Multiomic analysis reveals cellular and epigenetic plasticity in intestinal pouches of ulcerative colitis patients.

Background & Aims: Total proctocolectomy with ileal pouch anal anastomosis (IPAA) is the standard of care for patients with severe treatment resistant ulcerative colitis (UC). Despite improvements in patient outcomes, about 50% of patients will develop inflammation of the pouch within 1-2 years following surgery. Establishment of UC pouches is associated with profound histological changes of the mucosa. A detailed characterization of these changes on a cellular and molecular level is crucial for an improved understanding of pouch physiology and diseases management. Methods: We generated cell-type-resolved transcriptional and epigenetic atlases of UC pouches using scRNA-seq and scATAC-seq data from paired biopsy samples from the ileal pouch and ileal segment above the pouch (pre-pouch) of UC-IPAA patients (n=6, female=2) without symptoms. We also collected data from paired biopsies of the terminal ileum (TI) and ascending colon (AC) from healthy controls (n=6, female=3). Results: We identified novel populations of colon-like absorptive and secretory epithelial cells, constituting a significant proportion of the epithelial cell fraction in the pouch but not in matched pre-pouch samples. Pouch-specific enterocytes expressed colon-specific genes, including CEACAM5, CA2. However, in contrast to normal colonic epithelium, these cells also expressed a range of inflammatory and secretory genes, similar to previously detected gene expression signatures in IBD patients. Comparison to longitudinal bulk RNA-seq data from UC pouches demonstrated that colon-like epithelial cells are present early after pouch functionalization and independently of subsequent pouchitis. Finally, single cell chromatin accessibility revealed activation colonic transcriptional regulators, including CDX1, NFIA, and EHF. Conclusion: UC pouches are characterized by partial colonic metaplasia of the epithelium. These data constitute a resource of transcriptomic and epigenetic signatures of cell populations in the pouch and provide an anchor for understanding the underlying molecular mechanisms of pouchitis.

Colitis-induced upregulation of tumor necrosis factor receptor-2 (TNFR2) terminates epithelial regenerative signaling to restore homeostasis.

Colonic epithelial repair is a key determinant of health. Repair involves changes in epithelial differentiation, an extensive proliferative response, and upregulation of regeneration-associated "fetal-like" transcripts, including Ly6a (Sca-1), that represent Yap1 and interferon targets. However, little is known about how this regenerative program terminates and how homeostasis is restored during injury and inflammation. Here we show that, after the initial entry into the regenerative state, the subsequent upregulation of tumor necrosis factor (TNF) receptor 2 (R2, TNFR2, Tnfrsf1b) clears the regenerative signaling and restores homeostatic patterns of epithelial differentiation. Targeted deletion of epithelial TNFR2 in vivo and in colonoid cultures revealed persistent expression of Ly6a, hyperproliferation, and reduced secretory differentiation. Moreover, mice lacking epithelial TNFR2 also failed to complete colon ulcer healing, suggesting that partial resolution of regenerative signaling is essential for the completion of the repair process. These results demonstrate how epithelial cells dynamically leverage a colitis-associated cytokine to choreograph repair.

Wound-healing plasticity enables clonal expansion of founder progenitor cells in colitis.

Chronic colonic injury and inflammation pose high risks for field cancerization, wherein injury-associated mutations promote stem cell fitness and gradual clonal expansion. However, the long-term stability of some colitis-associated mutational fields could suggest alternate origins. Here, studies of acute murine colitis reveal a punctuated mechanism of massive, neutral clonal expansion during normal wound healing. Through three-dimensional (3D) imaging, quantitative fate mapping, and single-cell transcriptomics, we show that epithelial wound repair begins with the loss of structural constraints on regeneration, forming fused labyrinthine channels containing epithelial cells reprogrammed to a non-proliferative plastic state. A small but highly proliferative set of epithelial founder progenitor cells (FPCs) subsequently emerges and undergoes extensive cell division, enabling fluid-like lineage mixing and spreading across the colonic surface. Crypt budding restores the glandular organization, imprinting the pattern of clonal expansion. The emergence and functions of FPCs within a critical window of plasticity represent regenerative targets with implications for preneoplasia.

Deletion of Endogenous Neuregulin-4 Limits Adaptive Immunity During Interleukin-10 Receptor-Neutralizing Colitis.

