymal Function for miR-143/145 in Intestinal Epithelial Regeneration

Downregulation of the miR-143/145 microRNA (miRNA) cluster has been repeatedly reported in colon cancer and other epithelial tumors. In addition, overexpression of these miRNAs inhibits tumorigenesis, leading to broad consensus that they function as cell-autonomous epithelial tumor suppressors. We generated mice with deletion of miR-143/145 to investigate the functions of these miRNAs in intestinal physiology and disease in vivo. Although intestinal development proceeded normally in the absence of these miRNAs, epithelial regeneration after injury was dramatically impaired. Surprisingly, we found that miR-143/145 are expressed and function exclusively within the mesenchymal compartment of intestine. Defective epithelial regeneration in miR-143/145-deficient mice resulted from the dysfunction of smooth muscle and myofibroblasts and was associated with derepression of the miR-143 target Igfbp5, which impaired IGF signaling after epithelial injury. These results provide important insights into the regulation of epithelial wound healing and argue against a cell-autonomous tumor suppressor role for miR-143/145 in colon cancer.

Canonical Wnt signaling regulates the proliferative expansion and differentiation of fibrocytes in the murine inner ear

Otic fibrocytes tether the cochlear duct to the surrounding otic capsule but are also critically involved in maintenance of ion homeostasis in the cochlea, thus, perception of sound. The molecular pathways that regulate the development of this heterogenous group of cells from mesenchymal precursors are poorly understood. Here, we identified epithelial Wnt7a and Wnt7b as possible ligands of Fzd-mediated β-catenin (Ctnnb1)-dependent (canonical) Wnt signaling in the adjacent undifferentiated periotic mesenchyme (POM). Mice with a conditional deletion of Ctnnb1 in the POM exhibited a complete failure of fibrocyte differentiation, a severe reduction of mesenchymal cells surrounding the cochlear duct, loss of pericochlear spaces, a thickening and partial loss of the bony capsule and a secondary disturbance of cochlear duct coiling shortly before birth. Analysis at earlier stages revealed that radial patterning of the POM in two domains with highly condensed cartilaginous precursors and more loosely arranged inner mesenchymal cells occurred normally but that proliferation in the inner domain was reduced and cytodifferentiation failed. Cells with mis/overexpression of a stabilized form of Ctnnb1 in the entire POM mesenchyme sorted to the inner mesenchymal compartment and exhibited increased proliferation. Our analysis suggests that Wnt signals from the cochlear duct epithelium are crucial to induce differentiation and expansion of fibrocyte precursor cells. Our findings emphasize the importance of epithelial-mesenchymal signaling in inner ear development.

Macrophage/epithelium cross-talk regulates cell cycle progression and migration in pancreatic progenitors.

Macrophages populate the mesenchymal compartment of all organs during embryogenesis and have been shown to support tissue organogenesis and regeneration by regulating remodeling of the extracellular microenvironment. Whether this mesenchymal component can also dictate select developmental decisions in epithelia is unknown. Here, using the embryonic pancreatic epithelium as model system, we show that macrophages drive the epithelium to execute two developmentally important choices, i.e. the exit from cell cycle and the acquisition of a migratory phenotype. We demonstrate that these developmental decisions are effectively imparted by macrophages activated toward an M2 fetal-like functional state, and involve modulation of the adhesion receptor NCAM and an uncommon “paired-less” isoform of the transcription factor PAX6 in the epithelium. Over-expression of this PAX6 variant in pancreatic epithelia controls both cell motility and cell cycle progression in a gene-dosage dependent fashion. Importantly, induction of these phenotypes in embryonic pancreatic transplants by M2 macrophages in vivo is associated with an increased frequency of endocrine-committed cells emerging from ductal progenitor pools. These results identify M2 macrophages as key effectors capable of coordinating epithelial cell cycle withdrawal and cell migration, two events critical to pancreatic progenitors' delamination and progression toward their differentiated fates.

