Sarcopenia predicts postoperative complications in head and neck cancer: a systematic review and meta-analysis.

PURPOSE: We performed this systematic review and meta-analysis to explore the impact of preoperative sarcopenia on postoperative complication risks after head and neck cancer (HNC) surgery. METHODS: We identified eligible studies by searching Ovid-MEDLINE, Ovid-Embase, EBM Reviews-Cochrane Central Register of Controlled Trials, Web of Science Core Collection, and Scopus. This systematic review was performed in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidance. RESULTS: Twenty-one studies with a total of 3480 patients met our inclusion criteria. The presence of sarcopenia significantly increased the incidence of overall postoperative complications (OR = 1.72, 95% CI 1.23, 2.41; P = 0.002; I2 = 59%). Subgroup analyses showed a higher risk of postoperative complications in the populations in which sarcopenia was diagnosed with low L3-skeletal muscle index (L3-SMI) or low cross-sectional area of the rectus femoris, but not in the group that sarcopenia was diagnosed with low C3-SMI. Preoperative sarcopenia also substantially increased the risk of severe postoperative complications (OR = 2.26), pharyngocutaneous fistulas (OR = 2.15), free flap-related complications (OR = 1.63), and surgical site infections (OR = 1.84). We also found a tendency toward a higher incidence of wound complications and 30-day mortality in patients with sarcopenia. CONCLUSION: Preoperative sarcopenia is a negative prognostic indicator for postoperative complications in patients with HNC after surgery. To reduce the incidence of postoperative complications and improve poor prognosis, further attention needs to be paid to the evaluation and management of preoperative sarcopenia.

Cuproptosis facilitates immune activation but promotes immune escape, and a machine learning-based cuproptosis-related signature is identified for predicting prognosis and immunotherapy response of gliomas.

AIMS: Cell death, except for cuproptosis, in gliomas has been extensively studied, providing novel targets for immunotherapy by reshaping the tumor immune microenvironment through multiple mechanisms. This study aimed to explore the effect of cuproptosis on the immune microenvironment and its predictive power in prognosis and immunotherapy response. METHODS: Eight glioma cohorts were included in this study. We employed the unsupervised clustering algorithm to identify novel cuproptosis clusters and described their immune microenvironmental characteristics, mutation landscape, and altered signaling pathways. We verified the correlation among FDX1, SLC31A1, and macrophage infiltration in 56 glioma tissues. Next, based on multicenter cohorts and 10 machine learning algorithms, we constructed an artificial intelligence-driven cuproptosis-related signature named CuproScore. RESULTS: Our findings suggested that glioma patients with high levels of cuproptosis had a worse prognosis owing to immunosuppression caused by unique immune escape mechanisms. Meanwhile, we experimentally validated the positive association between cuproptosis and macrophages and its tumor-promoting mechanism in vitro. Furthermore, our CuproScore exhibited powerful and robust prognostic predictive ability. It was also capable of predicting response to immunotherapy and chemotherapy drug sensitivity. CONCLUSIONS: Cuproptosis facilitates immune activation but promotes immune escape. The CuproScore could predict prognosis and immunotherapy response in gliomas.

Evaluation of risk factors for surgical site infections in osteoarthritis patients undergoing total knee arthroplasty.

This research sought to delineate risk factors associated with surgical site infections (SSIs) post-total knee arthroplasty (post-TKA) in elderly osteoarthritis patients, aiming to enhance post-surgical outcomes. A retrospective examination was conducted on a cohort of 650 elderly patients who underwent unilateral TKA between January 2018 and August 2022. Data procurement was from the hospital's Electronic Health Record, and a comprehensive statistical evaluation was performed using IBM SPSS Statistics version 24.0. Both univariate and multivariate techniques assessed a spectrum of risk determinants such as age, body mass index (BMI), coexisting medical conditions and surgical variables. The univariate examination spotlighted age, BMI, diabetes prevalence, chronic corticosteroid consumption and American Society of Anesthesiologists (ASA) physical status classification as notable predictors of SSIs. The multivariate logistic regression pinpointed age, BMI, history of smoking and diabetes diagnosis as salient risk attributors for post-TKA infections. Concurrently, parameters like ASA classification, surgical duration and intraoperative haemorrhage further enriched the risk landscape. Geriatric patients undergoing TKA for knee osteoarthritis manifest a tangible infection susceptibility post-surgery. Precision interventions concentrating on amendable risk components, including meticulous preoperative evaluations and strategic postoperative care, are imperative to attenuate SSI incidence, thereby amplifying surgical efficacy and optimizing patient recuperation trajectories.