BACKGROUND: Growth factors are essential for maintenance of intestinal health. We previously showed that exogenous neuregulin-4 (NRG4) promotes colonocyte survival during cytokine challenge and is protective against acute models of intestinal inflammation. However, the function(s) of endogenous NRG4 are not well understood. Using NRG4-/- mice, we tested the role of endogenous NRG4 in models of colitis skewed toward either adaptive (interleukin-10 receptor [IL-10R] neutralization) or innate (dextran sulfate sodium [DSS]) immune responses. METHODS: NRG4-/- and wild-type cage mate mice were subjected to chronic IL-10R neutralization colitis and acute DSS colitis. Disease was assessed by histological examination, inflammatory cytokine levels, fecal lipocalin-2 levels, and single cell mass cytometry immune cell profiling. Homeostatic gene alterations were evaluated by RNA sequencing analysis from colonic homogenates, with real-time quantitative polymerase chain reaction confirmation in both tissue and isolated epithelium. RESULTS: During IL-10R neutralization colitis, NRG4-/- mice had reduced colonic inflammatory cytokine expression, histological damage, and colonic CD8+ T cell numbers vs wild-type cage mates. Conversely, in DSS colitis, NRG4-/- mice had elevated cytokine expression, fecal lipocalin-2 levels, and impaired weight recovery. RNA sequencing showed a loss of St3gal4, a sialyltransferase involved in immune cell trafficking, in NRG4-null colons, which was verified in both tissue and isolated epithelium. The regulation of St3gal4 by NRG4 was confirmed with ex vivo epithelial colon organoid cultures from NRG4-/- mice and by induction of St3gal4 in vivo following NRG4 treatment. CONCLUSIONS: NRG4 regulates colonic epithelial ST3GAL4 and thus may allow for robust recruitment of CD8+ T cells during adaptive immune responses in colitis. On the other hand, NRG4 loss exacerbates injury driven by innate immune responses.

Neuregulin-4 (NRG4) is a growth factor that protects the epithelial cells lining the colon from injury and restrains innate (non-specific) immune responses. Here we show that NRG4's role in inflammation is context-specific, and mice that lack NRG4 have impaired adaptive immunity in a model of chronic immune-mediated colitis.

Deep Crypt Secretory Cell Differentiation in the Colonic Epithelium Is Regulated by Sprouty2 and Interleukin 13.

BACKGROUND & AIMS: Deep crypt secretory (DCS) cells are a critical component of the colonic stem cell niche. However, the regulatory mechanisms controlling DCS cell numbers and function are not well understood. Sprouty2 is an inflammation-responsive regulator of intracellular signaling that influences colonic secretory cell numbers in colitis via an epithelial-stromal interleukin (IL)33/IL13 signaling loop. Here, we tested the hypothesis that IL13, induced by epithelial Sprouty2 down-regulation, promotes DCS cell differentiation and function. METHODS: Distal colons from mice with an intestinal epithelial-specific Sprouty2 deletion (Spry2ΔIE) and littermate controls were analyzed by in situ hybridization for Reg4+ DCS cells. Single-cell RNA sequencing and immunostaining were used to identify DCS cell-derived host defense peptides (HDPs) and localization of IL13 and IL13 receptor; bulk RNA sequencing and quantitative polymerase chain reaction were used to quantify changes in expression of identified HDPs. Cytokine-treated colonoids were assessed for DCS cells. A requirement for an IL33/IL13 signaling loop in the regulation of DCS cells was assessed in vivo using IL13 null mice. RESULTS: Reg4+ DCS cell numbers were increased 2-fold in distal colons of Spry2ΔIE mice with a concomitant overall increase in DCS cell marker expression (Reg4, Spink4, and Agr2). Single-cell transcriptomics showed the HDP Retnlb/Resistin Like Beta (RELMß) is highly enriched in DCS cells. Retnlb/RELMß expression was increased in Spry2ΔIE colons. IL13, but not IL33, induced Reg4 and Retnlb expression in colonic epithelial organoids, and IL33-mediated expansion of the DCS cell population in vivo was dependent on IL13, which was expressed predominantly by type II innate lymphoid cells in the colonic mucosa. CONCLUSIONS: Sprouty2 limits colonic DCS cell differentiation through suppression of IL13 signaling. At homeostasis, DCS cells are marked by high levels of the HDP RELMß. Loss of epithelial Sprouty2 activates type II innate lymphoid cells to release IL13, promoting expansion of the DCS cell population and increased colonic RELMß levels.