MiR-142-3pbalances proliferation and differentiation of mesenchymal cells during lung development

The regulation of the balance between proliferation and differentiation in the mesenchymal compartment of the lung is largely uncharacterized, unlike its epithelial counterpart. In this study, we determined that miR-142-3p contributes to the proper proliferation of mesenchymal progenitors by controlling the level of WNT signaling. miR-142-3p can physically bind to adenomatous polyposis coli mRNA, functioning to regulate its expression level. In miR-142-3p loss-of-function experiments, proliferation of parabronchial smooth muscle cell progenitors is significantly impaired, leading to premature differentiation. Activation of WNT signaling in the mesenchyme, or Apc loss of function, can both rescue miR-142-3p knockdown. These findings show that in the embryonic lung mesenchyme, the microRNA machinery modulates the level of WNT signaling, adding an extra layer of control in the feedback loop between FGFR2C and β-catenin-mediated WNT signaling.

Downregulation of p300 gene expression in airway mesenchyme of nitrofen-induced hypoplastic lungs

Congenital diaphragmatic hernia (CDH) is a relatively common developmental abnormality causing life-threatening respiratory distress at birth. The nitrofen model has been widely used to investigate the pathogenesis of hypoplastic lungs associated with CDH. Embryos lacking p300 and CBP genes are significantly smaller in lung formation. We hypothesized that pulmonary gene expression of p300 and CBP is downregulated during late gestation in the nitrofen-induced CDH model. Time-pregnant rats were treated with either nitrofen or vehicle on gestational day 9 (D9). Fetal lungs were harvested on D18 and D21 (n = 8 at each time point). Pulmonary gene expression of p300 and CBP was analyzed by quantitative real-time PCR. Immunohistochemistry was performed to investigate expression and localization of pulmonary p300 and CBP proteins. Relative mRNA expression levels of p300 were significantly decreased in nitrofen-induced hypoplastic lungs on D18 compared to controls (3.00 ± 0.20 vs. 3.76 ± 0.14; p = 0.0039), while CBP levels were not altered. p300 immunoreactivity was markedly diminished in surrounding mesenchymal compartments and nuclei of proximal and distal airway cells, while CBP expression was not altered. Downregulation of p300 gene expression during the early canalicular stage may disrupt epithelial-mesenchymal signaling interactions, contributing to the development of hypoplastic lungs in the nitrofen-induced CDH model.

47: Differential expression of Rac1 in common aneuploidies: a model for altered placentation

Inadequate formation and regeneration of the placenta contribute to several pregnancy syndromes such as IUGR & preeclampsia, which may lead to iatrogenic preterm delivery with the goal of preventing fetal death and maternal complications. We investigated the effects of commonly occurring aneuploidies (Trisomy 21,18, and 13; T21,T18 and T13, respectively) on placentation. Specifically, we focused on the role of Rac1, which our microarray data showed was upregulated in all three trisomies. We collected second trimester samples of the maternal-fetal (decidua-placental) interface: 20 trisomy cases (T21, n=10; T18, n=6; T13, n=4) and 4 gestational age-matched, euploid controls (14-22 weeks). Fluorescent in situ hybridization was used to confirm ploidy. Microarray approach enabled global transcriptional profiling in trisomies vs. controls. For immunolocalization, samples were fixed in 3% paraformaldehyde and embedded in OCT. Cryosections were stained with an antibody specific for Rac1 to validate its differential expression at the protein level. Microarray analyses revealed upregulation of RAC1 mRNA in the trisomies that were studied (T13, T18, T21). Rac1, a small G protein, is involved in a wide variety of signaling pathways and in the migration of extravillous trophoblasts. We observed a marked increase in staining intensity for the Rac1 protein in trisomic placental biopsies as compared to control. RAC1 localized primarily to the mesenchymal cores of floating/anchoring chorionic villi, with expression generally absent from the basal plate and invasive trophoblasts (Fig 1.). Our findings showed altered expression, in trisomy-affected pregnancies, of a protein that is involved in normal placental development. These pregnancies have increased rates of miscarriage and adverse pregnancy outcomes. Overexpression of Rac1 could explain these problems and points to the possible role of the mesenchymal compartment in the placental defects that are associated with these aneuploidies.

