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The intestinal epithelium has high intrinsic turnover rate, and the precise renewal of the epithelium is dependent on the microenvironment. The intestine is innervated by a dense network of peripheral nerves that controls various aspects of intestinal physiology. However, the role of neurons in regulating epithelial cell regeneration remains largely unknown. Here, we investigated the effects of gut-innervating adrenergic nerves on epithelial cell repair following irradiation (IR)-induced injury. We observed that adrenergic nerve density in the small intestine increased post IR, while chemical adrenergic denervation impaired epithelial regeneration. Single-cell RNA sequencing experiments revealed a decrease in IL-22 signaling post IR in denervated animals. Combining pharmacologic and genetic tools, we demonstrate that β-adrenergic receptor signaling drives IL-22 production from type 3 innate lymphoid cells (ILC3s) post IR, which in turn promotes epithelial regeneration. These results define an adrenergic-ILC3 axis important for intestinal regeneration., Competing Interests: Declaration of interests All authors are current employees of Genentech Inc., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Self-renewal and differentiation of stem and progenitor cells are tightly regulated to ensure tissue homeostasis. This regulation is enabled both remotely by systemic circulating cues, such as cytokines and hormones, and locally by various niche-confined factors. R-spondin 3 (RSPO3) is one of the most potent enhancers of Wnt signaling, and its expression is usually restricted to the stem cell niche where it provides localized enhancement of Wnt signaling to regulate stem cell expansion and differentiation. Disruption of this niche-confined expression can disturb proper tissue organization and lead to cancers. Here, we investigate the consequences of disrupting the niche-restricted expression of RSPO3 in various tissues, including the hematopoietic system. We show that normal Rspo3 expression is confined to the perivascular niche in the bone marrow. Induction of increased systemic levels of circulating RSPO3 outside of the niche results in prominent loss of early B-cell progenitors and anemia but surprisingly has no effect on hematopoietic stem cells. Using molecular, pharmacologic, and genetic approaches, we show that these RSPO3-induced hematopoietic phenotypes are Wnt and RSPO3 dependent and mediated through noncanonical Wnt signaling. Our study highlights a distinct role for a Wnt/RSPO3 signaling axis in the regulation of hematopoiesis, as well as possible challenges related to therapeutic use of RSPOs for regenerative medicine., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Repair of the intestinal epithelium is tightly regulated to maintain homeostasis. The response after epithelial damage needs to be local and proportional to the insult. How different types of damage are coupled to repair remains incompletely understood. We report that after distinct types of intestinal epithelial damage, IL-1R1 signaling in GREM1 + mesenchymal cells increases production of R-spondin 3 (RSPO3), a Wnt agonist required for intestinal stem cell self-renewal. In parallel, IL-1R1 signaling regulates IL-22 production by innate lymphoid cells and promotes epithelial hyperplasia and regeneration. Although the regulation of both RSPO3 and IL-22 is critical for epithelial recovery from Citrobacter rodentium infection, IL-1R1-dependent RSPO3 production by GREM1 + mesenchymal cells alone is sufficient and required for recovery after dextran sulfate sodium-induced colitis. These data demonstrate how IL-1R1-dependent signaling orchestrates distinct repair programs tailored to the type of injury sustained that are required to restore intestinal epithelial barrier function., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Fibroblastic reticular cells (FRCs) are specialized stromal cells that define tissue architecture and regulate lymphocyte compartmentalization, homeostasis, and innate and adaptive immunity in secondary lymphoid organs (SLOs). In the present study, we used single-cell RNA sequencing (scRNA-seq) of human and mouse lymph nodes (LNs) to identify a subset of T cell-zone FRCs defined by the expression of Gremlin1 (Grem1) in both species. Grem1-CreER T2 knock-in mice enabled localization, multi-omics characterization and genetic depletion of Grem1 + FRCs. Grem1 + FRCs primarily localize at T-B cell junctions of SLOs, neighboring pre-dendritic cells and conventional dendritic cells (cDCs). As such, their depletion resulted in preferential loss and decreased homeostatic proliferation and survival of resident cDCs and compromised T cell immunity. Trajectory analysis of human LN scRNA-seq data revealed expression similarities to murine FRCs, with GREM1 + cells marking the endpoint of both trajectories. These findings illuminate a new Grem1 + fibroblastic niche in LNs that functions to maintain the homeostasis of lymphoid tissue-resident cDCs.

