Hedgehog (HH) signaling participates in hepatobiliary repair after injury and is activated in patients with cholangiopathies. Cholangiopathies are associated with bile duct (BD) hyperplasia, including expansion of peribiliary glands, the niche for biliary progenitor cells. The inflammation-associated cytokine interleukin (IL)-33 is also up-regulated in cholangiopathies, including cholangiocarcinoma. We hypothesized that HH signaling synergizes with IL-33 in acute inflammation-induced BD hyperplasia. We measured extrahepatic BD (EHBD) thickness and cell proliferation with and without an IL-33 challenge in wild-type mice, mice overexpressing Sonic HH (pCMV-Shh), and mice with loss of the HH pathway effector glioma-associated oncogene 1 (Gli1lacZ/lacZ ). LacZ reporter mice were used to map the expression of HH effector genes in mouse EHBDs. An EHBD organoid (BDO) system was developed to study biliary progenitor cells in vitro. EHBDs from the HH overexpressing pCMV-Shh mice showed increased epithelial cell proliferation and hyperplasia when challenged with IL-33. In Gli1lacZ/lacZ mice, we observed a decreased proliferative response to IL-33 and decreased expression of Il6. The HH ligands Shh and Indian HH (Ihh) were expressed in epithelial cells, whereas the transcriptional effectors Gli1, Gli2, and Gli3 and the HH receptor Patched1 (Ptch1) were expressed in stromal cells, as assessed by in situ hybridization and lacZ reporter mice. Although BDO cells lacked canonical HH signaling, they expressed the IL-33 receptor suppression of tumorigenicity 2. Accordingly, IL-33 treatment directly induced BDO cell proliferation in a nuclear factor κB-dependent manner. Conclusion: HH ligand overexpression enhances EHBD epithelial cell proliferation induced by IL-33. This proproliferative synergism of HH and IL-33 involves crosstalk between HH ligand-producing epithelial cells and HH-responding stromal cells.

Loss-of-function mutations in the human ICK (intestinal cell kinase) gene cause dysfunctional primary cilia and perinatal lethality which are associated with human ciliopathies. The enzyme that we herein call CAPK (ciliopathy-associated protein kinase) is a serine/threonine protein kinase that has a highly conserved MAPK-like N-terminal catalytic domain and an unstructured C-terminal domain (CTD) whose functions are completely unknown. In this study, we demonstrate that truncation of the CTD impairs the ability of CAPK to interact with and phosphorylate its substrate, kinesin family member 3A (KIF3A). We also find that deletion of the CTD of CAPK compromises both localization to the primary cilium and negative regulation of ciliogenesis. Thus, CAPK substrate recognition, ciliary targeting, and ciliary function depend on the non-catalytic CTD of the protein which is predicted to be intrinsically disordered.

Intestinal crypts have great capacity for repair and regeneration after intestinal stem cell (ISC) injury. Here, we define the cellular remodeling process resulting from ISC niche interruption by transient Notch pathway inhibition in adult mice. Although ISCs were retained, lineage tracing demonstrated a marked reduction in ISC function after Notch disruption. Surprisingly, Notch ligand-expressing Paneth cells were rapidly lost by apoptotic cell death. The ISC-Paneth cell changes were followed by a regenerative response, characterized by expansion of cells expressing Notch ligands Dll1 and Dll4, enhanced Notch signaling, and a proliferative surge. Lineage tracing and organoid studies showed that Dll1-expressing cells were activated to function as multipotential progenitors, generating both absorptive and secretory cells and replenishing the vacant Paneth cell pool. Our analysis uncovered a dynamic, multicellular remodeling response to acute Notch inhibition to repair the niche and restore homeostasis. Notably, this crypt regenerative response did not require ISC loss.