Hoxa5/Cre transgenic mice: Novel tools for regional deletion along the anterior‐posterior axis

The Hoxa5 homeobox gene encodes a transcription factor that plays a critical role in specifying the identity of the cervico-thoracic region along the anterior-posterior embryo axis and in orchestrating organ morphogenesis. The loss of Hoxa5 function results in skeletal transformations, lethality at birth due to lung defects, and organ anomalies affecting the digestive tract, the mammary gland and the ovary. Study of Hoxa5 gene regulation has revealed the interplay of several control regions that direct Hoxa5 developmental expression. Enhancers targeting expression in the CNS, the paraxial and lateral plate mesoderm at the cervico-thoracic level, and in the mesenchymal compartment of the respiratory and digestive tracts have been identified. Using these molecular tools, we have generated two Hoxa5/Cre transgenic mouse lines carrying different combinations of Hoxa5 regulatory enhancers and allowing site-specific recombination in subsets of Hoxa5 expression sites as tested with the Rosa26/lacZ reporter mice. Further validation of the recombination efficiency of the Hoxa5/Cre transgenic lines was performed with mice carrying a Hoxa5 conditional allele. Hoxa5 deletion with the Hoxa5/Cre mouse lines recapitulates Hoxa5 mutant phenotypes, such as skeletal defects, neonatal lethality, and lung malformations. Hoxa5/Cre mouse lines provide novel genetic tools for gene function analysis in defined tissues along the anterior-posterior axis.

Notch signaling and new therapeutic options in liver disease

Notch signaling is a crucial determinant of cell fate decision during development and disease in several organs. Notch effects are strictly dependent on the cellular context in which it is activated. In the liver, Notch signaling is involved in biliary tree development and tubulogenesis. Recent advances have shed light on Notch as a critical player in liver regeneration and repair, as well as in liver metabolism and inflammation and cancer. Notch signaling is finely regulated at several levels. The complexity of the pathway provides several possible targets for development of therapeutic agents able to inhibit Notch. Recent reports have shown that persistent activation of Notch signaling is associated with liver malignancies, particularly hepatocellular with stem cell features and cholangiocarcinoma. These novel findings suggest that interfering with the aberrant activation of the Notch pathway may have therapeutic relevance. However, further studies are needed to clarify the mechanisms regulating physiologic and pathologic Notch activation in the adult liver, to better understand the mechanistic role(s) of Notch in liver diseases and to develop safe and specific therapeutic agents.

Endodermal and mesenchymal cross talk: a crossroad for the maturation of foregut organs

The developmental stages of each foregut organ are intimately linked to the development of the other foregut organs such that the ultimate function of any one foregut organ, such as the metabolic function of the liver, depends on organizational changes associated with the maturation of multiple foregut organs. These changes include: (i) proliferation of the intrahepatic bile ducts and hepatoblasts within the liver coinciding with parenchymal expansion, (ii) elongation of extrahepatic bile ducts, which allows for proper gallbladder (GB) formation, and (iii) duodenal elongation and rotation, which coincides with all of the above to connect the intrahepatic, extrahepatic, and pancreatic ductal systems with the intestine. It is well established that cross talk between endodermal and mesenchymal components of the foregut occurs, particularly regarding the vascularization of developing organs. Furthermore, genetic mutations in mesenchymal and hepatic compartments of the developing foregut result in similar foregut pathologies: hypoplastic liver, absence of GB, biliary atresia (intrahepatic and/or extrahepatic), and failure of gut elongation and rotation. Finally, these shared pathologies can be linked to deficiencies in genes specific to the septum transversum mesenchyme (Hes1, Hlx, and Foxf1) or liver (Hhex and Hnf6), illustrating the complexity of such cross talk.

