Sox4 Promotes Atoh1-Independent Intestinal Secretory Differentiation Toward Tuft and Enteroendocrine Fates

BACKGROUND & AIMS: The intestinal epithelium is maintained by intestinal stem cells (ISCs), which produce postmitotic absorptive and secretory epithelial cells. Initial fate specification toward enteroendocrine, goblet, and Paneth cell lineages requires the transcription factor Atoh1, which regulates differentiation of the secretory cell lineage. However, less is known about the origin of tuft cells, which participate in type II immune responses to parasite infections and appear to differentiate independently of Atoh1. We investigated the role of Sox4 in ISC differentiation. METHODS: We performed experiments in mice with intestinal epithelial-specific disruption of Sox4 (Sox4fl/fl:vilCre; SOX4 conditional knockout [cKO]) and mice without disruption of Sox4 (control mice). Crypt- and single-cell-derived organoids were used in assays to measure proliferation and ISC potency. Lineage allocation and gene expression changes were studied by immunofluorescence, real-time quantitative polymerase chain reaction, and RNA-seq analyses. Intestinal organoids were incubated with the type 2 cytokine interleukin 13 and gene expression was analyzed. Mice were infected with the helminth Nippostrongylus brasiliensis and intestinal tissues were collected 7 days later for analysis. Intestinal tissues collected from mice that express green fluorescent protein regulated by the Atoh1 promoter (Atoh1GFP mice) and single-cell RNA-seq analysis were used to identify cells that coexpress Sox4 and Atoh1. We generated SOX4-inducible intestinal organoids derived from Atoh1fl/fl:vilCreER (ATOH1 inducible knockout) mice and assessed differentiation. RESULTS: Sox4cKO mice had impaired ISC function and secretory differentiation, resulting in decreased numbers of tuft and enteroendocrine cells. In control mice, numbers of SOX4+ cells increased significantly after helminth infection, coincident with tuft cell hyperplasia. Sox4 was activated by interleukin 13 in control organoids; SOX4cKO mice had impaired tuft cell hyperplasia and parasite clearance after infection with helminths. In single-cell RNA-seq analysis, Sox4+/Atoh1- cells were enriched for ISC, progenitor, and tuft cell genes; 12.5% of Sox4-expressing cells coexpressed Atoh1 and were enriched for enteroendocrine genes. In organoids, overexpression of Sox4 was sufficient to induce differentiation of tuft and enteroendocrine cells-even in the absence of Atoh1. CONCLUSIONS: We found Sox4 promoted tuft and enteroendocrine cell lineage allocation independently of Atoh1. These results challenge the longstanding model in which Atoh1 is the sole regulator of secretory differentiation in the intestine and are relevant for understanding epithelial responses to parasitic infection

Structure and function analyses of the purified GPCR human vomeronasal type 1 receptor 1

The vomeronasal system is one of several fine-tuned scent-detecting signaling systems in mammals. However, despite significant efforts, how these receptors detect scent remains an enigma. One reason is the lack of sufficient purified receptors to perform detailed biochemical, biophysical and structural analyses. Here we report the ability to express and purify milligrams of purified, functional human vomeronasal receptor hVN1R1. Circular dichroism showed that purified hVN1R1 had an alpha-helical structure, similar to that of other GPCRs. Microscale thermophoresis showed that hVN1R1 bound its known ligand myrtenal with an EC50 ∼1 µM. This expression system can enable structural and functional analyses towards understanding how mammalian scent detection works

Diagnostic tools in Rhinology EAACI position paper

This EAACI Task Force document aims at providing the readers with a comprehensive and complete overview of the currently available tools for diagnosis of nasal and sino-nasal disease. We have tried to logically order the different important issues related to history taking, clinical examination and additional investigative tools for evaluation of the severity of sinonasal disease into a consensus document. A panel of European experts in the field of Rhinology has contributed to this consensus document on Diagnostic Tools in Rhinology

Vomeronasal organ and human pheromones

SummaryFor many organisms, pheromonal communication is of particular importance in managing various aspects of reproduction. In tetrapods, the vomeronasal (Jacobson's) organ specializes in detecting pheromones in biological substrates of congeners. This information triggers behavioral changes associated, in the case of certain pheromones, with neuroendocrine correlates. In human embryos, the organ develops and the nerve fibers constitute a substrate for the migration of GnRH-secreting cells from the olfactory placode toward the hypothalamus. After this essential step for subsequent secretion of sex hormones by the anterior hypophysis, the organ regresses and the neural connections disappear. The vomeronasal cavities can still be observed by endoscopy in some adults, but they lack sensory neurons and nerve fibers. The genes which code for vomeronasal receptor proteins and the specific ionic channels involved in the transduction process are mutated and nonfunctional in humans. In addition, no accessory olfactory bulbs, which receive information from the vomeronasal receptor cells, are found. The vomeronasal sensory function is thus nonoperational in humans. Nevertheless, several steroids are considered to be putative human pheromones; some activate the anterior hypothalamus, but the effects observed are not comparable to those in other mammals. The signaling process (by neuronal detection and transmission to the brain or by systemic effect) remains to be clearly elucidated

Direct influence of the sodium pump on the membrane potential of vomeronasal chemoreceptor neurones in frog.

1. Whole-cell measurements were made from microvillous receptor neurones isolated from the frog vomeronasal organ. We examined the mechanisms that determined the value of the resting membrane potential. 2. Cells recorded in Ringer solution containing 4 mM K+ showed a resting membrane potential of -88 +/- 20 mV (mean +/- 1 S.D., n = 56). Sixty-six per cent of the cells had stable resting potentials more negative than the calculated equilibrium potentials for K+ (EK, -82 mV) indicating the presence of a hyperpolarizing outward pump current. 3. Cells recorded with an intracellular solution containing Na+ instead of K+, to set EK at 0 mV, presented stable membrane potentials in the range -65 to -119 mV when bathed in a normal Ringer solution. 4. Ouabain, a specific inhibitor of the Na+,K(+)-ATPase, blocked the outward sodium pump current (Ip) and depolarized the membrane. 5. The sodium pump current, measured as the current blocked by 0.5 mM dihydro-ouabain, was linearly related to the membrane potential in the range -60 to -120 mV. The reversal potential measured with a calculated free energy of ATP hydrolysis of -36.2 kJ mol-1 was estimated to be -143 mV. 6. Reduction of the external K+ concentration to 0 mM depolarized the membrane to less than -40 mV. Voltage-clamp observations in this condition indicated a reduction of Ip. Ouabain added to the bath reduced the blocking effect of low external K+. The addition of external K+ activated Ip and induced a rapid hyperpolarization of the cell membrane. 7. At membrane potentials more negative than -80 mV, an inward rectifying depolarizing current characterized as Ih was activated. When Ih was blocked by 5 mM external Cs+ the resting membrane potential increased. 8. These data indicate that the membrane potential of the vomeronasal receptor neurones is not generated by a passive diffusion of K+ ions but by the hyperpolarizing current created by the Na+,K(+)-ATPase. We propose that the resting potential is set by a balance between Ip and Ih. The physiological implications of these mechanisms for setting the resting potential are discussed