Rheumatoid arthritis-associated pain is poorly managed, often persisting when joint inflammation is pharmacologically controlled. Comparably, in the mouse K/BxN serum-transfer model of inflammatory arthritis, hind paw nociceptive hypersensitivity occurs with ankle joint swelling (5 days after immunisation) persisting after swelling has resolved (25 days after immunisation). In this study, lipid mediator (LM) profiling of lumbar dorsal root ganglia (DRG), the site of sensory neuron cell bodies innervating the ankle joints, 5 days and 25 days after serum transfer demonstrated a shift in specialised proresolving LM profiles. Persistent nociception without joint swelling was associated with low concentrations of the specialised proresolving LM Maresin 1 (MaR1) and high macrophage numbers in DRG. MaR1 application to cultured DRG neurons inhibited both capsaicin-induced increase of intracellular calcium ions and release of calcitonin gene-related peptide in a dose-dependent manner. Furthermore, in peritoneal macrophages challenged with lipopolysaccharide, MaR1 reduced proinflammatory cytokine expression. Systemic MaR1 administration caused sustained reversal of nociceptive hypersensitivity and reduced inflammatory macrophage numbers in DRG. Unlike gabapentin, which was used as positive control, systemic MaR1 did not display acute antihyperalgesic action. Therefore, these data suggest that MaR1 effects observed after K/BxN serum transfer relate to modulation of macrophage recruitment, more likely than to direct actions on sensory neurons. Our study highlights that, in DRG, aberrant proresolution mechanisms play a key role in arthritis joint pain dissociated from joint swelling, opening novel approaches for rheumatoid arthritis pain treatment.

Top-predators play stabilising roles in island food webs, including Fraser Island, Australia. Subsidising generalist predators with human-sourced food could disrupt this balance, but has been proposed to improve the overall health of the island's dingo (Canis lupus dingo) population, which is allegedly 'starving' or in 'poor condition'. We assess this hypothesis by describing the diet and health of dingoes on Fraser Island from datasets collected between 2001 and 2015. Medium-sized mammals (such as bandicoots) and fish were the most common food items detected in dingo scat records. Stomach contents records revealed additional information on diet, such as the occurrence of human-sourced foods. Trail camera records highlighted dingo utilisation of stranded marine fauna, particularly turtles and whales. Mean adult body weights were higher than the national average, body condition scores and abundant-excessive fat reserves indicated a generally ideal-heavy physical condition, and parasite loads were low and comparable to other dingo populations. These data do not support hypotheses that Fraser Island dingoes have restricted diets or are in poor physical condition. Rather, they indicate that dingoes on Fraser Island are capable of exploiting a diverse array of food sources which contributes to the vast majority of dingoes being of good-excellent physical condition.

Top-predators can be important components of resilient ecosystems, but they are still controlled in many places to mitigate a variety of economic, environmental and/or social impacts. Lethal control is often achieved through the broad-scale application of poisoned baits. Understanding the direct and indirect effects of such lethal control on subsequent movements and behaviour of survivors is an important pre-requisite for interpreting the efficacy and ecological outcomes of top-predator control. In this study, we use GPS tracking collars to investigate the fine-scale and short-term movements of dingoes (Canis lupus dingo and other wild dogs) in response to a routine poison-baiting program as an example of how a common, social top-predator can respond (behaviourally) to moderate levels of population reduction. We found no consistent control-induced differences in home range size or location, daily distance travelled, speed of travel, temporal activity patterns or road/trail usage for the seven surviving dingoes we monitored immediately before and after a typical lethal control event. These data suggest that the spatial behaviour of surviving dingoes was not altered in ways likely to affect their detectability, and if control-induced changes in dingoes' ecological function did occur, these may not be related to altered spatial behaviour or movement patterns.

The water mouse is a small and vulnerable rodent present in coastal areas of south-west Papua New Guinea, and eastern Queensland and the Northern Territory of Australia. Current knowledge regarding the distribution of the water mouse is incomplete and the loss of one local population has been documented in southeast Queensland, a region where pressures from urban and industrial development are increasing. Water mouse populations have not been studied intensively enough to enable the primary factors responsible for the local decline to be identified. We surveyed the distribution and density of the water mouse along the Maroochy River of southeast Queensland, near the southern extent of the species' range, to gather baseline data that may prove valuable for detecting any future decline in this population's size or health. All areas of suitable habitat were surveyed on foot or by kayak or boat over a three-year period. We found 180 water mouse nests, of which ~94% were active. Permanent camera monitoring of one nest and limited supplementary live trapping suggested that up to three individual mice occupied active nests. Water mouse density was estimated to be 0.44 per hectare of suitable habitat along the Maroochy River. Should future monitoring reveal an adverse change in the water mouse population on the Maroochy River, a concerted effort should be made to identify contributing factors and address proximate reasons for the decline.

