text:
Perturbation of nucleotide metabolism--the driving force of oncogene-induced senescence.

Induction of premature (stress induced) cellular senescence of normal cells provides the mechanism eliminating proliferative capability of potentially carcinogenic cells and thereby is considered to be a barrier preventing neoplastic tranformation [1]. On the other hand, induction of senescence in tumor cells provides a therapeutic opportunity to treat cancers that may be potentially susceptible [2]. Among factors inducing senescence are oxidative DNA damage leading to persistent replication stress, activation of oncogenes and loss of tumor suppressor genes [1]. It was recently reported however that perturbation of the nucleotide metabolism can be added into these already “classic” inducers. The early observations by Mannava et al.,[3]and Liu et al [4], established evidence of a cause-effect relationship between C-MYC, the oncogene that is capable of suppressing senescence and nucleotide metabolism. The authors reported that downregulation of the C-MYC in several human metastatic melanoma lines via lentivirus-based shRNA led to repression of several genes encoding enzymes related to dNTP metabolism such as thymidylate synthase (TS), inosine monophosphate dehydrogenase 2 (IMPDH2) and phosphoribosyl pyrophosphate synthetase 2 (PRPS2). This observation provided a rationale to explore a potential link between dNTP metabolism, DNA damage and the oncogene induced senescence (OIS).

Additional evidence on the role of perturbed dNTP pools in induction of DNA damage and replication stress was provided by Bester et al.,[5]. Their studies were focused on oncogenes regulating cell cycle progression, specifically the Rb-E2F pathway. The authors observed that aberrant activation of this pathway by HPV-16 E6/E7 or cyclin E oncogenes led to a reduction of cellular nucleotide levels. Interestingly, when C-MYC was coexpressed with cyclin E, its presence led to an increase in transcription of nucleotide biosynthesis genes, prevented the reduction of nucleotide pool and rescued the replication-stress induced DNA damage [5].

In the subsequent studies Mannava et al.,[6] provided direct evidence that down-regulation of nucleoside pools is a cause of DNA damage and senescence in normal human fibroblasts (NHFs) overexpressing HRASG12V, a classical model of oncogene-induced senescence. Among the enzymes down-regulated by activated HRAS were TS, ribonucleotide reductase subunits M1 and M2 (RRM1 and RRM2). Ectopic coexpression of TS, RRM1 and RRM2 or addition of deoxyribonucleosides to a large extent prevented DNA damage, decreased expression of senescence-associated phenotypes as well as attenuated the extent of proliferation arrest. Interestingly, overexpression of TS, RRM1 and RRM2 in quiescent NHFs had no effect on their proliferation arrest suggesting that unlike the OIS, quiescence is not induced by perturbation of dNTP pools. Moreover, in a separate study, Mannava et al.,[7]demonstrated that shRNA-mediated depletion of RRM2 and TS was sufficient to induce senescence in melanoma cells. The observation that knockdown of RRM2 and TS in cells with ctivated oncogenes drives them to senescence provides further evidence that abundance of dNTPs promotes melanoma by suppressing cellular senescence.

Most of the above findings were reproduced in a study of Aird et al.,[8] on a role of suppressed nucleotide pools in DNA damage and oncogene-induced senescence. Additionally the authors reported that RRM2 repression correlated with senescent status of melanocytes in human benign nevi and melanoma harboring oncogenic BRAF or NRAS. Furthermore, high expression of RRM2 correlates with poor survival of melanoma patiens[8].

The seminal findings on different cellular and oncogene models that perturbation of dNTP pools leads to DNA damage, replication stress, irreversible senescence [5-8],may have important clinical implications. The example that with similar oncogene activation the senescence of melanocytes induced by shortage of dNTPs results in nevi remaining benign for years whereas abundance of dNTPs leads to malignant melanoma, opens new possibilities to explore potential role of dNTPs as the target in cancer treatment. In light of these findings the commonly used anticancer drug hydroxyurea which targets dNTPs or 5-fluorouracil that affects thymidylates should be revaluated in terms of its possible senescence-promoting efficiency. For years, acceptance of antitumor drugs by the FDA was based on response criteria assessed by tumor shrinkage that most likely occurs as a result of rapid cell death by apoptosis. Assessment of the tumor response by its size reduction criteria precluded the detection of potential treatment benefits that could be mediated by induction of senescence of tumor cells which may remain alive but reproductively dead. It is tempting to explore whether a combined treatment of cancer initially by cytotoxic regimen, relying on induction of apoptosis, be followed by prolonged treatment promoting senescence of cancer cells, is more effective than the traditional chemotherapy.

Perturbation of nucleotide metabolism -the driving force of oncogene-induced senescence Zbigniew Darzynkiewicz
Induction of premature (stress induced) cellular senescence of normal cells provides the mechanism eliminating proliferative capability of potentially carcinogenic cells and thereby is considered to be a barrier preventing neoplastic tranformation [1]. On the other hand, induction of senescence in tumor cells provides a therapeutic opportunity to treat cancers that may be potentially susceptible [2]. Among factors inducing senescence are oxidative DNA damage leading to persistent replication stress, activation of oncogenes and loss of tumor suppressor genes [1]. It was recently reported however that perturbation of the nucleotide metabolism can be added into these already "classic" inducers. The early observations by Mannava et al., [3]and Liu et al [4], established evidence of a cause-effect relationship between C-MYC, the oncogene that is capable of suppressing senescence and nucleotide metabolism. The authors reported that downregulation of the C-MYC in several human metastatic melanoma lines via lentivirus-based shRNA led to repression of several genes encoding enzymes related to dNTP metabolism such as thymidylate synthase (TS), inosine monophosphate dehydrogenase 2 (IMPDH2) and phosphoribosyl pyrophosphate synthetase 2 (PRPS2). This observation provided a rationale to explore a potential link between dNTP metabolism, DNA damage and the oncogene induced senescence (OIS).
Additional evidence on the role of perturbed dNTP pools in induction of DNA damage and replication stress was provided by Bester et al., [5]. Their studies were focused on oncogenes regulating cell cycle progression, specifically the Rb-E2F pathway. The authors observed that aberrant activation of this pathway by HPV-16 E6/E7 or cyclin E oncogenes led to a reduction of cellular nucleotide levels. Interestingly, when C-MYC was coexpressed with cyclin E, its presence led to an increase in transcription of nucleotide biosynthesis genes, prevented the reduction of nucleotide pool and rescued the replication-stress induced DNA damage [5].
In the subsequent studies Mannava et al., [6] provided direct evidence that down-regulation of nucleoside pools is a cause of DNA damage and senescence in normal human fibroblasts (NHFs) overexpressing HRAS G12V , a classical model of oncogene-induced senescence. Among the enzymes downregulated by activated HRAS were TS, ribonucleotide reductase subunits M1 and M2 (RRM1 and RRM2). Ectopic coexpression of TS, RRM1 and RRM2 or addition of deoxyribonucleosides to a large extent prevented DNA damage, decreased expression of senescence-associated phenotypes as well as attenuated the extent of proliferation arrest. Interestingly, overexpression of TS, RRM1 and RRM2 in quiescent NHFs had no effect on their proliferation arrest suggesting that unlike the OIS, quiescence is not induced by perturbation of dNTP pools. Moreover, in a separate study, Mannava et al.[7]demonstrated that shRNAmediated depletion of RRM2 and TS was sufficient to induce senescence in melanoma cells. The observation that knockdown of RRM2 and TS in cells with activated oncogenes drives them to senescence provides further evidence that abundance of dNTPs promotes melanoma by suppressing cellular senescence.
Most of the above findings were reproduced in a study of Aird et al.,[8] on a role of suppressed nucleotide pools in DNA damage and oncogene-induced senescence. Additionally the authors reported that RRM2 repression correlated with senescent status of melanocytes in human benign nevi and melanoma harboring oncogenic BRAF or NRAS. Furthermore, high expression of RRM2 correlates with poor survival of melanoma patiens[8].
The seminal findings on different cellular and oncogene models that perturbation of dNTP pools leads to DNA damage, replication stress, irreversible senescence [5][6][7][8],may have important clinical implications. The example that with similar oncogene activation the senescence of melanocytes induced by shortage of dNTPs results in nevi remaining benign for years whereas abundance of dNTPs leads to malignant melanoma, opens new possibilities to explore potential role of dNTPs as the target in cancer treatment. In light of these findings the commonly used anticancer drug hydroxyurea which targets dNTPs or 5-fluorouracil that affects thymidylates should be revaluated in terms of its possible senescence-promoting efficiency. For years, acceptance of antitumor drugs by the FDA was based on response criteria assessed by tumor shrinkage that most likely occurs as a result of rapid cell death by apoptosis. Assessment of the tumor response by its size reduction criteria precluded the detection of potential treatment benefits that could be mediated by induction of senescence of tumor cells which may remain alive but reproductively dead. It is tempting to explore whether a combined treatment of cancer initially by cytotoxic regimen, relying on induction of apoptosis, be followed by prolonged treatment promoting senescence of cancer cells, is more effective than the traditional chemotherapy.


extfieldsofstudy: Biology Medicine

text:
HDACIs and the inhibition of invasive potential

A major problem in the treatment of cancer and prolongation of patient survival is the dissemination of cells from a defined tumor site into a loco-regional disease and ultimately to full metastatic spread into distant organs. In the manuscript by Ierano et al. multiple chemically diverse histone deacetylase inhibitors (HDACIs) in tumor cell types of many diverse origins were shown to increase expression of the receptor CXCR4; a receptor whose expression promotes metastatic spread of tumor cells and that is correlated with a stage independent poor prognosis.1,2 The ligand of CXCR4, CXCL12, also called stromal cell-derived factor (SDF1), stimulates signaling through multiple pathways downstream of the CXCR4 receptor including SRC kinases, ERK1/2, and STAT3. Inhibition of SRC, ERK, or STAT3 can all suppress tumor cell migration and reduce the threshold at which tumor cells undergo apoptosis.3-8 The authors noted that despite increased CXCR4 expression following HDACI treatment, exogenous CXCL12 ligand had a reduced ability to stimulate cell signaling processes, with the phosphorylation of both SRC and STAT3 at activating sites declining. This resulted in less induced migration of HDACI-treated tumor cells. No studies were undertaken to determine whether HDACI-treated cells transduced to express activated forms of SRC or STAT3 or retained their invasive phenotype; however a loss of SRC and STAT3 signaling would predict for a less invasive phenotype.