Enpp2/Autotaxin in Dermal Papilla Precursors Is Dispensable for Hair Follicle Morphogenesis

Systematic ablation of previously identified dermal papilla (DP) signature genes in embryonic DP precursors will reveal their functional roles during hair follicle morphogenesis. In this study we validate Enpp2/Autotaxin as one of the highest expressed signature genes in postnatal DP, and demonstrate specific expression of this lysophosphatidic acid (LPA) generating enzyme in embryonic dermal condensates. We further identify dermal and epidermal expression of several LPA receptors suggesting that LPA signaling could contribute to follicle morphogenesis in both mesenchymal and epithelial compartments. We then utilize the recently characterized Cre-expressing Tbx18 knock-in line to conditionally ablate Enpp2 in embryonic DP precursors. Despite efficient gene knockout in E14.5 dermal condensates, morphogenesis proceeds regularly with normal numbers, lengths and sizes of all hair follicle types, suggesting that Enpp2 is not required for hair follicle formation. To interrogate DP signature gene expression, we finally isolate control and Enpp2 null DP precursors and identify the expression and upregulation of LIPH, an alternative LPA producing enzyme, suggesting that this gene could functionally compensate for the absence of Enpp2. We conclude that future co-ablation of both LPA producing enzymes or of several LPA receptors may reveal the functional role of LPA signaling during hair follicle morphogenesis.

Mesenchymal stromal cell turnover in the normal adult lung revisited

We have employed a simple and robust noninvasive method of continuous in vivo long-term bromodeoxyuridine (BrdU) labeling to analyze lung mesenchymal stromal cell turnover in adult mice in the steady state. Mathematical modeling of BrdU uptake in flow cytometrically sorted CD45(neg)CD31(neg)Sca-1(pos) lung cells following long-term feeding of BrdU to mice in their drinking water reveals that lung mesenchymal stromal cells cycle continuously throughout life. Analysis of BrdU incorporation during long-term feeding and during chasing (delabeling) following replacement of BrdU-water with normal water shows that the CD45(neg)CD31(neg)Sca-1(pos) lung mesenchymal stromal cell compartment turns over at a rate of ∼2.26% per day with a time to half-cycled of 44 days, an estimated cell proliferation rate of 0.004/day, and a cell death rate of 0.018/day.

Endothelial Jagged-1 Is Necessary for Homeostatic and Regenerative Hematopoiesis

The bone marrow (BM) microenvironment is composed of multiple niche cells that, by producing paracrine factors, maintain and regenerate the hematopoietic stem cell (HSC) pool (Morrison and Spradling, 2008). We have previously demonstrated that endothelial cells support the proper regeneration of the hematopoietic system following myeloablation (Butler et al., 2010; Hooper et al., 2009; Kobayashi et al., 2010). Here, we demonstrate that expression of the angiocrine factor Jagged-1, supplied by the BM vascular niche, regulates homeostatic and regenerative hematopoiesis through a Notch-dependent mechanism. Conditional deletion of Jagged-1 in endothelial cells (Jag1((ECKO)) mice) results in a profound decrease in hematopoiesis and premature exhaustion of the adult HSC pool, whereas quantification and functional assays demonstrate that loss of Jagged-1 does not perturb vascular or mesenchymal compartments. Taken together, these data demonstrate that the instructive function of endothelial-specific Jagged-1 is required to support the self-renewal and regenerative capacity of HSCs in the adult BM vascular niche.

Pulmonary FGF-18 gene expression is downregulated during the canalicular-saccular stages in nitrofen-induced hypoplastic lungs