Inhibition of CCR1 attenuates neuroinflammation via the JAK2/STAT3 signaling pathway after subarachnoid hemorrhage.

BACKGROUND AND PURPOSE: Neuroinflammation is an important mechanism underlying brain injury caused by subarachnoid hemorrhage (SAH). C-C chemokine receptor type 1 (CCR1)-mediated inflammation is involved in the pathology of many central nervous system diseases. Herein, we investigated whether inhibition of CCR1 alleviated neuroinflammation after experimental SAH and aimed to elucidate the mechanisms of its potential protective effects. METHODS: To analyze SAH transcriptome data R studio was used, and a mouse model of SAH was established using endovascular perforations. In this model, the selective CCR1 antagonist Met-RANTES (Met-R) and the CCR1 agonist recombinant CCL5 (rCCL5) were administered 1 h after SAH induction. To investigate the possible downstream mechanisms of CCR1, the JAK2 inhibitor AG490 and the JAK2 activator coumermycin A1 (C-A1) were administered 1 h after SAH induction. Furthermore, post-SAH evaluation, including SAH grading, neurological function tests, Western blot, the terminal deoxynucleotidyl transferase dUTP nick end labeling assay, and Fluoro-Jade B and fluorescent immunohistochemical staining were performed. Cerebrospinal fluid (CSF) samples were detected by ELISA. RESULTS: CCL5 and CCR1 expression levels increased significantly following SAH. Met-R significantly improved neurological deficits in mice, decreased apoptosis and degeneration of ipsilateral cerebral cortex neurons, reduced infiltrating neutrophils, and promoted microglial activation after SAH induction. Furthermore, Met-R inhibited the expression of p-JAK2, p-STAT3, interleukin-1ß, and tumor necrosis factor-α. However, the protective effects of Met-R were abolished by C-A1 treatment. Furthermore, rCCL5 injection aggravated neurological dysfunction and increased the expression of p-JAK2, p-STAT3, interleukin-1ß, and tumor necrosis factor-α in SAH mice, all of which were reversed by the administration of AG490. Finally, the levels of CCL5 and CCR1 were elevate in the CSF of SAH patient and high level of CCL5 and CCR1 levels were associated with poor outcome. CONCLUSION: The present results suggested that inhibition of CCR1 attenuates neuroinflammation after SAH via the JAK2/STAT3 signaling pathway, which may provide a new target for the treatment of SAH.

Establishment of Bactrian Camel Induced Pluripotent Stem Cells and Prediction of Their Unique Pluripotency Genes.

Induced pluripotent stem cells (iPSCs) can differentiate into all types of cells and can be used in livestock for research on biological development, genetic breeding, and in vitro genetic resource conservation. The Bactrian camel is a large domestic animal that inhabits extreme environments and holds value in the treatment of various diseases and the development of the local economy. Therefore, we transferred four mouse genes (Oct4, Sox2, Klf4, and c-Myc) into Bactrian camel fetal fibroblasts (BCFFs) using retroviruses with a large host range to obtain Bactrian camel induced pluripotent stem cells (bciPSCs). They were comprehensively identified based on cell morphology, pluripotency gene and marker expression, chromosome number, transcriptome sequencing, and differentiation potential. The results showed the pluripotency of bciPSCs. However, unlike stem cells of other species, late formation of stem cell clones was observed; moreover, the immunofluorescence of SSEA1, SSEA3, and SSEA4 were positive, and teratoma formation took four months. These findings may be related to the extremely long gestation period and species specificity of Bactrian camels. By mining RNA sequence data, 85 potential unique pluripotent genes of Bactrian camels were predicted, which could be used as candidate genes for the production of bciPSC in the future. Among them, ASF1B, DTL, CDCA5, PROM1, CYTL1, NUP210, Epha3, and SYT13 are more attractive. In conclusion, we generated bciPSCs for the first time and obtained their transcriptome information, expanding the iPSC genetic information database and exploring the applicability of iPSCs in livestock. Our results can provide an experimental basis for Bactrian camel ESC establishment, developmental research, and genetic resource conservation.