A major problem in the treatment of cancer and prolongation of patient survival is the dissemination of cells from a defined tumor site into a loco-regional disease and ultimately to full metastatic spread into distant organs. In the manuscript by Ierano et al. multiple chemically diverse histone deacetylase inhibitors (HDACIs) in tumor cell types of many diverse origins were shown to increase expression of the receptor CXCR4; a receptor whose expression promotes metastatic spread of tumor cells and that is correlated with a stage independent poor prognosis. 1,2 The ligand of CXCR4, CXCL12, also called stromal cell-derived factor (SDF1), stimulates signaling through multiple pathways downstream of the CXCR4 receptor including SRC kinases, ERK1/2, and STAT3. Inhibition of SRC, ERK or STAT3 can all suppress tumor cell migration and reduce the threshold at which tumor cells undergo apoptosis. [3][4][5][6][7][8] The authors noted that despite increased CXCR4 expression following HDACI treatment, exogenous CXCL12 ligand had a reduced ability to stimulate cell signaling processes, with the phosphorylation of both SRC and STAT3 at activating sites declining. This resulted in less induced migration of HDACI-treated tumor cells. No studies were undertaken to determine whether HDACI-treated cells transduced to express activated forms of SRC or STAT3 or retained their invasive phenotype; however a loss of SRC and STAT3 signaling would predict for a less invasive phenotype.
Precisely how and why signaling by CXCR4 was being altered by HDACIs was not investigated in the manuscript.

HDACIs and the inhibition of invasive potential
Paul Dent Department of Neurosurgery; Massey Cancer Center; Virginia Commonwealth University; Richmond, VA USA Of particular interest would be whether cell surface levels of CXCR4 are altered despite an overall increase in receptor protein levels. Another possibility is that the CXCR4 receptors or other docking proteins who function to mediate CXCR4 function have had their expression and/ or acetylation changed by HDACI treatment such that transduction of signals does not take place to SRC/STAT3. It is also possible that HDACIs, drugs known to increase reactive oxygen species levels and the basal level of ERK1/2 activity, may have also caused some form of uncoupling of the CXCR4 receptor from some of its downstream signaling intermediates. As protein tyrosine phosphatases are highly ROS-sensitive and the activity of SRC and STAT3 are both regulated by tyrosine phosphorylation, the HDACI effect on ROS/PTPase function may also be mechanistically involved in this process.
The data in the present manuscript also draw attention to the use of clinically relevant small molecule inhibitor drugs that could be used to suppress the invasive phenotype and simultaneously promote tumor cell killing. For example, there are a number of FDA-approved or late Phase II/ III trial clinically relevant SRC inhibitors, e.g., dasatinib, AZD0530; a JAK-STAT inhibitor e.g., Xeljanz; and MEK1/2 inhibitors, e.g., AZD6244, trametinib. The rational combination of such agents would likely both reduce the invasive potential of tumor cells as well as increase the levels of apoptosis or, in addition, cell radio-/chemo-sensitivity. Other groups are also attempting to block invasion using novel approaches, e.g., inhibition of mda-9. 9 At present, whether the combination of HDACIs with such signal modulators CommenTAry CommenTAry result in a less invasive phenotype will require additional experimentation.

Disclosure of Potential Conflicts of Interest
No potential conflict of interest was disclosed.


text:
Sweeten PAMPs: Role of Sugar Complexed PAMPs in Innate Immunity and Vaccine Biology

Innate sensors play a critical role in the early innate immune responses to invading pathogens through sensing of diverse biochemical signatures also known as pathogen associated molecular patterns (PAMPs). These biochemical signatures primarily consist of a major family of biomolecules such as proteins, lipids, nitrogen bases, and sugar and its complexes, which are distinct from host molecules and exclusively expressed in pathogens and essential to their survival. The family of sensors known as pattern recognition receptors (PRRs) are germ-line encoded, evolutionarily conserved molecules, and consist of Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), NOD-like receptors (NLRs), C-type lectin-like receptors (CLRs), and DNA sensors. Sensing of PAMP by PRR initiates the cascade of signaling leading to the activation of transcription factors, such as NF-κB and interferon regulatory factors (IRFs), resulting in a variety of cellular responses, including the production of interferons (IFNs) and pro-inflammatory cytokines. In this review, we discuss sensing of different types of glycosylated PAMPs such as β-glucan (a polymeric sugar) or lipopolysaccharides, nucleic acid, and so on (sugar complex PAMPs) by different families of sensors, its role in pathogenesis, and its application in development of potential vaccine and vaccine adjuvants.

www.frontiersin.org such as glycoproteins and glycolipids. These glycoconjugates are fundamental components of both the host and pathogens and play an important role in host-pathogen interactions and provide the molecular basis for discrimination and elicitation of appropriate immune responses in the host (1)(2)(3). Pathogen associated molecular patterns (PAMPs) are molecular signature of pathogens recognized by the host germ-line encoded pattern recognition receptors (PRRs), which are classified as toll-like receptors (TLRs), retinoic acid-inducible gene (RIG)-I-like receptors (RLRs), C-type lectin receptors (CLRs), and nucleotide oligomerization domain (NOD)-like receptors (NLRs), and DNA sensors (4)(5)(6)(7)(8). Most of the PAMPs such lipopolysaccharides (LPS) from gram-negative bacteria, bacterial/viral nucleic acid including single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), CpG rich DNA, β-glucans from fungus, and trehalose dimycolate (TDM) from Mycobacterium contain sugar as integral part of structure. PAMPs, which either constituted of sugar or contain sugar as a component of the complex structure can be given a new terminology as sugar complexed PAMPs (SCPs). The SCPs group of PAMPs is a largest group of PAMPs and almost all the PAMPs are grouped into it except pure lipid and protein such as flagellin. SCPs constitute essential structural and functional moieties of the pathogen that are essential for infection and establishment of disease in the host. On other hand these SCPs also play an important role in vaccine biology. The sensing of SCPs in various compartments of cells by different PRRs activates an array of biochemical reactions, leading to the activation of transcription factors such as nuclear factor kappa B (NF-κB) and interferon (IFN) regulatory factors (IRFs) for induction of inflammatory cytokine and type-I IFNs respectively. Additionally, these innate immune responses also play a pivotal role in initiation of pathogen-specific adaptive immunity via T and B lymphocytes. In this review, we will discuss different kinds of SCPs, their recognition by PRRs, and role in disease, and how these PAMPs can be explored for therapeutic application as vaccines and/or vaccine adjuvants.

SENSING OF SCPs BY PRRs
Large numbers of SCPs from different classes of pathogens (bacteria, virus, and fungi) are reported and sensing of these PAMPs by different families of PRRs is described below in detail.

SENSING OF SCPs BY TLRs
Toll-like receptors sense various kinds of SCPs such as LPS, ssRNA, and hypomethylated dsDNA (CpG) in different cellular compartments of the cells. The sensing of SCPs by TLRs is described in several review and listed in Tables 1 and 2 (7, 9-11). The recently discovered TLR13 is involved in sensing of bacterial 23S rRNA (12). TLR1, TLR2, TLR4, and TLR6 are plasma membrane localized TLRs, mainly sense hydrophobic SCPs ( Table 1) such as  LPS, while TLR3, TLR7, TLR8, and TLR9 are endosome localized  TLRs, sense hydrophilic SCPs (Table 2) such as 5 -ppp-ssRNA (9, 13). TLRs are comprised of N-terminal ligand binding extra cellular domain (ECD) with 19-25 leucine rich repeat (LRRs) motifs, single transmembrane domain, and C-terminal intracellular toll/interleukin receptor (TIR) domain (14, 15). LRRs are consists of "xLxxLxLxx"where"L"stands for leucine and"x"stands for any amino acid, and it is important for recognition of PAMPs.
Binding of PAMPs to LRRs leads to receptor dimerization that induces conformational changes to TIR domain for recruitment of an adaptor and activation of the signaling cascade (13, 15). Sensing of PAMPs by TLR2 induces oligomerization with TLR1  or TLR6, while TLR3, TLR7, TLR8, and TLR9 form homodimers; however, the sensing mechanism for TLR13 is not well known. It is possible that TLR13 forms a homodimer for the activation of a downstream signaling pathway. The glycolipid LPS, which is structurally constituted of lipid A, central oligosaccharide, and antigenic O-polysaccharide (16) is sensed by TLR4 (17, 18) ( Table 1) along with co-receptors known as myeloid differential factor 2 (MD-2) and cluster CD14 (17,19,20

SENSING OF SCPs BY RLRs
Retinoic acid-inducible gene-I-like receptors, retinoic acidinducible gene I (RIG-I), melanoma-differentiation-associated gene 5 (MDA5), and laboratory of genetics and physiology 2 (LGP2) (37), sense RNA SCPs ( Table 2). Studies on RIG-I and MDA5 knockout mice demonstrate that RIG-I and MDA5 sense viral RNA from different classes of RNA viruses (38, 39) and induce antiviral immune response; however, LGP2 functions as a regulatory molecule for RIG-I and MDA5 mediated sensing of viral RNA (40, 41). RIG-I and MDA5 are comprised of two N-terminal caspase activation and recruitment domains (CARD), middle ATPase containing DExD helicase domain (DExD helicase), and a Cterminal domain (CTD). The N-terminal CARD of RIG-I and MDA5 is necessary for recruitment of adaptor and downstream signaling. RIG-I mainly senses small size (<1 kb) RNA containing 5 ppp-ssRNA moieties (42), which could be ssRNA (43, 44) or dsRNA (45), while MDA5 mainly recognizes larger size RNA (>1 kb) from various kinds of viruses and discussed in reviews (46). MDA5 recognizes internal duplex structure of RNA, while RIG-I recognizes the terminus of RNA (47).