Terrestrial top-predators are expected to regulate and stabilise food webs through their consumptive and non-consumptive effects on sympatric mesopredators and prey. The lethal control of top-predators has therefore been predicted to inhibit top-predator function, generate the release of mesopredators and indirectly harm native fauna through trophic cascade effects. Understanding the outcomes of lethal control on interactions within terrestrial predator guilds is important for zoologists, conservation biologists and wildlife managers. However, few studies have the capacity to test these predictions experimentally, and no such studies have previously been conducted on the eclectic suite of native and exotic, mammalian and reptilian taxa we simultaneously assess. We conducted a series of landscape-scale, multi-year, manipulative experiments at nine sites spanning five ecosystem types across the Australian continental rangelands to investigate the responses of mesopredators (red foxes, feral cats and goannas) to contemporary poison-baiting programs intended to control top-predators (dingoes) for livestock protection.

CDK7 phosphorylates the RNA polymerase II (pol II) C-terminal domain CTD and activates the P-TEFb-associated kinase CDK9, but its regulatory roles remain obscure. Here, using human CDK7 analog-sensitive (CDK7as) cells, we observed reduced capping enzyme recruitment, increased pol II promoter-proximal pausing, and defective termination at gene 3' ends upon CDK7 inhibition. We also noted that CDK7 regulates chromatin modifications downstream of transcription start sites. H3K4me3 spreading was restricted at gene 5' ends and H3K36me3 was displaced toward gene 3' ends in CDK7as cells. Mass spectrometry identified factors that bound TFIIH-phosphorylated versus P-TEFb-phosphorylated CTD (versus unmodified); capping enzymes and H3K4 methyltransferase complexes, SETD1A/B, selectively bound phosphorylated CTD, and the H3K36 methyltransferase SETD2 specifically bound P-TEFb-phosphorylated CTD. Moreover, TFIIH-phosphorylated CTD stimulated SETD1A/B activity toward nucleosomes, revealing a mechanistic basis for CDK7 regulation of H3K4me3 spreading. Collectively, these results implicate a CDK7-dependent "CTD code" that regulates chromatin marks in addition to RNA processing and pol II pausing.

The trafficking of components within cilia, called intraflagellar transport (IFT), is powered by kinesin-2 and dynein-2 motors. Loss of function in any subunit of the heterotrimeric KIF3A/KIF3B/KAP kinesin-2 motor prevents ciliogenesis in mammalian cells and has hindered an understanding of how kinesin-2 motors function in cilium assembly and IFT. We used a chemical-genetic approach to generate an inhibitable KIF3A/KIF3B/KAP kinesin-2 motor (i3A/i3B) that is capable of rescuing wild-type (WT) motor function for cilium assembly and Hedgehog signaling in Kif3a/Kif3b double-knockout cells. We demonstrate that KIF3A/KIF3B function is required not just for cilium assembly but also for cilium maintenance, as inhibition of i3A/i3B blocks IFT within 2 min and leads to a complete loss of primary cilia within 8 h. In contrast, inhibition of dynein-2 has no effect on cilium maintenance within the same time frame. The kinetics of cilia loss indicate that two processes contribute to ciliary disassembly in response to cessation of anterograde IFT: a slow shortening that is steady over time and a rapid deciliation that occurs with stochastic onset. We also demonstrate that the kinesin-2 family members KIF3A/KIF3C and KIF17 cannot rescue ciliogenesis in Kif3a/Kif3b double-knockout cells or delay the loss of assembled cilia upon i3A/i3B inhibition. These results demonstrate that KIF3A/KIF3B/KAP is the sole and essential motor for cilium assembly and maintenance in mammalian cells. These findings highlight differences in how kinesin-2 motors were adapted for cilium assembly and IFT function across species.

The Notch-regulated transcription factor mouse atonal homolog 1 (Math1) is required for the development of intestinal secretory cells, as demonstrated by the loss of goblet, endocrine and Paneth cell types in null mice. However, it was unknown whether Math1 is sufficient to induce the program of secretory cell differentiation. To examine the function of Math1 in the differentiation of intestinal epithelial cells, intestinal morphology and epithelial and mesenchymal cell fate were examined by histological staining and marker gene expression in transgenic mice expressing a villin-regulated Math1 transgene. Late prenatal transgenic founders exhibited a gross cellular transformation into a secretory epithelium. The expansion of secretory cells coupled with the almost complete loss of absorptive enterocytes suggested reprogramming of a bipotential progenitor cell. Moreover, Math1 expression inhibited epithelial cell proliferation, as demonstrated by a marked reduction in Ki67 positive cells and blunted villi. Unexpectedly, the transgenic mesenchyme was greatly expanded with increased proliferation. Several mesenchymal cell types were amplified, including smooth muscle and neurons, with maintenance of basic radial patterning. Since transgenic Math1 expression was restricted to the epithelium, these findings suggest that epithelial-mesenchymal signaling is altered by the cellular changes induced by Math1. Thus, Math1 is a key effector directing multipotential precursors to adopt secretory and not absorptive cell fate.