NLRP3 Knockout Protects against Lung Injury Induced by Cerebral Ischemia-Reperfusion.

Methods: C57/BL6 wild-type (WT) and NLRP3-KO mice were used to construct middle cerebral artery occlusion (MCAO) models. 2,3,5-Triphenyltetrazolium chloride (TTC) was used to evaluate brain damage, and neurological deficits were assessed. Then, lung tissue injury was examined in the different groups of mice by hematoxylin-eosin (HE) staining. Inflammation (macrophage and neutrophil infiltration, NLRP3-associated inflammatory molecules) and oxidative stress (reactive oxygen species, ROS) in the lungs were comprehensively examined by immunofluorescence staining and Western blotting. Results: First, our findings demonstrated that NLRP3 knockout had a protective effect against cerebral ischemia-reperfusion injury after MCAO. Second, by reducing brain damage after MCAO, lung inflammation was also alleviated. Immunofluorescence staining showed that NLRP3-KO-MCAO mice had reduced inflammatory effector molecule (caspase-1 and IL-1ß) expression and macrophage and neutrophil infiltration in the lung, as well as remissive oxidative stress state in the lung, compared with WT-MCAO mice. We also observed a decrease in phosphorylated p65 (p-p65) (an NF-κB factor) in NLRP3-KO-MCAO mice, suggesting that the NF-κB pathway was involved in the protective effect of NLRP3 gene knockout on stroke-induced lung injury. Conclusions: NLRP3 inflammasome knockout not only is beneficial for cerebral ischemia-reperfusion injury but also reduces the severity of poststroke lung injury by reducing brain damage. It has been confirmed that there is a relationship between central insult and peripheral organ injury, and protecting the brain can prevent peripheral organ damage.

Systematic review with meta-analysis: effectiveness of hyperbaric oxygenation therapy for ulcerative colitis.

BACKGROUND AND AIMS: Hyperbaric oxygenation therapy has been used in the treatment of ulcerative colitis in the past few years. However, its efficacy still remains unclear. The aim of the study was to investigate the efficacy of hyperbaric oxygen combination therapy in patients with ulcerative colitis. METHODS: We conducted a comprehensive study search up to September 2020, from the online databases Embase, PubMed, Cochrane Library, China National Knowledge Infrastructure, WanFang and VIP. RESULTS: Thirteen studies comprising 780 patients were included. We found that compared with conventional therapy, hyperbaric oxygen combination therapy was superior in reaching clinical remission [risk ratio (RR)=1.62; 95% confidence interval (CI) 1.42 to 1.84; p < 0.001] and clinical response (RR=1.29; 95% CI 1.21 to 1.38; p < 0.001), with lower disease activity scores [standard mean difference (SMD)= -1.19; 95%CI -1.74 to -0.65; p < 0.001]. An obvious reduction of serum levels of tumor necrosis factor-α (SMD= -1.96; 95%CI -2.50 to -1.41; p < 0.001) and interleukin (IL)-6 (SMD= -2.49; 95% CI -2.84 to -2.15; p < 0.001), and elevation of IL-10 level (SMD=2.40; 95% CI 0.68 to 4.12; p = 0.006) were also observed. CONCLUSION: Hyperbaric oxygen combination therapy was effective in patients with ulcerative colitis, and has potential as a complementary method for its treatment.

Prognostic value of the pretreatment systemic immune-inflammation index (SII) in patients with non-small cell lung cancer: a meta-analysis.