PRR SIGNALING
After sensing of SCPs, PRRs recruit different adaptors for effector responses, which are mainly elicited through secretion of innate cytokines such as type I IFNs and inflammatory cytokines.

TLR SIGNALING
Sensing of SCPs by TLR induces conformational changes and oligomerization of receptors, which leads to the recruitment of various adaptor(s) molecules to the TIR domain, which include myeloid differentiation primary response gene 88 (MyD88), TIR domain containing adaptor-inducing IFN-β (TRIF), TIRcontaining adaptor protein (TIRAP), and TRIF-related adaptor molecule (TRAM). The signaling pathways activated by different TLRs are MyD88-dependent and MyD88-independent ( Figure 1) and described in several reviews in detail (7, 10, 11, 85). TLR signaling is regulated at three different levels, which include recruitment of adaptors, stability of signaling intermediates, and transcription regulation (86). A recent study shows that vitamin-D also plays a crucial role in regulation of TLR signaling (87).

NLR SIGNALING
Ligand binding leads to self-oligomerization of NOD1 and NOD2 and recruits an adaptor Receptor-interacting serine/threonineprotein kinase 2 (RIP2). RIP2 is a serine threonine kinase and activates transforming growth factor activated kinase-1 (TAK-1) that subsequently activates NF-κB and mitogen-activated protein kinases (MAPKs) such as p38 and c-Jun N-terminal kinases (JNKs) (Figure 2). MAPKs is activated through an adaptor CARD9 (7, [101][102][103][104]. NLR such as NLRP1 and NLRP3 induce IL-1 family cytokines, where ASC functions as a signaling adaptor and induces caspase-1-dependent maturation of IL-1 family cytokines and plays a crucial role in inflammatory responses and pyroptosis (103,105,106). Among all known inflammasome, NLRP3 is the most investigated inflammasome and it can be activated by multiple factors such as components of pathogenic microbes, tissue damage, autophagy, type-I IFNs, T cells, metabolic dysregulation, miRNA, and so on (107)(108)(109)(110). Furthermore, viruses also enhance the negative regulatory mechanism of hosts to promote survival within the host (111). Activation of NLRP1 induces pyroptosis in hematopoietic cells and inhibition of NLRP1 may play a protective role in individuals having blood disorders such as anemia and leukopenia and suffering from bacterial septic shock (112). Distinct from NLRP1 and NLRP3, inflammasome AIM2 activates caspase-1 and NF-κB in an ASC dependent manner (56).

CLR SIGNALING
Sensing of SCPs by CLRs activates multiple signaling cascades through their own ITAMs or interacting with ITAM-motif containing adaptor proteins such as FcRγ. Signaling cascades lead to activation of NF-κB through a spleen tyrosine kinase (Syk) and CARD9 dependent pathway(s). Binding of ligands to Dectin-1 promotes phosphorylation of YxxL motifs through Syk that promotes recruitment of adaptor CARD9, which exists as a complex B cell lymphoma-10 (Bcl-10) and mucosa-associated lymphoid tissue lymphoma gene-1 (MALT-1), and subsequently activates IKK kinase complex (Figure 2). The activated IKK complex activates canonical NF-κB subunits p65 and c-Rel to induce production of multiple cytokines such as IL-6, IL-10, IL-23, IL-1β, and TNF-α through a subset of T cells such as Th1, Th2, Th17, and www.frontiersin.org  Table 2), recruits an adaptor IPS-1 for induction of type-I IFNs and inflammatory cytokines. STING, a molecule plays a pivotal role in sensing of RNA. Several DNA sensors induce inflammatory cytokine and type-I IFNs in STING dependent and independent manner. NLRs family member, NOD1, NOD2, and inflammasome (mainly NLRP1 and NLRP3 and AIM2) induces inflammatory cytokine. NOD1 and NOD2 sense various SCPs (shown in Table 2) recruit an adaptor RIP2 and CARD9 for induction of inflammatory cytokines. Activation of inflammasome complex after stimulation with appropriate SCPs (shown in Table 2) process an inactive, proIL-1 family cytokine to active IL-1 family cytokine.

SCPs AND CYTOSOLIC DNA SENSORS
DNA-dependent activator of IFN-regulatory factors (DAI) is the first identified cytosolic DNA sensor that recognizes dsDNA and induces production of type-I IFNs (117); however, DAI-knockout cells display normal responses to poly(dA:dT), a synthetic analog of B-DNA, suggesting the existence of multiple sensors for cytosolic dsDNA (118). Nucleic acid binding protein known as leucine rich repeat Fli-I interacting protein (LRRFIPI) is a cytosolic sensor of AT rich B-DNA and GC rich Z-DNA and induces production of IFN-β, and the sensing is dependent on β-Catenin (119). Absent in melanoma 2 (AIM2), a member of the hematopoietic IFNinducible nuclear protein HIN-200 family, has been recognized as a cytosolic DNA sensor (57) and plays a crucial role against cytosolic bacteria and DNA viruses (59). IFN-γ-inducible protein-16 (IFI16; also known as p204) induces type-I IFNs in stimulator of IFN genes (STING) dependent manner (120). STING is an endoplasmic reticulum (ER) localized dsDNA sensor and induces activation of transcription factor NF-κB and IRF3 for downstream signaling (Figure 2) (121). Sensing of dsDNA by STING is positively regulated by IFN-inducible TRIM-56, which leads to production of IFN-β (122). Recently, a nucleotidyltransferase family member known as cyclic guanosine monophosphate-adenosine monophosphate (cyclic-GMP-AMP, or cGAMP) synthase was identified as a cytosolic DNA sensor that senses DNA in a STING dependent manner. During this process cGAMP synthase (cGAS) binds to microbial DNA and synthesizes secondary messenger cGAMP that interacts with STING and activates IRF3, which subsequently leads to transcription of type-I IFNs. The crystal structure of cGAS alone and bound with DNA, ATP, and GTP demonstrates that cGAS is structurally similar to dsRNA sensor 2 -5 oligoadenylate synthase (OAS1), but it contains a unique zincthumb structure through which it recognizes microbial B-DNA (123)(124)(125). DEAH (Asp-Glu-Ala-His) box containing polypeptide 9 and 36 (also known as DHX9 and DHX36) senses hypomethylated CpG DNA in Myd88 dependent manner; however, DDX41, a newly identified PRR, senses dsDNA in STING dependent manner and induces IFN-β (126). DDX41 is negatively regulated through E3 ubiquitin ligase TRIM21, where TRIM21 ubiquitinates DDX41 Frontiers in Immunology | Molecular Innate Immunity at K9, K48, and K115 positions, which leads to its degradation (127). Meiotic recombination 11 homolog A (MRE11) in association with DNA repair protein 50 (RAD50) recognizes various kinds of dsDNA in cytosol and induces production of type-I IFNs in a STING-IRF3 dependent manner (128). Although stimulation of granulocyte-monocyte-colony stimulating factor (GM-CSF) induces bone marrow-derived DCs (GM-DCs) with herpes simplex virus (HSV-1) and Listeria monocytogenes enhances type-I IFNs, siRNA mediated silencing of MRE11 does not abrogate the response, suggesting that further studies are required to understand the role of MRE11 in innate immunity (128). It can be concluded that sensing of DNA and RNA SCPs by cytosolic PRRs is more complicated than thought. Although several sensors have been discovered, the general consensus is that there may be some more cytosolic nucleic acid sensors that sense the respective DNA.