Transitional Anal Cells Mediate Colonic Re-epithelialization in Colitis.

BACKGROUND & AIMS: Epithelial wound healing is compromised and represents an unleveraged therapeutic target in inflammatory bowel disease (IBD). Intestinal epithelial cells exhibit plasticity that facilitates dedifferentiation and repair during the response to injury. However, it is not known whether epithelial cells of a neighboring organ can be activated to mediate re-epithelialization in acute colitis. Histological findings of a permanent squamous tissue structure in the distal colon in human IBD could suggest diverse cellular origins of repair-associated epithelium. Here, we tested whether skin-like cells from the anus mediate colonic re-epithelialization in murine colitis. METHODS: We studied dextran sulfate sodium-induced colitis and interleukin 10-deficient colitis in transgenic mice. We performed lineage tracing, 3-dimensional (3D) imaging, single-cell transcriptomics, and biophysical modeling to map squamous cell fates and to identify squamous cell types involved in colonic repair. RESULTS: In acute and chronic colitis, we found a large squamous epithelium, called squamous neo-epithelium of the colon (SNEC), near the anorectal junction. Neighboring squamous cells of the anus rapidly migrate into the ulcerated colon and establish this permanent epithelium of crypt-like morphology. These squamous cells derive from a small unique transition zone, distal to the border of colonic and anal epithelium, that resists colitic injury. The cells of this zone have a pre-loaded program of colonic differentiation and further upregulate key aspects of colonic epithelium during repair. CONCLUSION: Transitional anal cells represent unique reserve cells capable of rebuilding epithelial structures in the colon after colitis. Further study of these cells could reveal novel approaches to direct mucosal healing in inflammation and disease.

Persistence of Lgr5+ colonic epithelial stem cells in mouse models of inflammatory bowel disease.

Intestinal mucosal healing is the primary therapeutic goal of medical treatments for inflammatory bowel disease (IBD). Epithelial stem cells are key players in the healing process. Lgr5+ stem cells maintain cellular turnover during homeostasis in the colonic crypt. However, they are lost and dispensable for repair in a wide variety of injury models, including dextran sulfate sodium (DSS) colitis, radiation, helminth infection, and T-cell activation. The direct loss of Lgr5+ cells activates a plasticity response in the epithelium in which other cell types can serve as stem cells. Whether this paradigm applies to mouse models of IBD remains unknown. In contrast to previously tested models, IBD models involve an inflammatory response rooted in the loss of immunologic tolerance to intestinal luminal contents including the microbiome. Here, we show the persistence of Lgr5+ cells in oxazolone, 2,4,6-trinitrobenzene sulfonic acid (TNBS), and Il10-/-, and Il10-/- Tnfr1-/- IBD models. This contrasts with results obtained from DSS-induced injury. Through high-throughput expression profiling, we find that these colitis models were associated with distinct patterns of cytokine expression. Direct exposure of colonic epithelial organoids to DSS, oxazolone, or TNBS resulted in increased apoptosis and loss of Lgr5+ cells. Targeted ablation of Lgr5+ cells resulted in severe exacerbation of chronic, antibody-induced IL-10-deficient colitis, but had only modest effects in TNBS-induced colitis. These results show that distinct mouse models of IBD-like colitis induce different patterns of Lgr5+ stem cell retention and function.NEW & NOTEWORTHY Acute intestinal injury and epithelial repair are associated with the loss of fast-cycling Lgr5+ stem cells and plasticity in the activation of formerly quiescent cell populations. In contrast, here we show in murine inflammatory bowel disease the persistence of the Lgr5+ stem cell population and its essential role in restricting the severity of chronic colitis. This demonstrates a diversity of stem cell responses to colitis.

Epithelial wound healing in inflammatory bowel diseases: the next therapeutic frontier.