Intestinal stem cells (ISCs) are confined to crypt bottoms and their progeny differentiate near crypt-villus junctions. Wnt and bone morphogenic protein (BMP) gradients drive this polarity, and colorectal cancer fundamentally reflects disruption of this homeostatic signaling. However, sub-epithelial sources of crucial agonists and antagonists that organize this BMP gradient remain obscure. Here, we couple whole-mount high-resolution microscopy with ensemble and single-cell RNA sequencing (RNA-seq) to identify three distinct PDGFRA + mesenchymal cell types. PDGFRA(hi) telocytes are especially abundant at the villus base and provide a BMP reservoir, and we identified a CD81 + PDGFRA(lo) population present just below crypts that secretes the BMP antagonist Gremlin1. These cells, referred to as trophocytes, are sufficient to expand ISCs in vitro without additional trophic support and contribute to ISC maintenance in vivo. This study reveals intestinal mesenchymal structure at fine anatomic, molecular, and functional detail and the cellular basis for a signaling gradient necessary for tissue self-renewal., Competing Interests: Declaration of Interests E.E.S., V.N.K., C.C., S.K., F.J.d.S., and S.J.T. are employees of Genentech and own shares in Roche. The other authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Cancer stem cells (CSCs) have been hypothesized to represent the driving force behind tumour progression and metastasis, making them attractive cancer targets. However, conclusive experimental evidence for their functional relevance is still lacking for most malignancies. Here we show that the leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5) identifies intestinal CSCs in mouse tumours engineered to recapitulate the clinical progression of human colorectal cancer. We demonstrate that selective Lgr5 + cell ablation restricts primary tumour growth, but does not result in tumour regression. Instead, tumours are maintained by proliferative Lgr5 - cells that continuously attempt to replenish the Lgr5 + CSC pool, leading to rapid re-initiation of tumour growth upon treatment cessation. Notably, CSCs are critical for the formation and maintenance of liver metastasis derived from colorectal cancers. Together, our data highlight distinct CSC dependencies for primary versus metastasic tumour growth, and suggest that targeting CSCs may represent a therapeutic opportunity for managing metastatic disease.

Colorectal cancer remains a major unmet medical need, prompting large-scale genomics efforts in the field to identify molecular drivers for which targeted therapies might be developed. We previously reported the identification of recurrent translocations in R-spondin genes present in a subset of colorectal tumours. Here we show that targeting RSPO3 in PTPRK-RSPO3-fusion-positive human tumour xenografts inhibits tumour growth and promotes differentiation. Notably, genes expressed in the stem-cell compartment of the intestine were among those most sensitive to anti-RSPO3 treatment. This observation, combined with functional assays, suggests that a stem-cell compartment drives PTPRK-RSPO3 colorectal tumour growth and indicates that the therapeutic targeting of stem-cell properties within tumours may be a clinically relevant approach for the treatment of colorectal tumours.

Identifying and understanding changes in cancer genomes is essential for the development of targeted therapeutics. Here we analyse systematically more than 70 pairs of primary human colon tumours by applying next-generation sequencing to characterize their exomes, transcriptomes and copy-number alterations. We have identified 36,303 protein-altering somatic changes that include several new recurrent mutations in the Wnt pathway gene TCF7L2, chromatin-remodelling genes such as TET2 and TET3 and receptor tyrosine kinases including ERBB3. Our analysis for significantly mutated cancer genes identified 23 candidates, including the cell cycle checkpoint kinase ATM. Copy-number and RNA-seq data analysis identified amplifications and corresponding overexpression of IGF2 in a subset of colon tumours. Furthermore, using RNA-seq data we identified multiple fusion transcripts including recurrent gene fusions involving R-spondin family members RSPO2 and RSPO3 that together occur in 10% of colon tumours. The RSPO fusions were mutually exclusive with APC mutations, indicating that they probably have a role in the activation of Wnt signalling and tumorigenesis. Consistent with this we show that the RSPO fusion proteins were capable of potentiating Wnt signalling. The R-spondin gene fusions and several other gene mutations identified in this study provide new potential opportunities for therapeutic intervention in colon cancer.

Mouse embryos bearing hypomorphic and conditional null Fgf8 mutations have small and abnormally patterned telencephalons. We provide evidence that the hypoplasia results from decreased Foxg1 expression, reduced cell proliferation and increased cell death. In addition, alterations in the expression of Bmp4, Wnt8b, Nkx2.1 and Shh are associated with abnormal development of dorsal and ventral structures. Furthermore, nonlinear effects of Fgf8 gene dose on the expression of a subset of genes, including Bmp4 and Msx1, correlate with a holoprosencephaly phenotype and with the nonlinear expression of transcription factors that regulate neocortical patterning. These data suggest that Fgf8 functions to coordinate multiple patterning centers, and that modifications in the relative strength of FGF signaling can have profound effects on the relative size and nature of telencephalic subdivisions.

Mammals have developed patterns of social relationships that enhance the survival of individuals and maximize the reproductive success of species. Although social stimuli and social responses are highly complex, recent studies are providing substantial insights into their neural substrates. Neural pathways employing the nonapeptides vasopressin and oxytocin play a particularly prominent role both in social recognition and the expression of appropriate social responses. New insights into social neuroscience are discussed, along with the relevance of this rapidly developing field to human relationships and disease processes.