SCPs MEDIATED IMMUNE DYSREGULATION/DISEASE
Sensing of PAMPs by PRRs initiates innate immune responses through secretion of inflammatory cytokines, and subsequent B and T lymphocyte mediated humoral and cellular immune responses are essential for eradication of invading pathogens. However, excessive or hyper activation of innate and adaptive immune responses could be detrimental to the host (129, 130). SCPs are known to induce lethal immune response in the host, such as septic shock, which is characterized by a storm of inflammatory cytokines in the host after infection with gram bacteria.
The LPS (also known as endotoxin) induces activation of myeloid and non-myeloid lineage of cells (131) and leads to cytokine mediated immune shock that is generally known as septic shock; the mechanism of LPS mediated immune stimulation is largely studied in laboratory animals, which is a reflective process for humans. Administration of LPS in mice induces inflammatory lesions through excessive secretion of TNF-α, IL-1β from monocytes, and macrophages that ultimately culminates in septic shock mediated death (132). LPS induces lethal effects due to TLR4-CD14-MD12 receptor complex mediated recruitment of multiple signaling adaptors (MyD88, TRAF6, TRAM, and TRIF). Septic shock is associated with progression of kidney disease with incident microalbuminuria and induces degeneration of neurons in individuals with type-I diabetes, which ultimately leads to a condition known as diabetic neuropathy (133). LPS is also shown to aggravate polyclonal B cell activation, which is a similar kind of condition that occurs in systemic lupus erythematosus (SLE). The SLE kind of effect from LPS is exerted through increased level of IgG and IgM serotype and concurrent decrease of IgA specific isotopes (134). Receptors for advanced glycation (RAGE) are involved in negative regulation of LPS induced septic shock that is mediated through reduction in LPS induced inflammatory cytokines (135). The other glycolipid constituent LTA from bacteria induces inflammatory responses that are similar to LPS and plays a critical role in bacteria pathogenesis (130). TLR9 ligand, CpG-ODNs induces liver injury in d-Galactosamine sensitized mice that is mediated through excessive induction of TNF-α leading to mitochondria-mediated apoptosis of hepatic cells and ultimately culminates in death of the animal (129). The effect of CpG-ODNs is due to the organization of constitutive subunits, that is differential for different kind of CpG-ODNs, and some time it can be fatal to the host. Likewise, DC-SIGN receptor sensed gp120 from HIV virus inhibits secretion of inflammatory cytokine IL-6 and phosphorylation and signal transducer and activator of transcription-3 (STAT-3) that ultimately leads down regulation of host antiviral innate immune response and that further assist for viral internalization in host cell into non-endosomal compartments (136). Similarly, glycoprotein E1 and E2 from HCV modulate host cell entry factors and escape virus from neutralizing Abs. Hepatitis E virus (HEV) originated whole virion particles down regulate RIG-I mediated type-I IFNs production and employ IPS-1 in restricting antiviral inflammatory response (137). The HN series of influenza virus H1N1 can escape immune response through antigenic drift that is enabled by hemagglutinin (HA) head (138). It can be suggested that pathogens utilizes different strategies for down regulating host defense mechanism through targeting major innate immune pathways (129, 136-138). Herewith, these are the few examples to comprehend the role of SCPs in immune dysregulation and development of disease; however, proteins PAMPs are prime components involved in immune escape mechanism, which is beyond the scope of this review.

ROLE OF SCPs IN VACCINE BIOLOGY
The term vaccine was coined by Edward Jenner for cowpox, used for protection from small pox in humans. Formulation of vaccine can be synthetic or biological, and exerts its effect through activation of both arms of the immune system, namely, the innate and adaptive immune system (Figure 3) (139). Therapeutics agents or antigen alone do not mount appropriate immune responses for development of immunity against infection; therefore, an additional immune boosting agent known as adjuvant is required, which provides enough non-specific immune response to elicit appropriate immune response against antigenic determinants of the pathogen and protects the host from infection. Microbial PAMPs are potential candidates as vaccine adjuvants, which mainly activate innate immune cells such as macrophages and DCs. The DCs are pivotal for immunity as they link innate and adaptive immunity (140). How appropriately the immune system will be activated by a particular PAMP depends on its structural diversity and complexity. SCPs such as β-glucan (sensed by Dectin-1 and NLRP3) are made from the same basic unit of sugar but the bonds between monomeric subunits vary, which diversifies the structure. The β-glucan polymer with β 1,6 glycosidic bonds in polymer chain and β 1,3 glycosidic bonds at branching point is shown to be associated with immune stimulatory property (141). Formulation of β-glucan adjuvant with candidosis vaccine consists of α, β mannan-tetanus toxoid complex and enhances protection in mice against pathogenic C. albicans (142). Furthermore, β-glucan also functions as a vaccine candidate for immune clearance of fungal pathogens. Its formulation with immune adjuvant known as MF59 protects mice from vaginal candidosis infection through anti-β 1,3 d-glucan Abs (143). In addition to adjuvant and vaccine for infectious disease, β-glucan is also used for cancer therapeutics for example yeast derived β-glucan adjuvant potentially enhance antitumor effect of MUCIN-1 Abs in mice. Adjuvant and vaccine properties of β-glucan are exerted through induction of innate cytokines such as IL-6, IL-12, TGF-β, and IL-1β, and Th1 and Th2 www.frontiersin.org polarization through NLRP3, MyD88, and Syk (141)(142)(143). Aforementioned studies suggest that β-glucan can be used as a potential therapeutic candidate for both infectious and non-infectious disease. Mannans (α-mannan) from fungus are Dectin-2 ligands, impart their immune response through CARD9 dependent signaling, and are involved in activation of Th1 cells (73). C. albicans derived mannans with α 1,6 glycosidic backbone and α 1,2 or α 1,3 glycosidic branching as well β 1,2 linked oligomer in conjugation with bovine serum albumin protects mice from fungal infection. This study suggests that similar to β-glucan, α-mannans also possess vaccine adjuvant property and hence they can be a potential vaccine candidate for protection from fungal infection (144).
For decades, LPS was known for its adjuvant property; however, the endotoxic nature renders the use of LPS in immunotherapy (145). The modified derivative of LPS known as monophosphoryl lipid A (MPLA) is less toxic and retains its immunostimulatory activity, and is used as an immunotherapeutic vaccine adjuvant (146). Co-administration of MPLA along with basic fibroblast growth factor (bFGF) enhances IgG and IFN-γ level in the serum, and show antitumor activity in mice. Similarly, formulation of MPLA with IFN-γ induces secretion of IL-12 from DCs and shows antitumor activity in mice (147). The antitumor effects of bFGF growth factor and IFN-γ are exerted through cytokine induced by DCs for activation of cytotoxic T lymphocytes (CTL). In addition, these effects are very cell-specific; for example, antitumor effects of IFN-γ are mediated through CD4 + T cells and selectively eliminate human leukocyte antigen (HLA) positive tumor cells (148). Furthermore, a combination of MPLA and alum is used in humans as vaccine adjuvant and it has been shown to induce CD8 + T cell mediated immune responses. Alum induces long-term CD8 + T cells; however, due to programed death (PD)-1 molecule, differentiation of CD8 + T memory cells to effector cytotoxic T cells is inhibited. It has been shown that in the combination of MPLA and alum, MPLA inhibits PD1 activity through IL-6 that ultimately leads to differentiation of alum primed CD8 + T cells into effector CTLs, suggesting its application for protection from microbial pathogens (149). A polymeric form of LPS known as SP-LPS has immuno-modulatory functions without toxicity. The conjugate of cancer drug paclitaxel and SP-LPS known as PLC shows promising antitumor effects through induction of apoptosis. Additionally, the apoptotic bodies generated during apoptosis further stimulate antigen presenting cells (APCs) and promote more efficient tumor clearance (150). Another synthetic derivative of LPS known as glucopyranosyl lipid (GLA) is shown to function as immune adjuvant. GLA, along with Mycobacterium tuberculosis (MTB) antigen ID83 [combination of factor of virulence (Rv2608, Rv3620) and latency Rv1813)], induces protection against MTB infection in mice. Furthermore, a GLA and MTB Ag ID3 formulation induces Th1 cytokines, suggesting that this combination can be used in humans for protection against MTB infection (151).
Mincle and TLR2 agonist TDM (also known as MTB cord factor) is shown to function as immune adjuvant. Co-administration of TDM with type III pneumococcal polysaccharide (SSS-III) in mice augments Abs response to SSS-III antigen and further protects from pneumonia infection (152). A recent genetic study demonstrated that adjuvant action of TDM activates adaptive immune responses through MCL; however, Mincle is not essential (76). The use of TDM is limited due to its toxic nature; a synthetic analog of TDM known as trehalose-6,6-dibehenate (TDB) does not show cytotoxic effects in mice. The combination of dimethyl dioctadecyl ammonium (DDA) and TDB generates a complex DDA-TDB adjuvant; immunization of mice with DDA-TDB along with MTB fusion protein (Ag85B-ESAT) shows MTB specific Th1 adaptive immune response through elevation of IFN-γ secreting CD4 + T cells and production of isotype Abs IgG2b against the antigen (153)(154)(155), suggesting that DDA-TDB can serve as potential vaccine adjuvant against MTB. The size of DDA-TDB vesicle governs antigenic release time and overall it governs cell mediated adaptive immunity; however, the production of Abs is independent of vesicle size (156). Furthermore, TDB requires MyD88 for induction of IL-1β secretion and also activates adaptive immune response through Th17 cells (156). An in vitro study using mice bone marrow-derived DCs shows that TDB activates NLRP3 inflammasome in a caspase-1-dependent manner for production of IL-1β (157).
Lipoarabinomannan (sensed by TLR2 and Mincle) is shown to elicit protective immune response against Mycobacterium pathogenesis. Stimulation of cattle peripheral blood mononuclear cells (PBMCs) with LAM isolated from M. avium along with Freund's incomplete adjuvant (FIA) induces secretion of IFN-γ. The immune stimulatory function of LAM is exerted through a MyD88 directed CARD9/NF-κB pathway and NLRP3 inflammasome activation, suggesting that M. avium LAM could be used as a potential vaccine candidate against bovine tuberculosis (158). PGN of bacteria is not a potent immune adjuvant; however, peptidoglycan monomer (PGM) induces appropriate immune responses without any pyrogenic and toxic effects (159). Mice sensitized with PGM adjuvant and ovalbumin antigen (OVN) formulation enhance secretion of IFN-γ and IL-4 cytokines and specific anti-OVN Abs. Similarly, PGM derived from Staphylococcus known as A170PG protect mice from Staphylococcus infection (160). The adjuvant effects of PGM are initiated through TLR2 and culminate in activation of Th1 and Th2 mediated adaptive immune responses. Most of the studies related to PGM adjuvant are performed using OVN antigen (159)(160)(161). Therefore further studies are required with a wide range of antigens from pathogens to establish the adjuvant properties of PGM.
Peptidoglycan teichoic acid (PG-TA) and LTA from Bacillus subtilis are non-pyrogenic at low concentration and show adjuvant activity by enhancing the number of granulocyte-monocytecolony forming cells in bone marrow in mice. The immune stimulatory property of these SCPs is possibly mediated through GM-CSF (162), but the application of PG-TA and LTA as an immune adjuvant is limited due to poor immunostimulatory functions. The synthetic adjuvant OK-PSA prepared from the cell wall of non-virulent Streptococcus pyogenes strain OK-432 is structurally similar to LTA and shows potent antitumor effect in mice. Antitumor effects of OK-PSA are mediated through induction of IFN-γ, TNF-α, IL-2, IL-12, and IL-18 cytokines from Th1 cells (163), suggesting its utility as a potent immune adjuvant. Similarly, GPI anchored proteins (sensed by TLR2/TLR1) are also shown to possess adjuvant activity (164). Immunization of mice with enzymatically cleaved GPI anchored proteins along with membrane molecules from Schistosoma mansoni potentially protects mice from worm infection. The adjuvant effect of GPI anchored proteins induces Th1/Th2 responses via IFN-γ and TNF-α production (164), suggesting the potential application of GPI anchored proteins as an adjuvant for protection from parasite infection.
The unmethylated CpG rich DNA from bacteria and virus is a well-established deoxy pentose sugar containing SCPs. The cellular responses to CpG are mediated through its sensing by the TLR9 receptor (29). The synthetic analogs of CpG DNA known as CpG oligodeoxynucleotides (CpG-ODNs) are functionally similar to bacterial CpG DNA and show potent immune stimulatory property through induction of innate cytokines (IL-6 and type-I IFNs), improve antigen presentation by DCs, and enhance humoral and cellular immune responses (165)(166)(167)(168). The CpG-ODNs are structurally different from bacterial DNA; backbone of bacterial DNA contains phosphodiester while CpG-ODN has a phosphorothioated (PS) backbone, which protects them from DNAse in cells. CpG-ODN contains poly G tail at 3 and 5 ends, due to which CpG-ODNs form high molecular weight aggregates, which is responsible for enhanced cellular uptake and potent immune responses (169,170). Based on sequence, CpG-ODNs are classified as type A, B, and C (171,172); however, another report shows five types of CpG-ODNs (173). Type A CpG-ODNs contain phosphodiester backbone, a central CpG dinucleotides palindrome, and a PS 3 poly G string, activates natural killer (NK) cells, and induces high IFN-α production from pDCs but weakly induces NF-κB dependent inflammatory cytokines. Type B CpG-ODNs contain full PS backbone with one or more CpG dinucleotides and are weak inducers of cytokine IFN-α. Type C CpG-ODNs (with complete PS backbone and CpG palindrome motif) show combined property of both A and B class CpG-ODNs and efficiently stimulate B and NK cells and also induce IFN-α from pDCs. CpG-ODNs from all classes have been used as immune adjuvants. Immunization of mice with hepatitis B virus surface antigen (HBsAg) along with type B CpG-ODN (ODN 1826) enhanced HBsAg-specific IgG2a Abs (174). Similarly, CpG-ODN 1758 enhances production of tumor surface associated Id-38C13 antigen-specific IgG2a Abs in mice (175). CpG-ODN 7909 enhances hepatitis B virus (HBV) induced PBMCs proliferation (176). A study shows that CpG-ODN stimulated secretion of type-I IFNs from DCs is a dose and time dependent mechanism (177). In addition to these applications, CpG-ODNs have a wide range of application in therapeutics and discussed in several reviews (167,178,179).
HN series of influenza virus such as H1N1 and H5N1 lyse PBMCs in IFN-α dependent manner (180). Priming of the host immune system prior to infection can potentially protect the host from pathogenesis from influenza. Immunization of mice with β-propiolactone inactivated (this inactivation does not affect ssRNA genome of virus) H5N1 and H1N1 virus known as whole inactivated virus (WIV) protects mice from viral infection. The immune protection is mediated through TLR7 directed activation Th1 and Th2 response, as mice lacking TLR7 are susceptible to www.frontiersin.org H5N1 infection (181). Furthermore, in vitro stimulation of human respiratory bronchial epithelial cells with GNR (gold nano rods) complexed 5 ppp-ssRNA activates RIG-I and MDA5 mediated antiviral immune memory in these cells, which further protects from infection with pandemic influenza H1N1 and common influenza A/Solomon Island/03/06 (182), suggesting the potential application of viral 5 ppp-ssRNA and WIV as vaccine candidates for protection from influenza pandemics. Poly(I:C), the synthetic analog of viral dsRNA (sensed by TLR3, RIG-I, and MDA5), shows potential antiviral immune response (183). Co-administration of poly-l-Lysine and carboxymethylcellulose along with poly(I:C) and with Plasmodium falciparum circumsporozoite protein (CSP) antigen increase the frequency of activation of CD8 + Th1 cells and anti-CSP-Abs production (184). Furthermore, immunization of mice with CpG-ODNs or poly(I:C) and alum adjuvant with HBV vaccine rAdSS1 [adenoviral vector encoding HBV S (1-223aa) and pre S (1-47aa) fusion gene] shows immune protection through CD4 + T cells and CD8 + T cells via IFN-γ and IL-12 production and inducing cytotoxic effects, suggesting their potential application in vaccine biology for protection from HBV (185,186). Since it is known that poly(I:C) and CpG-ODNs are potent immune adjuvant, their use in combination has been shown as potential immune adjuvant functions. Mice immunized with poly(I:C) or CpG-ODN 2006 with low dose of salmonid alphavirus (SAV) antigen preparations show potent induction of type-I IFNs and further protection from SAV (187). Recently in mice and ferrets, it was shown that oral administration of an enteric capsule containing adenovirus vector expressing HA from H5N1 virus and a dsRNA adjuvant enhances antiviral immunity through anti-HA Abs; however, there is no Abs against adjuvant. Furthermore, this combination ensures the stability and slow release of viral HA antigen and dsRNA into the gastrointestinal tract, suggesting the potential application of enteric capsules as a vaccine candidate against avian influenza; however, further research work is required to understand the mechanism for immune activation (188).
Carbohydrate based targeting of CLRs (189) in APCs is an interesting strategy for therapeutic application of synthetic glucans and glycopolymer. Virus utilizes DC-SIGN for immune escape can be targeting through synthetic glyconanoparticles such as Man-LAM (190)(191)(192). DC-SIGN mediated internalized glucans further activate CD4 + and CD8 + cells of adaptive immune system. Furthermore the strategy can be utilized for non-pathogenic disease like cancer (191) These synthetic glycopolymers can mimic viral glycoprotein and can be utilized efficiently as antiviral therapeutic agents (193). In summary, there are potential roads of adjuvant based vaccine development using formulations of both intact pathogen and pathogen derived SCPs or their synthetic derivatives.