BACKGROUND: The objective of this study is to explore the association between the pretreatment systemic immune-inflammation index (SII) and prognosis in non-small cell lung cancer (NSCLC) patients. METHODS: A systemic literature search of PubMed, EMBASE, the Web of Science, the Cochrane Library, China National Knowledge Infrastructure (CNKI), Wanfang, VIP and SinoMed databases was performed from January 1, 1966 to April 15, 2019, to identify potential studies that assessed the prognostic role of the pretreatment SII in NSCLC. The hazard ratio (HR) and 95% confidence interval (CI) were combined to evaluate the correlation of the pretreatment SII with overall survival (OS), disease-free survival (DFS), progression-free survival (PFS) and cancer-specific survival (CSS) in NSCLC patients. RESULTS: A total of 9 studies involving 2,441 patients were eventually included. An elevated pretreatment SII indicated significantly poorer OS (HR =1.88, 95% CI: 1.50-2.36; P<0.001) with high heterogeneity (I2=60.6%, P=0.019), DFS/PFS (HR =2.50, 95% CI: 1.20-5.20; P=0.014) with high heterogeneity (I2=58.2%, P=0.092) and CSS (HR =1.852, 95% CI: 1.185-2.915; P=0.007). Subgroup analyses further verified the above results. In addition, compared with the neutrophil to lymphocyte ratio (NLR) and the platelet to lymphocyte ratio (PLR), the SII showed a much higher prognostic value in NSCLC. CONCLUSIONS: The pretreatment SII may serve as a useful prognostic indicator in NSCLC and contribute to prognosis evaluation and treatment strategy formulation. However, more well-designed studies are warranted to verify our findings.

Tissue-Specific Oncogenic Activity of KRASA146T.

KRAS is the most frequently mutated oncogene. The incidence of specific KRAS alleles varies between cancers from different sites, but it is unclear whether allelic selection results from biological selection for specific mutant KRAS proteins. We used a cross-disciplinary approach to compare KRASG12D, a common mutant form, and KRASA146T, a mutant that occurs only in selected cancers. Biochemical and structural studies demonstrated that KRASA146T exhibits a marked extension of switch 1 away from the protein body and nucleotide binding site, which activates KRAS by promoting a high rate of intrinsic and guanine nucleotide exchange factor-induced nucleotide exchange. Using mice genetically engineered to express either allele, we found that KRASG12D and KRASA146T exhibit distinct tissue-specific effects on homeostasis that mirror mutational frequencies in human cancers. These tissue-specific phenotypes result from allele-specific signaling properties, demonstrating that context-dependent variations in signaling downstream of different KRAS mutants drive the KRAS mutational pattern seen in cancer. SIGNIFICANCE: Although epidemiologic and clinical studies have suggested allele-specific behaviors for KRAS, experimental evidence for allele-specific biological properties is limited. We combined structural biology, mass spectrometry, and mouse modeling to demonstrate that the selection for specific KRAS mutants in human cancers from different tissues is due to their distinct signaling properties.See related commentary by Hobbs and Der, p. 696.This article is highlighted in the In This Issue feature, p. 681.

Orthodontic tooth movement: The biology and clinical implications.

Orthodontic tooth movement relies on coordinated tissue resorption and formation in the surrounding bone and periodontal ligament. Tooth loading causes local hypoxia and fluid flow, initiating an aseptic inflammatory cascade culminating in osteoclast resorption in areas of compression and osteoblast deposition in areas of tension. Compression and tension are associated with particular signaling factors, establishing local gradients to regulate remodeling of the bone and periodontal ligament for tooth displacement. Key regulators of inflammation and tissue turnover include secreted factors like RANK ligand and osteoprotegerin, transcription factors such as RUNX2 and hypoxia-inducible factor, cytokines, prostaglandins, tissue necrosis factors, and proteases, among others. Inflammation occurred during tooth movement needs to be well controlled, as dysregulated inflammation leads to tissue destruction manifested in orthodontic-induced root resorption and periodontal disease. Understanding the biology has profound clinical implications especially in the area of accelerating orthodontic tooth movement. Surgical, pharmacological, and physical interventions are being tested to move teeth faster to reduce treatment times and time-dependent adverse outcomes. Future developments in acceleratory technology and custom appliances will allow orthodontic tooth movement to occur more efficiently and safely.

Continuous and scalable fabrication of stable and biocompatible MOF@SiO2 nanoparticles for drug loading.