FGF8 is known to be an important regulator of forebrain development. Here, we investigated the effects of varying the level of Fgf8 expression in the mouse forebrain. We detected two distinct responses, one that was proportionate with Fgf8 expression and another that was not. The latter response, which led to effects on cell survival, displayed a paradoxical relationship to Fgf8 dosage. Either eliminating or increasing Fgf8 expression increased apoptosis, whereas reducing Fgf8 expression had the opposite effect. To explain these counterintuitive observations, we suggest that an FGF8-dependent cell-survival pathway is negatively regulated by intracellular inhibitors produced in proportion to FGF8 concentration. Our data provide insight into the function of FGF8 in forebrain development and underscore the value of using multiple alleles and different experimental approaches to unravel the complexities of gene function in vertebrate development.

A functional skeletal system requires the coordinated development of many different tissue types, including cartilage, bones, joints, and tendons. Members of the Bone morphogenetic protein (BMP) family of secreted signaling molecules have been implicated as endogenous regulators of skeletal development. This is based on their expression during bone and joint formation, their ability to induce ectopic bone and cartilage, and the skeletal abnormalities present in animals with mutations in BMP family members. One member of this family, Growth/differentiation factor 5 (GDF5), is encoded by the mouse brachypodism locus. Mice with mutations in this gene show reductions in the length of bones in the limbs, altered formation of bones and joints in the sternum, and a reduction in the number of bones in the digits. The expression pattern of Gdf5 during normal development and the phenotypes seen in mice with single or double mutations in Gdf5 and Bmp5 suggested that Gdf5 has multiple functions in skeletogenesis, including roles in joint and cartilage development. To further understand the function of GDF5 in skeletal development, we assayed the response of developing chick and mouse limbs to recombinant GDF5 protein. The results from these assays, coupled with an analysis of the development of brachypodism digits, indicate that GDF5 is necessary and sufficient for both cartilage development and the restriction of joint formation to the appropriate location. Thus, GDF5 function in the digits demonstrates a link between cartilage development and joint development and is an important determinant of the pattern of bones and articulations in the digits., (Copyright 1999 Academic Press.)

The mouse brachypodism locus encodes a bone morphogenetic protein (BMP)-like molecule called growth/differentiation factor 5 (GDF5). Here we show that Gdf5 transcripts are expressed in a striking pattern of transverse stripes within many skeletal precursors in the developing limb. The number, location and time of appearance of these stripes corresponds to the sites where joints will later form between skeletal elements. Null mutations in Gdf5 disrupt the formation of more than 30% of the synovial joints in the limb, leading to complete or partial fusions between particular skeletal elements, and changes in the patterns of repeating structures in the digits, wrists and ankles. Mice carrying null mutations in both Gdf5 and another BMP family member, Bmp5, show additional abnormalities not observed in either of the single mutants. These defects include disruption of the sternebrae within the sternum and abnormal formation of the fibrocartilaginous joints between the sternebrae and ribs. Previous studies have shown that members of the BMP family are required for normal development of cartilage and bone. The current studies suggest that particular BMP family members may also play an essential role in the segmentation process that cleaves skeletal precursors into separate elements. This process helps determine the number of elements in repeating series in both limbs and sternum, and is required for normal generation of the functional articulations between many adjacent structures in the vertebrate skeleton.

Using video-enhanced microscopy and a pulse-radiolabeling paradigm, we show that proteins synthesized in the medial giant axon cell body of the crayfish (Procambarus clarkii) are delivered to the axon via fast (approximately 62 mm/day) and slow (approximately 0.8 mm/day) transport components. These data confirm that the medial giant axon cell body provides protein to the axon in a manner similar to that reported for mammalian axons. Unlike mammalian axons, the distal (anucleate) portion of a medial giant axon remains intact and functional for > 7 months after severance. This axonal viability persists long after fast transport has ceased and after the slow wave front of radiolabeled protein has reached the terminals. These data are consistent with the hypothesis that another source (i.e., local glial cells) provides a significant amount of protein to supplement that delivered to the medial giant axon by its cell body.

The mutation brachypodism (bp) alters the length and number of bones in the limbs of mice but spares the axial skeleton. It illustrates the importance of specific genes in controlling the morphogenesis of individual skeletal elements in the tetrapod limb. We now report the isolation of three new members of the transforming growth factor-beta (TGF-beta) superfamily (growth/differentiation factors (GDF) 5,6 and 7) and show by mapping, expression patterns and sequencing that mutations in Gdf5 are responsible for skeletal alterations in bp mice. GDF5 and the closely related GDF6 and GDF7 define a new subgroup of factors related to known bone- and cartilage-inducing molecules, the bone morphogenetic proteins (BMPs). Studies of Bmp5 mutations in short ear mice have shown that at least one other BMP gene is also required for normal skeletal development. The highly specific skeletal alterations in bp and short ear mice suggest that different members of the BMP family control the formation of different morphological features in the mammalian skeleton.