FUTURE PERSPECTIVES
The complexity of the transcription networks that operate during SCP sensing and subsequent immune response is a major spotlight of present research. Although there is enormous progress in the field of innate immune sensing of pathogenic SCPs, there is still a lot to understand about the evolving dynamicity of protein-sugar complex (glycoproteins). Glycoproteins are the most dynamic PAMPs that pathogens utilize to escape from immune recognition and therapeutic targets. Synthetic drugs or Abs targeting protein epitopes can be masked by pathogens through protein PTM; for example, the HN series of influenza virus has evolved immune escape mechanism through the glycosylation in HA head, and during recurrence these viruses show drug resistance. Other pathogens also utilize such strategies against therapeutic targets; for example, evolution of drug resistant MTB. Therefore, it is all about the glycobiome diversity and dynamicity that pathogens explore as a survival mechanism. Though sugars are the simplest biomolecules, the bonding flexibility with the same sugar, other sugar, lipids, and proteins generates very complex structures. Understanding the sugar moieties dynamicity in pathogens is very crucial for identification of therapeutic targets. It is also necessary to understand what carbohydrate metabolic pathways are specific to pathogen survival and are not present in the host. It is also interesting to implement bio-informatics prediction method to identify putative PTM sites in structural and non-structural proteins of viruses. Pathogens target particular cells types in the host, so understanding the epigenetic changes that are modulated by pathogens in infected cells but are not occurring in uninfected cells can provide new insights in designing potential therapeutic targets. Mutation incorporated in viral genomes, genetic polymorphism of the host, and dynamicity of carbohydrate metabolism in both the host and pathogens generate a condition that decides latent or virulent stage of pathogens; for example, a third of the world population is infected with MTB, but virulence cannot be seen in all individuals. Therefore, it is very important to consider multiple factors while designing a therapeutic agent, which mainly includes comparative glycobiome analysis of the host and the pathogen and analysis of genetic makeup of the host.


text:
Cells Derived from the Coelomic Epithelium Contribute to Multiple Gastrointestinal Tissues in Mouse Embryos

Gut mesodermal tissues originate from the splanchnopleural mesenchyme. However, the embryonic gastrointestinal coelomic epithelium gives rise to mesenchymal cells, whose significance and fate are little known. Our aim was to investigate the contribution of coelomic epithelium-derived cells to the intestinal development. We have used the transgenic mouse model mWt1/IRES/GFP-Cre (Wt1cre) crossed with the Rosa26R-EYFP reporter mouse. In the gastrointestinal duct Wt1, the Wilms’ tumor suppressor gene, is specific and dynamically expressed in the coelomic epithelium. In the embryos obtained from the crossbreeding, the Wt1-expressing cell lineage produces the yellow fluorescent protein (YFP) allowing for colocalization with differentiation markers through confocal microscopy and flow cytometry. Wt1cre-YFP cells were very abundant throughout the intestine during midgestation, declining in neonates. Wt1cre-YFP cells were also transiently observed within the mucosa, being apparently released into the intestinal lumen. YFP was detected in cells contributing to intestinal vascularization (endothelium, pericytes and smooth muscle), visceral musculature (circular, longitudinal and submucosal) as well as in Cajal and Cajal-like interstitial cells. Wt1cre-YFP mesenchymal cells expressed FGF9, a critical growth factor for intestinal development, as well as PDGFRα, mainly within developing villi. Thus, a cell population derived from the coelomic epithelium incorporates to the gut mesenchyme and contribute to a variety of intestinal tissues, probably playing also a signaling role. Our results support the origin of interstitial cells of Cajal and visceral circular muscle from a common progenitor expressing anoctamin-1 and SMCα-actin. Coelomic-derived cells contribute to the differentiation of at least a part of the interstitial cells of Cajal.