Patients with one of the many chronic inflammatory disorders broadly classified as inflammatory bowel disease (IBD) now have a diverse set of immunomodulatory therapies at their disposal. Despite these recent medical advances, complete sustained remission of disease remains elusive for most patients. The full healing of the damaged intestinal mucosa is the primary goal of all therapies. Achieving this requires not just a reduction of the aberrant immunological response, but also wound healing of the epithelium. No currently approved therapy directly targets the epithelium. Epithelial repair is compromised in IBD and normally facilitates re-establishment of the homeostatic barrier between the host and the microbiome. In this review, we summarize the evidence that epithelial wound healing represents an important yet underdeveloped therapeutic modality for IBD. We highlight 3 general approaches that are promising for developing a new class of epithelium-targeted therapies: epithelial stem cells, cytokines, and microbiome engineering. We also provide a frank discussion of some of the challenges that must be overcome for epithelial repair to be therapeutically leveraged. A concerted approach by the field to develop new therapies targeting epithelial wound healing will offer patients a game-changing, complementary class of medications and could dramatically improve outcomes.

NRG4-ErbB4 signaling represses proinflammatory macrophage activity.

Proinflammatory macrophages are essential drivers of colitis and express the growth factor receptor ErbB4. This study tested the role of ErbB4 and its specific ligand, NRG4, in regulating macrophage function. We show that endogenous NRG4-ErbB4 signaling limits macrophage production of proinflammatory cytokines in vitro and limits colitis severity in vivo and thus is a potential target for therapeutic intervention.

Sprouty2 limits intestinal tuft and goblet cell numbers through GSK3ß-mediated restriction of epithelial IL-33.

Dynamic regulation of intestinal cell differentiation is crucial for both homeostasis and the response to injury or inflammation. Sprouty2, an intracellular signaling regulator, controls pathways including PI3K and MAPKs that are implicated in differentiation and are dysregulated in inflammatory bowel disease. Here, we ask whether Sprouty2 controls secretory cell differentiation and the response to colitis. We report that colonic epithelial Sprouty2 deletion leads to expanded tuft and goblet cell populations. Sprouty2 loss induces PI3K/Akt signaling, leading to GSK3ß inhibition and epithelial interleukin (IL)-33 expression. In vivo, this results in increased stromal IL-13+ cells. IL-13 in turn induces tuft and goblet cell expansion in vitro and in vivo. Sprouty2 is downregulated by acute inflammation; this appears to be a protective response, as VillinCre;Sprouty2F/F mice are resistant to DSS colitis. In contrast, Sprouty2 is elevated in chronic colitis and in colons of inflammatory bowel disease patients, suggesting that this protective epithelial-stromal signaling mechanism is lost in disease.

TNF Receptor 1 Promotes Early-Life Immunity and Protects against Colitis in Mice.

Neutralization of tumor necrosis factor (TNF) represents a widely used therapeutic strategy for autoimmune diseases including inflammatory bowel disease (IBD). However, the fact that many patients with IBD are non-responsive to anti-TNF therapies suggests the need for a better understanding of TNF signaling in IBD. Here, we show that co-deletion of TNF receptor 1 (TNFR1, Tnfrsf1a) in the Il10-/- spontaneous colitis model exacerbates disease, resulting in very-early-onset inflammation after weaning. The disease can be interrupted by treatment with antibiotics. The single deletion of TNFR1 induces subclinical colonic epithelial dysfunction and mucosal immune abnormalities, including accumulation of neutrophils and depletion of B cells. During the pre-disease period (before weaning), both Tnfr1-/- and Il10-/-Tnfr1-/- animals exhibit impaired expression of pro-inflammatory cytokines compared with wild-type and Il10-/- controls, respectively. Collectively, these results demonstrate the net anti-inflammatory functions of TNF/TNFR1 signaling through the regulation of colonic immune homeostasis in early life.

Cellular maps of gastrointestinal organs: getting the most from tissue clearing.

The development of modern methods to induce optical transparency ("clearing") in biological tissues has enabled the three-dimensional (3D) reconstruction of intact organs at cellular resolution. New capabilities in visualization of rare cellular events, long-range interactions, and irregular structures will facilitate novel studies in the alimentary tract and gastrointestinal systems. The tubular geometry of the alimentary tract facilitates large-scale cellular reconstruction of cleared tissue without specialized microscopy setups. However, with the rapid pace of development of clearing agents and current relative paucity of research groups in the gastrointestinal field using these techniques, it can be daunting to incorporate tissue clearing into experimental workflows. Here, we give some advice and describe our own experience bringing tissue clearing and whole mount reconstruction into our laboratory's investigations. We present a brief overview of the chemical concepts that underpin tissue clearing, what sorts of questions whole mount imaging can answer, how to choose a clearing agent, an example of how to clear and image alimentary tissue, and what to do after obtaining the image. This short review will encourage other gastrointestinal researchers to consider how utilizing tissue clearing and creating 3D "maps" of tissue might deepen the impact of their studies.