In extrahepatic bile duct (EHBD) cholangiopathies, including primary sclerosing cholangitis, a reactive cholangiocyte phenotype is associated with inflammation and epithelial hyperproliferation. The signaling pathways involved in EHBD injury response are poorly understood. In this study, we investigated the role of Hedgehog (HH) signaling and its downstream effectors in controlling biliary proliferation and inflammation after EHBD injury.

The tumor microenvironment is critical to cancer growth and therapy resistance. We previously characterized human ovarian carcinoma-associated mesenchymal stem cells (CA-MSCs). CA-MSCs are multi-potent cells that can differentiate into tumor microenvironment components including fibroblasts, myofibroblasts and adipocytes. We previously reported CA-MSCs, compared to normal MSCs, express high levels of BMP proteins and promote tumor growth by increasing numbers of cancer stem-like cells (CSCs). We demonstrate here that ovarian tumor cell-secreted Hedgehog (HH) induces CA-MSC BMP4 expression. CA-MSC-derived BMP4 reciprocally increases ovarian tumor cell HH expression indicating a positive feedback loop. Interruption of this loop with a HH pathway inhibitor or BMP4 blocking antibody decreases CA-MSC-derived BMP4 and tumor-derived HH preventing enrichment of CSCs and reversing chemotherapy resistance. The impact of HH inhibition was only seen in CA-MSC-containing tumors, indicating the importance of a humanized stroma. These results are reciprocal to findings in pancreatic and bladder cancer, suggesting HH signaling effects are tumor tissue specific warranting careful investigation in each tumor type. Collectively, we define a critical positive feedback loop between CA-MSC-derived BMP4 and ovarian tumor cell-secreted HH and present evidence for the further investigation of HH as a clinical target in ovarian cancer.

Vertebrate Hedgehog (HH) signaling is controlled by several ligand-binding antagonists including Patched-1 (PTCH1), PTCH2, and HH-interacting protein 1 (HHIP1), whose collective action is essential for proper HH pathway activity. However, the molecular mechanisms used by these inhibitors remain poorly understood. In this paper, we investigated the mechanisms underlying HHIP1 antagonism of HH signaling. Strikingly, we found evidence that HHIP1 non-cell-autonomously inhibits HH-dependent neural progenitor patterning and proliferation. Furthermore, this non-cell-autonomous antagonism of HH signaling results from the secretion of HHIP1 that is modulated by cell type-specific interactions with heparan sulfate (HS). These interactions are mediated by an HS-binding motif in the cysteine-rich domain of HHIP1 that is required for its localization to the neuroepithelial basement membrane (BM) to effectively antagonize HH pathway function. Our data also suggest that endogenous, secreted HHIP1 localization to HS-containing BMs regulates HH ligand distribution. Overall, the secreted activity of HHIP1 represents a novel mechanism to regulate HH ligand localization and function during embryogenesis.

The Hedgehog signaling pathway is part of the ancient developmental-evolutionary animal toolkit. Frequently co-opted to pattern new structures, the pathway is conserved among eumetazoans yet flexible and pleiotropic in its effects. The Hedgehog receptor, Patched, is transcriptionally activated by Hedgehog, providing essential negative feedback in all tissues. Our locus-wide dissections of the cis-regulatory landscapes of fly patched and mouse Ptch1 reveal abundant, diverse enhancers with stage- and tissue-specific expression patterns. The seemingly simple, constitutive Hedgehog response of patched/Ptch1 is driven by a complex regulatory architecture, with batteries of context-specific enhancers engaged in promoter-specific interactions to tune signaling individually in each tissue, without disturbing patterning elsewhere. This structure-one of the oldest cis-regulatory features discovered in animal genomes-explains how patched/Ptch1 can drive dramatic adaptations in animal morphology while maintaining its essential core function. It may also suggest a general model for the evolutionary flexibility of conserved regulators and pathways.