Introduction
Intestinal motility disorders include a collection of pathologic processes secondary to alterations in the intestinal wall. These alterations may be congenital or secondary to degenerative changes due to systemic illness. Congenital disorders in the development of the enteric nervous system have been described in Hirschsprung disease, intestinal neuronal dysplasia, ganglioneuromatosis or chronic intestinal pseudoobstruction. Familial visceral myopathy is characterized by alterations in the smooth muscle fibers of the muscular layers of the intestinal wall causing a clinical picture of chronic intestinal pseudoobstruction. Some of these pathologies have been correlated with alterations in the network of interstitial cells of Cajal (ICC) [1]. Despite the potential importance of congenital factors in the pathogeny of these anomalies, the knowledge on the embryonic development of the mesodermal cell populations that contribute to the different intestinal tissues is still insufficient.
The coelomic epithelium of the mammalian embryo is composed of a single, squamous cell layer that invests the external surface of all the viscera and the inner surface of the body wall. The embryonic coelomic epithelium has recently revealed to be a very active tissue participating in visceral organogenesis. This is due to its ability to generate mesenchymal cells through a process of epithelial-mesenchymal transition (EMT). In this way, the embryonic epicardium gives rise to epicardially-derived cells that contribute to the cardiac vascularization and also play a key signaling role for myocardial growth and differentiation [2][3][4]. Migration of coelomic cells into the lung buds to give rise to vascular smooth muscle, has also been reported [5]. The mesothelium of the developing liver is also involved in the origin of sinusoidal endothelial and stellate cells [6]. In the testicles, progenitors of Sertoli cells migrate from the coelomic epithelium of the genital ridges [7].
However, the role played by the coelomic epithelium of the intestine during its development is far less known. The digestive tract develops by association of the endodermal tube with the surrounding splanchnic mesenchyme, which is later colonized by the neural crest cells [8]. It has been reported that, as in other viscera, the coelomic epithelium transforms into migrating cells which incorporate to the mesenchymal compartment and contribute at least to the vascular smooth muscle [9]. These authors used the Wilms' tumor suppressor gene Wt1 as a lineage marker of the coelomic and coelomic-derived cells. In fact, Wt1 is dynamically expressed in the coelomic epithelium as well as in coelomic epithelium-derived cells in many organs [10]. We have developed a model based on the same mWt1/IRES/GFP-Cre (Wt1 cre ) recombinase system from other studies [11,12] but using the Rosa26R-EYFP mouse line as reporter. In our hands, this model has shown a much higher sensitivity than the Rosa26R-LacZ reporter. Cells that express YFP in the reporter line after recombination with the Wt1 cre mouse (herein referred to as Wt1 cre-YFP cells) could be easily immunolabelled with a number of differentiation markers. This has allowed us to describe how coelomic epithelium-derived cells play multiple and hitherto littleknown roles in intestinal development and contribute to many cell populations. The embryonic origin of two of these populations, Cajal and Cajal-like interstitial cells (ICC and ICC-like, respectively), was poorly known. ICC-like have been described as cells closely related to ICC in the gut, but lacking of c-Kit expression [13]. Their embryonic origin and precise function are unknown. ICC are closely associated to the gut musculature and neurons and they act as pacemakers for gastrointestinal contractility [14]. The hypothesis of a common progenitor for ICC and the visceral musculature has received experimental support [15]. This hypothesis is supported by the findings herein shown, that also point out to the role played by coelomic-derived cells in these developmental processes.

Methods
The animals used in our research program were handled in compliance with the institutional and European Union guidelines for animal care and welfare. The experimental procedures were approved by the Committee on the Ethics of Animal Experiments of the University of Málaga (procedure code 2009-0037).
The mWt1/IRES/GFP-Cre (Wt1 cre ) transgenic mouse line is the same used for previous studies of the Wt1 lineage [11,12]. The endogenous expression of GFP in embryos of this line was not detectable by confocal microscopy after the fixation procedure used in our study. Homozygote (Cre+/+) mice were crossed with Rosa26R-EYFP (B6.129X1-Gt(ROSA)26Sortm1(EYFP)Cos/J).
Both homozygote mouse lines were maintained and bred at the UMA facility.
Embryos were staged from the time point of vaginal plug observation, which was designated as the stage E0.5. Whole embryos and the viscera of neonates were excised, washed in PBS and fixed in 4% fresh paraformaldehyde solution in PBS for 2-8 h. Then, the embryos were washed in PBS, cryoprotected in sucrose solutions, embedded in OCT and frozen in liquid N 2cooled isopentane. Ten mm cryosections were stored at 220uC until use.
Immunofluorescence was performed using routine protocols. Cryosections were rehydrated in Tris-PBS (TPBS) and blocked for non-specific binding with SB (16% sheep serum, 1% bovine albumin in TPBS) or SBT (the same solution plus 0.1% Triton X-100) for membrane-bound and intracellular antigens, respectively. When biotinylated secondary antibodies were used, endogenous biotin was blocked with the Avidin-Biotin blocking kit from Vector. Single immunofluorescence was performed incubating the sections with the primary antibody overnight at 4uC, washing in TPBS and incubating with the corresponding fluorochromeconjugated secondary antibody. Secondary antibodies were not used in the case of the anti-CD34 antibody, which was conjugated to eFluor660. Nuclei were counterstained with DAPI (Sigma). Double immunofluorescence was performed by mixing both primary antibodies (rabbit polyclonal and mouse or rat monoclonal), and incubating overnight at 4uC. We then used a Cy5conjugated and a biotin-conjugated secondary antibody, followed by a 45 min incubation with TRITC-conjugated streptavidin. No nuclear counterstaining was made on these slides. In the case of the double CD34/SMCa-actin immunostaining we incubated overnight the sections with the anti-CD34 antibody conjugated to eFluor660, then we blocked the sections with monovalent donkey anti-mouse IgG or Mouse-on-Mouse blocking kit (Vector), and we incubated the sections again with the anti-SMCa-actin antibody. TRITC-conjugated rabbit anti-mouse IgG was used as secondary antibody. C-kit immunostaining was performed on paraformalde- hyde-fixed intestinal tissue cryosections using a polyclonal anti-ckit antibody (Dako) and a secondary antibody (Cy5-conjugated donkey anti-rabbit IgG). Negative controls were always performed incubating with non-immune rat isotype, mouse or rabbit IgG instead of the primary antibody. Wt1 immunohistochemistry was performed on unfixed, cryoprotected embryos frozen as described above. Cryosections were fixed in cool methanol/acetone (1:1) for 10 min. The sections were incubated with a rabbit polyclonal anti-Wt1, biotinylated antirabbit IgG and extravidin-peroxidase. Finally they were revealed with a diaminobenzidine kit (Sigma-Aldrich).
Details of the antibodies used in this study are provided in Table 1.
For flow cytometry analysis, intestines from Wt1 cre-YFP embryos were excised, dissociated for 15 min at 37uC in pre-warmed Accutase (Millipore) and homogenized by repeated pippeting. Cell suspension was washed in PBS plus 2% fetal bovine serum and 10 mM HEPES. Then, cells were incubated on ice at the dark with Cy5-conjugated rat Anti-mouse CD31 (Pecam-1) or APCconjugated anti C-Kit (clone ACK2). After washing, the cells were analyzed in a MoFlo cell sorter.
For RT-PCR, total RNA was isolated from the Wt1 cre-YFP cell fraction of the intestines obtained from three E15.5 embryos. The cells were dissociated as described above and purified by cell sorting. RNA isolation was performed using NucleoSpin RNA XS kit (Macherey-Nagel KG, Germany). Two-step RT-PCR was performed with the total RNA obtained with the First Strand Amplification Kit (Roche Diagnostica, Spain). C-Kit PCR amplification was performed with GoTaq Flexi Polymerase (Promega, Madison, USA) and with QuantiTech Primer Assays (Qiagen, Hilden, Germany). RNA from an E15.5 whole embryo was used as positive control, and water instead of cDNA was the negative control.