Microbiomes through the Looking Glass: What Do UC?

Pediatric ulcerative colitis incidence is rapidly rising, yet improved prognostic and therapeutic strategies are needed. In this issue of Cell Host & Microbe, Schirmer et al. (2018) reveal the dynamism of pediatric patient microbiomes through initial diagnosis and treatments, providing insights into microbial targets that predict therapeutic response and disease outcomes.

Pharmacological activation of epidermal growth factor receptor signaling inhibits colitis-associated cancer in mice.

Current treatments for inflammatory bowel disease (IBD) target the overactive immune response of the intestinal mucosa. However, epidermal growth factor (EGF), an activating ligand of the EGF receptor (EGFR), has been shown to induce disease remission through direct targeting of intestinal mucosal healing. Despite promising preclinical and clinical results, this EGFR-activating therapy has not progressed, in part due to the potential for carcinogenesis associated with long-term use and the increased risk of colitis-associated cancer (CAC) in IBD. Here we tested whether pharmacological modulation of EGFR altered outcomes of CAC in the murine azoxymethane/dextran sulfate sodium model. We found that administering EGF during the period of maximum colitis severity ("early"), coincident with the initiation and early promotion of tumors, improved outcomes of colitis and reduced tumor size. In contrast, daily EGF administration beginning ~2 months after tumor initiation ("late") increased tumor size. Administration of the EGFR kinase inhibitor gefitinib increased the tumor size when the drug was given early and decreased the tumor size when the drug was administered late. EGF administration not only reduced colonic cytokine and chemokine expression during injury, but also baseline chemokine expression in homeostasis. These results suggest that EGFR activation during acute bouts of colitis may reduce the long-term burden of CAC.

Tumor Necrosis Factor Receptor 2 Restricts the Pathogenicity of CD8(+) T Cells in Mice With Colitis.

BACKGROUND & AIMS: Tumor necrosis factor receptor 2 (TNFR2, Tnfrsf1b) regulates multiple aspects of immune function, but little is known about its role in the immunopathogenesis of inflammatory bowel disease (IBD). We investigated whether TNFR2 restricts the activity of specific immune cell subtypes to protect against the development of colitis in mice. METHODS: Tnfr2(-/-) mice were crossed with interleukin (Il) 10(-/-) mice, which spontaneously develop colitis, to generate Il10(-/-)Tnfr2(-/-) mice. Colonic tissues were collected from Il10(-/-)Tnfr2(-/-) mice along with Il10(-/-) mice (controls) and analyzed by flow cytometry and histology. Bone marrow was transplanted into Il10(-/-) and Il10(-/-)Tnfr2(-/-) mice from Il10(-/-) or Il10(-/-)Tnfr2(-/-) donors by intravenous injection. CD8(+) T cells were neutralized in Il10(-/-)Tnfr2(-/-) mice by intraperitoneal injection of anti-CD8 or isotype control antibodies. Colitis was induced in Rag2(-/-) mice by intravenous injections of naïve CD8(+) T cells isolated from C57BL/6 or Tnfr2(-/-) mice. RESULTS: Il10(-/-)Tnfr2(-/-) mice spontaneously developed more severe colitis compared with Il10(-/-) controls, characterized by selective expansion of colonic CD8(+) T cells. Transplantation of TNFR2-deficient bone marrow resulted in significantly increased incidence and severity of colitis. Transcriptome analyses showed that the expression of genes regulated by TNFR2 were specific to CD8(+) T cells and included genes associated with risk for IBD. Depletion of CD8(+) T cells from Il10(-/-)Tnfr2(-/-) mice prevented colonic inflammation. Adoptive transfer of TNFR2-null naïve CD8(+) T cells compared with CD8(+) T cells from control mice increased the severity of colitis that developed in Rag2(-/-) mice. CONCLUSIONS: TNFR2 protects mice from colitis by inhibiting the expansion of colonic CD8(+) T cells. TNFR2 regulates expression of genes that regulate CD8(+) T cells and have been associated with susceptibility to IBD. Disruption in TNFR2 signaling might therefore be associated with pathogenesis. Strategies to increase levels or activity of TNFR2 and thereby reduce the activity of CD8(+) T cells might be developed to treat IBD patients with CD8(+) T cell dysfunction.