Metal-organic frameworks (MOFs) have been widely used for drug/dye loading, but the large scale continuous production of these nanoparticles with good stability and biocompatibility still faces great challenges. Herein, a thermal-assisted microfluidic system was developed for the continuous and scalable fabrication of drug loaded MOFs@SiO2 nanoparticles with uniform shape, narrow size distribution, good stability and excellent biocompatibility. This facile and general strategy is readily employed in the encapsulation of many dyes and drugs. In vivo photodynamic therapy for cancers, based on rose bengal-loaded nanoparticles, demonstrates the great potential of this novel fabrication strategy in biomedical fields.

Dopaminergic enhancement of cellular adhesion in bone marrow derived mesenchymal stem cells (MSCs).

Dopamine (DA) is a well-known neurotransmitter and critical element in the mussel adhesive protein that has gained increasing attention for its role in cellular growth enhancement in biomaterials, including cellular adhesion improvement. As the mechanism underlying this remains unclear, the objective of this study was to explore the effects of DA on the adhesion properties of bone marrow derived rat mesenchymal stem cells (rMSCs) using an hydroxyapatite gelatin nanocomposite biomaterial and to test whether the effects are mediated through various endogenously expressed DA receptors. Primary rMSCs were pretreated with D1-like antagonist, D2-like antagonist, or a combination of these antagonists followed by treatment with 50 µM DA and cellular adhesion quantification at 0.5, 1, 2 and 4 hours post DA addition. DA was found to increase rMSC adhesion and spreading at the 0.5 hour time-point and the dopaminergic effect on cell adhesion was partially blocked by DA antagonists. In addition, the D1-like and D2-like antagonists appeared to have a similar effect on rMSCs. Immunofluorescent staining indicated that the rMSC spreading area was significantly increased in the DA treated group versus the control group. Treatment of the D1-like DA antagonists with DA revealed that the actin filaments of rMSCs could not connect the membrane with the nucleus. In summary, DA was found to enhance early rMSC adhesion partially via DA receptor activation.

Using a new Lrig1 reporter mouse to assess differences between two Lrig1 antibodies in the intestine.

Lrig1 is an intestinal stem cell marker important for epithelial homeostasis. However, the position of the Lrig1(+) population in the intestinal crypt has been debated, largely due to discrepant staining patterns using two Lrig1 antibodies. Here, we set out to decipher the differences between these Lrig1 antibodies to clarify their use for Lrig1-related studies. We confirmed that the commercially available Lrig1-R&D antibody stained the bottom third of the colonic crypt, whereas an independently generated Lrig1-VU antibody recognized a subset of anti-Lrig1-R&D(+) cells. Biochemically, we found that anti-Lrig1-VU recognized a non-glycosylated form of Lrig1; in contrast, anti-Lrig1-R&D recognized both glycosylated and non-glycosylated forms of Lrig1. In addition, we generated a reporter mouse (Lrig1-Apple) as an independent readout of Lrig1 transcriptional activity. Flow cytometry of isolated colonic epithelial cells from Lrig1-Apple mice demonstrated anti-Lrig1-R&D recognized mostly RFP-hi cells, while anti-Lrig1-VU recognized cells that were largely RFP-mid. Of note, by qRT-PCR, Lgr5 was expressed in the RFP-hi population, but not in the RFP-mid population. We conclude that anti-Lrig1-R&D appears to recognize all Lrig1(+) cells, while anti-Lrig1-VU recognizes a subpopulation of Lrig1(+) cells.

The pan-ErbB negative regulator Lrig1 is an intestinal stem cell marker that functions as a tumor suppressor.

Lineage mapping has identified both proliferative and quiescent intestinal stem cells, but the molecular circuitry controlling stem cell quiescence is incompletely understood. By lineage mapping, we show Lrig1, a pan-ErbB inhibitor, marks predominately noncycling, long-lived stem cells that are located at the crypt base and that, upon injury, proliferate and divide to replenish damaged crypts. Transcriptome profiling of Lrig1(+) colonic stem cells differs markedly from the profiling of highly proliferative, Lgr5(+) colonic stem cells; genes upregulated in the Lrig1(+) population include those involved in cell cycle repression and response to oxidative damage. Loss of Apc in Lrig1(+) cells leads to intestinal adenomas, and genetic ablation of Lrig1 results in heightened ErbB1-3 expression and duodenal adenomas. These results shed light on the relationship between proliferative and quiescent intestinal stem cells and support a model in which intestinal stem cell quiescence is maintained by calibrated ErbB signaling with loss of a negative regulator predisposing to neoplasia.