Cells of the Wt1 Lineage are Already Abundant Since the Earliest Stages of Intestinal Development
Wt1 protein expression is present in the coelomic epithelium and mesenchymal cells of the intestine by E10 (Fig. 1A-D), weakly in the fore and hindgut and stronger in the midgut (Fig. 1C). High Wt1 expression is detected by this stage in the coelomic epithelium of the developing liver (Fig. 1A). Wt1 expression becomes uniformly observed throughout all the intestinal coelomic epithelium by E10.5 ( Fig. 1E-F) when Wt1 cre-YFP mesenchymal cells are already abundant, especially in the foregut and anterior midgut (Fig. 1H, I). Wt1 cre-YFP mesenchymal cells are scarcer in the posterior midgut, where many Wt1 cre-YFP mesenchymal cells can be seen in the mesentery (Fig. 1J). These cells are much scarcer or even absent in the hindgut (Fig. 1K). On the other hand, cytokeratin immunoreactivity exhibited a dot-like pattern in most intestinal mesenchymal cells, either Wt1 cre-YFP or YFP-negative ( Figure 1L, M). As discussed below, this pattern suggests an epithelial origin of these cells. This cytokeratin staining is transient and it has disappeared from mesenchymal cells by E13.5 (Fig. 1N).

Wt1 cre-YFP Cells Contribute to the Early Steps of Intestinal Vascularization
By E11.5, Wt1 cre-YFP cells become very abundant in all the mesodermal layer surrounding the endoderm, including the hindgut ( Fig. 2A-C). Most mesenchymal Wt1 cre-YFP cells seem to remain undifferentiated throughout these stages. Cells expressing the endothelial markers PECAM and CD34 appear around the endoderm, either isolated or forming small vessels. A few Wt1 cre-YFP cells also express these endothelial markers ( Fig. 2A-C). SMCa-actin expressing Wt1 cre-YFP cells are also found in the vascular walls ( Fig. 3A and 3C). Colocalization of YFP with the pericyte marker NG2 is found in embryos at the stage E13.5 (Fig. 2D). However, colocalization of YFP with the fibroblastic markers 5B5 and FSP1was infrequent (Fig. 2E, F).
The colocalization of PECAM-1 with YFP was confirmed by flow cytometry of dissociated intestines at E11.5. This colocalization accounted for 2.9% of all the Wt1 cre-YFP cells of the intestine (0.3% of total cells) in the representative experiment shown in Fig. 2G. Colocalization of YFP with endothelial markers was not found in later stages of development.

Wt1 is Dynamically Expressed in the Intestinal Coelomic Epithelium by Midgestation
Changes in Wt1 immunoreactivity were detected in the coelomic epithelium in embryos of stages E12.5 and E14.5 ( Fig. 1O-R). By E12.5, Wt1 expression was high in the mesothelium of the foregut and midgut (Fig. 1O), but the hindgut shows a much reduced Wt1 immunoreactivity (Fig. 1P). By E14.5, however, all the gastrointestinal duct showed a Wt1 immunoreactive mesothelium, including the hindgut (Fig. 1Q, R).

Wt1 cre-YFP Cells Contribute to All the Layers of Visceral Smooth Muscle and also to a Submucosal Layer of Fibroblastoid CD34+ Cells
SMCa-actin expressing cells form a ventral crescent by the stage E11.5 (Fig. 3A) and a continuous ring around the endoderm by E12.5. This ring is located at a middle level, and it is sandwiched between two layers of SMCa-actin negative cells (Fig. 3B). All these layers show a number of Wt1 cre-YFP cells, colocalizing with SMCa-actin in the middle layer. In later stages (from E15.5 on) the band of SMCa-actin+ cells becomes more peripherical and the difference between the prospective muscularis layer and the submucosal layer is well established. The longitudinal muscular layer appears by these stages at a submesothelial level, where cells expressing variable levels of SMCa-actin immunoreactivity are appearing (Fig. 3C). This process is more patent in the small intestine, where SMCa-actin+ cells are already arranged in differentiated circular and longitudinal layers by E15.5, while a single, middle layer of SMCa-actin+ cells is still present in the large intestine. The coelomic epithelium shows a strong immunoreactivity for RALDH2 by E15.5, although it seems to be weaker in earlier stages (Fig. 3D, E).
Interestingly, a layer of non-endothelial, CD34+ cells can be observed between the muscular and the submucosal layers (Fig. 3F). CD34+ cells show long filaments and a fibroblastoid appearance. Many Wt1 cre-YFP cells are located within this layer and show CD34+ immunoreactivity in their surface.
In addition to the circular and longitudinal muscular layers a loose submucosal SMCa-actin positive cell layer appear in the mucosa of the late embryos and neonates (Fig. 3F). Wt1 cre-YFP cells are also integrated in this muscular layer.
A Number of Wt1 cre-YFP Cells Show Expression of the Chloride Channel Anoctamin-1 and Differentiate into c-Kit+ Interstitial Cells of Cajal Cells from the muscular layer, especially the circular one, show anoctamin-1 (ANO1) immunoreactivity ( Fig. 4A-C). This immunostaining becomes strong by E16.5. Colocalization of YFP with ANO1 is observed in many cells of both, the circular and the longitudinal layer. Colocalization of ANO1 and SMCa-actin is frequent in the circular layer, especially by the E16.5 stage. Colocalization is more scarce in the longitudinal layer and completely absent in the submucosal muscular layer, where all cells are SMCa-actin+/ ANO1- (Fig. 4B). In neonates, the colocalization of these markers is very occasional, suggesting a decrease of ANO1 immunoreactivity in muscular cells (Fig. 4C). ANO1+/SMCa-actin-cells are mainly found in the space between the circular and longitudinal layers, the place where the myenteric plexus is developing (Fig. 4B,  C).  In neonates, the ICC marker c-Kit was detected in cells from the submesothelial layer and also in cells located between both muscular layers. The elongated morphology of these cells was typical of the ICC. Some of these c-Kit+ cells were also Wt1 cre-YFP and appeared closely connected with other c-Kit+/YFP-cells (Fig. 4D, E). Colocalization of c-Kit and YFP was confirmed by flow cytometry of a dissociated intestine from an E15.5 embryo. As shown in the representative experiment shown in Fig. 4F, 3.8% of all the Wt1 cre-YFP cells were also c-Kit immunoreactive. Furthermore, expression of c-Kit was detected by RT-PCR in Wt1 cre-YFP cells purified by cell sorting of dissociated intestines obtained from E15.5 embryos (Fig. 5).

Wt1 cre-YFP Cells are Transiently Detected within the Endodermal Epithelium and they are Released to the Gut Lumen
By midgestation Wt1 cre-YFP cells are present in the gut endoderm. This event starts by E12.5 in the dorsal and anterior part of the gut, and spreads along all the digestive tube (Fig. 3B,  6A, B, D). Some Wt1 cre-YFP cells are apparently migrating through the basal lamina of the endoderm. Migration of Wt1 cre-YFP cells can be boosted by the high expression of fibronectin in the submucosa (Fig. 6A). In the areas where this phenomenon is observed, the basal lamina of the endoderm shows discontinuities, as suggested by the decrease of laminin immunoreactivity (Fig. 6B,  D). However, areas of the mucosa where Wt1 cre-YFP cells were absent from the epithelium show an intact, laminin-immunoreactive basal lamina (Fig. 6C). Wt1 immunostaining did not revealed Wt1 expression in the endoderm throughout these stages (Fig. 1J-L). Furthermore, the study of homozygous ROSA26R-EYFP embryos showed no YFP-expressing cells in the digestive tract or elsewhere, thus discarding an artifact due to spontaneous recombination in absence of cre-recombinase (not shown).
Wt1 cre-YFP cells apparently delaminate from the gut endodermal epithelium and they are released to the intestinal lumen from E15.5 on, as suggested by some images. In the lumen, Wt1 cre-YFP cells show pyknotic and fragmented nuclei, indicating cell death (Fig. 6A). Wt1 cre-YFP cells become progressively more scarce in the endodermal mucosa by late developmental stages, and they have disappeared from the intestinal epithelium of neonates. The basal surface of the mucosa is still laminin-negative in some areas, especially where Wt1 cre-YFP cells are still present (Fig. 6E). However, in neonates, laminin immunoreactivity is continuous along all the basal lamina of the mucosa (Fig. 6F). Wt1 cre-YFP Cells Express FGF9 and PDGFRa FGF9 immunoreactivity is strong in large cells located between the circular and longitudinal muscle layers (Fig. 7A, B) of late embryos. These cells are probably neural, given their strong btubulin immunoreactivity (not shown). Immunoreactive cells can also be observed in the coelomic epithelium and in both muscle layers. YFP/FGF9 colocalization is observed in the coelomic epithelium, circular muscle layer and also in the Wt1 cre-YFP cells located within the endodermal epithelium (Fig. 7A, B).
The tyrosine-kinase receptor PDGFRa was also expressed by mesenchymal cells (including Wt1 cre-YFP cells) within developing villi of the small intestine (Fig. 7C, D) with a strongest immunoreactivity just in the tip of the villus.