For homeostasis, lingual taste papilla organs require regulation of epithelial cell survival and renewal, with sustained innervation and stromal interactions. To investigate a role for Hedgehog/GLI signaling in adult taste organs we used a panel of conditional mouse models to manipulate GLI activity within epithelial cells of the fungiform and circumvallate papillae. Hedgehog signaling suppression rapidly led to taste bud loss, papilla disruption, and decreased proliferation in domains of papilla epithelium that contribute to taste cells. Hedgehog responding cells were eliminated from the epithelium but retained in the papilla stromal core. Despite papilla disruption and loss of taste buds that are a major source of Hedgehog ligand, innervation to taste papillae was maintained, and not misdirected, even after prolonged GLI blockade. Further, vimentin-positive fibroblasts remained in the papilla core. However, retained innervation and stromal cells were not sufficient to maintain taste bud cells in the context of compromised epithelial Hedgehog signaling. Importantly taste organ disruption after GLI blockade was reversible in papillae that retained some taste bud cell remnants where reactivation of Hedgehog signaling led to regeneration of papilla epithelium and taste buds. Therefore, taste bud progenitors were either retained during epithelial GLI blockade or readily repopulated during recovery, and were poised to regenerate taste buds once Hedgehog signaling was restored, with innervation and papilla connective tissue elements in place. Our data argue that Hedgehog signaling is essential for adult tongue tissue maintenance and that taste papilla epithelial cells represent the key targets for physiologic Hedgehog-dependent regulation of taste organ homeostasis. Because disruption of GLI transcriptional activity in taste papilla epithelium is sufficient to drive taste organ loss, similar to pharmacologic Hedgehog pathway inhibition, the findings suggest that taste alterations in cancer patients using systemic Hedgehog pathway inhibitors result principally from interruption of signaling activity in taste papillae.

Intestinal crypts have a remarkable capacity to regenerate after injury from loss of crypt base columnar (CBC) stem cells. After injury, facultative stem cells (FSCs) are activated to replenish the epithelium and replace lost CBCs. Our aim was to assess the role of insulin-like growth factor-1 (IGF-1) to activate FSCs for crypt repair.

aISCs (aISCs) are sensitive to acute insults including chemotherapy and irradiation. Regeneration after aISC depletion has primarily been explored in irradiation (IR). However, the cellular origin of epithelial regeneration after doxorubicin (DXR), a common chemotherapeutic, is poorly understood.

Growth arrest-specific 1 (GAS1) acts as a co-receptor to patched 1, promoting sonic hedgehog (SHH) signaling in the developing nervous system. GAS1 mutations in humans and animal models result in forebrain and craniofacial malformations, defects ascribed to a function for GAS1 in SHH signaling during early neurulation. Here, we confirm loss of SHH activity in the forebrain neuroepithelium in GAS1-deficient mice and in induced pluripotent stem cell-derived cell models of human neuroepithelial differentiation. However, our studies document that this defect can be attributed, at least in part, to a novel role for GAS1 in facilitating NOTCH signaling, which is essential to sustain a persistent SHH activity domain in the forebrain neuroepithelium. GAS1 directly binds NOTCH1, enhancing ligand-induced processing of the NOTCH1 intracellular domain, which drives NOTCH pathway activity in the developing forebrain. Our findings identify a unique role for GAS1 in integrating NOTCH and SHH signal reception in neuroepithelial cells, and they suggest that loss of GAS1-dependent NOTCH1 activation contributes to forebrain malformations in individuals carrying GAS1 mutations.

Proper Hedgehog (HH) signaling is essential for embryonic development, while aberrant HH signaling drives pediatric and adult cancers. HH signaling is frequently dysregulated in pancreatic cancer, yet its role remains controversial, with both tumor-promoting and tumor-restraining functions reported. Notably, the GLI family of HH transcription factors (GLI1, GLI2, GLI3), remain largely unexplored in pancreatic cancer. We therefore investigated the individual and combined contributions of GLI1-3 to pancreatic cancer progression. At pre-cancerous stages, fibroblast-specific Gli2/Gli3 deletion decreases immunosuppressive macrophage infiltration and promotes T cell infiltration. Strikingly, combined loss of Gli1/Gli2/Gli3 promotes macrophage infiltration, indicating that subtle changes in Gli expression differentially regulate immune infiltration. In invasive tumors, Gli2/Gli3 KO fibroblasts exclude immunosuppressive myeloid cells and suppress tumor growth by recruiting natural killer cells. Finally, we demonstrate that fibroblasts directly regulate macrophage and T cell migration through the expression of Gli-dependent cytokines. Thus, the coordinated activity of GLI1-3 directs the fibroinflammatory response throughout pancreatic cancer progression.