Pulmonary hypoplasia (PH) associated with congenital diaphragmatic hernia (CDH) represents one of the major challenges in neonatal intensive care. However, the molecular pathogenesis of PH is still poorly understood. In developing fetal lungs, fibroblast growth factor 18 (FGF-18) plays a crucial role in distal airway maturation. FGF-18 knockouts show smaller lung sizes with reduced alveolar spaces and thicker interstitial mesenchymal compartments, highlighting its important function for fetal lung growth and differentiation. We hypothesized that pulmonary FGF-18 gene expression is downregulated during late gestation in nitrofen-induced hypoplastic lungs. Pregnant rats were exposed to either olive oil or nitrofen on day 9 of gestation (D9). Fetuses were harvested on D18 and D21, and lungs were divided into three groups: controls, hypoplastic lungs without CDH [CDH(-)], and hypoplastic lungs with CDH [CDH(+)] (n = 24 at each time-point). Pulmonary FGF-18 gene expression levels were analyzed by qRT-PCR. Immunohistochemistry was performed to investigate FGF-18 protein expression/distribution. Relative mRNA levels of pulmonary FGF-18 gene expression were significantly decreased in CDH(-) and CDH(+) on D18 and D21 compared to controls (p < 0.05 and p < 0.01, respectively). Immunoreactivity of FGF-18 was markedly diminished in mesenchymal cells surrounding the airway epithelium on D18 and D21 compared to controls. Downregulation of FGF-18 gene expression in nitrofen-induced hypoplastic lungs suggests that decreased FGF-18 expression during the canalicular-saccular stages may interfere with saccular-alveolar differentiation and distal airway maturation resulting in PH.

Multiple Shh signaling centers participate in fungiform papilla and taste bud formation and maintenance

The adult fungiform taste papilla is a complex of specialized cell types residing in the stratified squamous tongue epithelium. This unique sensory organ includes taste buds, papilla epithelium and lateral walls that extend into underlying connective tissue to surround a core of lamina propria cells. Fungiform papillae must contain long-lived, sustaining or stem cells and short-lived, maintaining or transit amplifying cells that support the papilla and specialized taste buds. Shh signaling has established roles in supporting fungiform induction, development and patterning. However, for a full understanding of how Shh transduced signals act in tongue, papilla and taste bud formation and maintenance, it is necessary to know where and when the Shh ligand and pathway components are positioned. We used immunostaining, in situ hybridization and mouse reporter strains for Shh, Ptch1, Gli1 and Gli2-expression and proliferation markers to identify cells that participate in hedgehog signaling. Whereas there is a progressive restriction in location of Shh ligand-expressing cells, from placode and apical papilla cells to taste bud cells only, a surrounding population of Ptch1 and Gli1 responding cells is maintained in signaling centers throughout papilla and taste bud development and differentiation. The Shh signaling targets are in regions of active cell proliferation. Using genetic-inducible lineage tracing for Gli1-expression, we found that Shh-responding cells contribute not only to maintenance of filiform and fungiform papillae, but also to taste buds. A requirement for normal Shh signaling in fungiform papilla, taste bud and filiform papilla maintenance was shown by Gli2 constitutive activation. We identified proliferation niches where Shh signaling is active and suggest that epithelial and mesenchymal compartments harbor potential stem and/or progenitor cell zones. In all, we report a set of hedgehog signaling centers that regulate development and maintenance of taste organs, the fungiform papilla and taste bud, and surrounding lingual cells. Shh signaling has roles in forming and maintaining fungiform papillae and taste buds, most likely via stage-specific autocrine and/or paracrine mechanisms, and by engaging epithelial/mesenchymal interactions.

Dual requirement of ectodermal Smad4 during AER formation and termination of feedback signaling in mouse limb buds

BMP signaling is pivotal for normal limb bud development in vertebrate embryos and genetic analysis of receptors and ligands in the mouse revealed their requirement in both mesenchymal and ectodermal limb bud compartments. In this study, we genetically assessed the potential essential functions of SMAD4, a mediator of canonical BMP/TGFß signal transduction, in the mouse limb bud ectoderm. Msx2-Cre was used to conditionally inactivate Smad4 in the ectoderm of fore- and hindlimb buds. In hindlimb buds, the Smad4 inactivation disrupts the establishment and signaling by the apical ectodermal ridge (AER) from early limb bud stages onwards, which results in severe hypoplasia and/or aplasia of zeugo- and autopodal skeletal elements. In contrast, the developmentally later inactivation of Smad4 in forelimb buds does not alter AER formation and signaling, but prolongs epithelial-mesenchymal feedback signaling in advanced limb buds. The late termination of SHH and AER-FGF signaling delays distal progression of digit ray formation and inhibits interdigit apoptosis. In summary, our genetic analysis reveals the temporally and functionally distinct dual requirement of ectodermal Smad4 during initiation and termination of AER signaling.