Discussion
In recent years, the study of coelomic epithelium-derived cells lining developing organs has revealed important roles in organogenesis, as described in the introduction. The case of the intestine seems not to be different. The existence of an EMT of the coelomic epithelium lining the intestine, and the importance of the Wilms' tumor suppressor gene product as a lineage marker of the coelomic-derived cells had already been demonstrated. Our findings have revealed a substantial contribution of the Wt1expressing cell lineage to the development of multiple intestinal tissues including some, such as the Cajal and Cajal-like interstitial cells, whose developmental origin was poorly known.
Wt1 expression in the coelomic epithelium of the gut starts by E10. It is conceivable that this early activation of Wt1 in the splanchnopleural coelomic epithelium triggers the expression of Snail, a main effector of the EMT [4] promoting the transformation of epithelial cells into mesenchyme. In fact, the embryonic gut already contains an abundant population of  Wt1 cre-YFP , coelomic-derived mesenchymal cells at least since E10.5, first in the fore and midgut, and later in the hindgut. The epithelial origin of these cells is confirmed by the transient, dot-like cytokeratin immunoreactivity. The presence of collapsed cytokeratin filaments in mesenchymal cells is an evidence of their recent epithelial origin [16]. Interestingly, the expression of the Wt1 seems to be highly dynamic, as suggested by the downregulation observed in the hindgut by E12.5 (Fig. 1P). At later stages of the development this expression is recovered, and thus the whole coelomic epithelium of the gastrointestinal duct is highly Wt1 immunoreactive by E14.5. This upregulation of Wt1 expression seems to be unrelated with a new epithelial-mesenchymal transition, and it precedes to an upregulation of Raldh2, the main mesodermal retinoic acid (RA) synthesizing enzyme, and the differentiation of the longitudinal muscle layer. Raldh2 is a target of Wt1 [17] and RA promotes smooth muscle differentiation in mesenchymal stem cells [18]. Thus, the late expression of Wt1 could be related with the differentiation of the longitudinal musculature through a RA-mediated signaling mechanism.
Only during the earliest stages of the intestinal development Wt1 cre-YFP cells were able to differentiate into endothelial cells. The sharp decrease of the proportion of endothelium originating from the Wt1-expressing cell lineage can be explained by the recruitment of angioblasts or circulating endothelial progenitor cells. Anyway, our observation supports the concept that cells derived from the coelomic epithelium have vasculogenic potential. This observation correlates our findings with the roles played by cells derived from the epicardium during cardiac development [4]. In both cases, coelomic cells express Wt1, undergo an EMT and give rise to a multipotential mesenchyme, contributing to organ vascularization. We think that this can be a generalized mechanism for vascularization of developing viscera, based on the vasculogenic potential of the mesenchymal cells derived from the coelomic epithelium, a potential that seem to be locally activated as the visceral primordia develop. Besides the vasculogenic fate, the multipotentiality acquired by the cells derived from the coelomic epithelium would allow these cells to differentiate in organ-specific derivatives, such as Cajal interstitial cells, as shown in this paper, or Sertoli cells in the testicle [7].
Smooth muscle differentiation starts in the ventral aspect of the digestive tract. A complete muscular ring is observed by E12-13. Later, an outer, submesothelial layer of SMCa-actin positive cells appears. Our observations point to an independent origin of both muscle layers. Circular arrangement of the muscle cells is already evident when the submesothelial SMCa-actin positive cells have not yet appeared. On the other hand, SMCa-actin immunoreactivity is weak and scattered in submesothelial cells before the appearance of the submesothelial layer. Furthermore, expression of markers seems to be different among both layers. For example, Anoctamin-1 (ANO1) is strongly expressed by E16.5 in the circular layer, but not in submesothelial mesenchyme. Thus, the middle SMCa-actin positive ring of cells seems to contribute only to the circular muscle, being the longitudinal muscle layer a result of the differentiation of submesothelial mesenchymal cells, possibly related with the expression of RALDH2 in the coelomic epithelium, as discussed above. An early differentiation of the circular layer in mice had been also suggested [19,20], although the differentiation of circular and longitudinal cell layers is described as simultaneous in humans [21]. Finally, the origin of the third muscular layer, called muscularis mucosae, seems to be also independent of the other muscular layers. It appears in late developmental stages and neonates and it never expresses ANO1.
Wt1 cre-YFP cells contribute to the smooth muscle of all these muscle layers. Wt1 cre-YFP cells also express the ANO1 marker, mainly by E16.5, although this colocalization is rare in neonates, where ANO1 immunoreactive cells, most probably progenitors of ICC, have become scarcer and they localize preferentially between the circular and longitudinal muscle layers. This pattern of differentiation is consistent with the descriptions made on human foetuses [22].
We think that our results strongly support the hypothesis of an origin of the ICC from a common progenitor shared with the lineage of the visceral smooth muscle [15]. ANO1 is a calciumdependent chloride channel and its expression has been associated with the generation of ICC slow waves. ANO1 labels all classes of ICC and represents a highly specific marker for ICC in mouse and human tissues [23,24]. Probably, the ANO1+/SMCa-actin+ cells of the circular layer can lose one of these markers differentiating either in ICC or visceral smooth muscle. It is important to remark that c-Kit, another ICC marker, is expressed in the circular layer by midgestation [19] supporting this possibility. The ANO1+/ SMCa-actin negative cells observed in the developing longitudinal layer are an alternative source. Anyhow, since some ANO1+/ SMCa-actin negative cells are also Wt1 cre-YFP , and also given the YFP/c-Kit immunocolocalization observed in presumptive ICC and the expression of c-Kit detected by RT-PCR in purified Wt1 cre-YFP cells, we can conclude that cells from the Wt1 lineage are involved at least in the earliest stages of ICC differentiation. Colocalization with YFP is observed in cells of the circular muscle layer as well as in Wt1 cre-YFP cells within the endodermal mucosa (white arrows). Note the large FGF9+ cells, probably neural, between the circular and the longitudinal muscle layers (arrowheads). The coelomic epithelium is also FGF9+ (yellow arrow in B. C,D. Immunolocalization of PDGFRa in the small intestine of an E18.5 embryo. Positive mesenchymal cells are located within the developing villi (arrows in C). Colocalization of PDGFRa with YFP is shown at higher magnification in D (arrows). Scale bars A-C = 25 mm; D = 10 mm. doi:10.1371/journal.pone.0055890.g007 We have also observed a cell population, containing an important component of Wt1 cre-YFP cells, that is constituted of cells with long prolongations and characterized by CD34 immunoreactivity, lacking of SMCa-actin, ANO1 and c-Kit expression. These cells are preferentially located in the submucosal layer. This population probably correspond to the CD34+ Cajallike or fibroblast-like cells described by other authors [25,26]. This cell population decreases in some motility disorders such as chronic intestinal pseudoobstruction, suggesting some role related with ICC function [27]. The relation of these CD34+ cells with the so-called intestinal ''telocytes'' [28] is unclear, since telocytes have been mainly described on morphological criteria in multiple tissues, but their immunophenotyping is needed of clarification.
Colocalization of YFP with PDGFRa into the developing villi demonstrates that cells from the Wt1-expressing lineage are also involved in villus morphogenesis. In fact the clusters of PDGFRa positive cells had been already described, and the essential role played by PDGF-A and its receptor PDGFRa was well known [29]. Together with the expression of FGF9 (discussed below) this observation suggests that the cells derived from the coelomic epithelium are not only contributing to a number of tissues but also playing a signaling role for intestinal tissue morphogenesis.
It is interesting to remark that colocalization of Wt1 cre-YFP cells with the fibroblast markers 5B5 (prolin hydroxylase subunit) and FSP1 was rare in the embryos studied. This might reveal a lineage restriction of the Wt1 cre-YFP cells towards the muscular/vascular rather than to the connective tissue, but it is also possible that the timing of differentiation towards fully-differentiated, collagenproducing cells be delayed in Wt1 cre-YFP cells in relation to other developmental fates. Thus, the ontogenetically older splanchnopleural cells could be predominant in the differentiation of the early fibroblasts. Studies in young Wt1 cre-YFP mice would be necessary to confirm this.
Finally, our most intriguing observation has been the presence of Wt1 cre-YFP cells within the endoderm, given the lack of expression of Wt1 in this tissue. The signs of migration of Wt1 cre-YFP cells throughout discontinuities of the basal lamina suggest that mesodermal cells can move throughout the endoderm coinciding with the transition from the pluristratified to the columnar epithelium. The process starts by E12.5 and seems to decline after E16.5. What seems to be clear is the release of all the mucosal Wt1 cre-YFP cells to the intestinal lumen where they show signs of cell death. We have found only a paper describing the migration of mesenchymal cells through the gut endoderm in the human fetus, by stages corresponding to our observations in mouse [30]. The question remains open but we think that it deserves further research. The hypothetical migration seems to involve degradation of the endodermal basal lamina. This could be physiologically relevant since proliferation and differentiation of the intestinal epithelium is dependent on paracrine signals from mesenchymal cells. If migration of mesenchymal cells into the endodermal compartment were confirmed, this would allow a paracrine release of these factors within the endoderm. FGF9 has been related with intestinal differentiation [31,32], and we have observed intraendodermal Wt1 cre-YFP /FGF9+ cells. Significantly, cardiac ventricle proliferation and differentiation is promoted by FGF9 secreted by epicardially-derived cells (the developmental equivalent of the coelomic-derived cells of the intestine) migrating throughout the myocardiocytes [2].
In summary, cells from the Wt1 lineage seem to play multiple and dynamic roles in intestinal development. Furthermore, the cells from the Wt1 lineage seem to show no difference in their distribution or developmental fate with respect to other splanchnopleural mesodermal cells. In contrast with previously published data [9] we show evidence that cells derived from the coelomic epithelium can differentiate into endothelium, vascular and visceral smooth muscle, Cajal and Cajal-like interstitial cells. Thus, it would suggest that the only difference between the Wt1expressing lineage cells and other splanchnopleural cells is their late origin. It is conceivable that the role of Wt1 would be to promote and to temporally extend the EMT of the splanchnopleura in order to increase the population of mesodermal cells that contribute to different intestinal tissues. The differentiation of this pool of splanchnopleural cells would be dependent from local signals. These findings will raise many new questions whose investigation can contribute to our knowledge of the development of the digestive tract and its congenital motility disorders.