Optical reconstruction of murine colorectal mucosa at cellular resolution.

The mucosal layer of the colon is a unique and dynamic site where host cells interface with one another and the microbiome, with major implications for physiology and disease. However, the cellular mechanisms mediating colonic regeneration, inflammation, dysplasia, and dysbiosis remain undercharacterized, partly because the use of thin tissue sections in many studies removes important volumetric context. To address these challenges in visualization, we have developed the deep mucosal imaging (DMI) method to reconstruct continuous extended volumes of mouse colorectal mucosa at cellular resolution. Use of ScaleA2 and SeeDB clearing agents enabled full visualization of the colonic crypt, the fundamental unit of adult colon. Confocal imaging of large colorectal expanses revealed epithelial structures involved in repair, inflammation, tumorigenesis, and stem cell function, in fluorescent protein-labeled, immunostained, paraffin-embedded, or human biopsy samples. We provide freely available software to reconstruct and explore on computers with standard memory allocations the large DMI datasets containing in toto representations of distal colonic mucosal volume. Extended-volume imaging of colonic mucosa through the novel, extensible, and readily adopted DMI approach will expedite mechanistic investigations of intestinal physiology and pathophysiology at intracrypt to multicrypt length scales.

Electrophysiological characterization of Grueneberg ganglion olfactory neurons: spontaneous firing, sodium conductance, and hyperpolarization-activated currents.

Mammals rely on their acute olfactory sense for their survival. The most anterior olfactory subsystem in the nose, the Grueneberg ganglion (GG), plays a role in detecting alarm pheromone, cold, and urinary compounds. GG neurons respond homogeneously to these stimuli with increases in intracellular [Ca(2+)] or transcription of immediate-early genes. In this electrophysiological study, we used patch-clamp techniques to characterize the membrane properties of GG neurons. Our results offer evidence of functional heterogeneity in the GG. GG neurons fire spontaneously and independently in several stable patterns, including phasic and repetitive single-spike modes of discharge. Whole cell recordings demonstrated two distinct voltage-gated fast-inactivating Na(+) currents with different steady-state voltage dependencies and different sensitivities to tetrodotoxin. Hodgkin-Huxley simulations showed that these Na(+) currents confer dual mechanisms of action potential generation and contribute to different firing patterns. Additionally, GG neurons exhibited hyperpolarization-activated inward currents that modulated spontaneous firing in vitro. Thus, in GG neurons, the heterogeneity of firing patterns is linked to the unusual repertoire of ionic currents. The membrane properties described here will aid the interpretation of chemosensory function in the GG.

Grueneberg ganglion olfactory subsystem employs a cGMP signaling pathway.

The mammalian olfactory sense employs several olfactory subsystems situated at characteristic locations in the nasal cavity to detect and report on different classes of odors. These olfactory subsystems use different neuronal signal transduction pathways, receptor expression repertoires, and axonal projection targets. The Grueneberg ganglion (GG) is a newly appreciated olfactory subsystem with receptor neurons located just inside of the nostrils that project axons to a unique domain of interconnected glomeruli in the caudal olfactory bulb. It is not well understood how the GG relates to other olfactory subsystems in contributing to the olfactory sense. Furthermore, the range of chemoreceptors and the signal transduction cascade utilized by the GG have remained mysterious. To resolve these unknowns, we explored the molecular relationship between the GG and the GC-D neurons, another olfactory subsystem that innervates similarly interconnected glomeruli in the same bulbar region. We found that mouse GG neurons express the cGMP-associated signaling proteins phosphodiesterase 2a, cGMP-dependent kinase II, and cyclic nucleotide gated channel subunit A3 coupled to a chemoreceptor repertoire of cilia-localized particulate guanylyl cyclases (pGC-G and pGC-A). The primary cGMP signaling pathway of the GG is shared with the GC-D neurons, unifying their target glomeruli as a unique center of olfactory cGMP signal transduction. However, the distinct chemoreceptor repertoire in the GG suggests that the GG is an independent olfactory subsystem. This subsystem is well suited to detect a unique set of odors and to mediate behaviors that remained intact in previous olfactory perturbations.