Mutational analysis of NOG in esophageal atresia and tracheoesophageal fistula patients.

PURPOSE: The NOG protein is a secretory antagonist of bone morphogenetic proteins (BMPs). Nog-/- mouse embryos demonstrate proximal esophageal atresia (EA) and distal tracheoesophageal fistula (TEF) compatible with the most common configuration of EA/TEF observed in humans. Four microdeletions that span the NOG locus at 17q22 have been described in human patients having EA/TEF. We investigated the incidence of point mutations in the coding region of the NOG gene in human EA/TEF. METHODS: DNA was collected from 50 patients previously treated for EA/TEF. PCR was used to amplify the coding region of NOG. To detect single nucleotide polymorphisms (SNPs), amplicons were subjected to temperature gradient capillary electrophoresis (TGCE). Candidate SNPs were directly sequenced. RESULTS: TGCE analysis revealed a SNP in the coding region of NOG in 1 of 50 patients (2%). DNA sequencing revealed a synonymous SNP at position 468 (C-T) of the NOG coding region. CONCLUSION: SNPs in the coding region of the NOG gene are identified infrequently in human cases of EA/TEF. Further investigation of SNPs in the promoter region of NOG is warranted, as is the effect of synonymous SNPs on NOG mRNA stability.

Bmp4 is required for tracheal formation: a novel mouse model for tracheal agenesis.

Tracheal agenesis/atresia (TA) is a rare but fatal congenital disease in which the breathing tube fails to grow. The etiology of this serious condition remains largely unknown. We found that Bmp signaling is prominently present in the anterior foregut where the tracheal primordium originates and targeted ablation of Bmp4 (Bmp4(cko)) resulted in a loss-of-trachea phenotype that closely resembles the Floyd type II pathology, the most common form of TA in humans. In Bmp4(cko) embryos, tracheal specification was not affected; however, its outgrowth was severely impaired due to reduced epithelial and mesenchymal proliferation. In agreement, we also observed significant reduction in the expression of Cyclin D1, a key cell cycle regulator associated with cellular proliferation. However, the proliferative effect of Bmp signaling appears to be independent of Wnt signaling. Interestingly, we found significantly reduced expression of activated extracellular signal-regulated kinase (Erk) in the Bmp4(cko) ventral foregut, suggesting that Bmp signaling promotes Erk phosphorylation which has been associated with cellular proliferation. This study provides the first evidence linking Bmp signaling to tracheal formation by regulating the proliferative response of the anterior ventral foregut. Our finding sheds light on human tracheal malformations by providing a novel mouse model implicating Bmp signaling, non-canonical Erk activation and cellular proliferation.

Cholesterol modification restricts the spread of Shh gradient in the limb bud.

Sonic hedgehog (Shh) produced in the zone of polarizing activity is the major determinant of anteroposterior development of the amniote limb. The mature and active Shh protein is cholesterol-modified at its C terminus, and the hydrophobic nature of the modification requires the function of Dispatched (mDispA), a seven-pass transmembrane protein, for Shh release from its source. The current model suggests that the cholesterol moiety promotes the spread of Shh gradient in the limb bud. However, this model is inconsistent with findings in Drosophila and not in line with current thoughts on the role of the cholesterol moiety in Shh multimerization. Therefore, it remains unclear how the cholesterol moiety affects the postrelease extracellular behavior of Shh that relates to the shape of its activity gradient in responsive tissues. Here, we report functional analyses in mice showing that Shh lacking cholesterol modification (ShhN) has an increased propensity to spread long-distance, eliciting ectopic Shh pathway activation consistent with target gene expressions and modulating the level of Gli3 processing in the anterior limb mesoderm. These molecular alterations are reflected in the mispatterning of digits in ShhN mutants. Additionally, we provide direct evidence for the long-distance movement of ShhN across the anteroposterior axis of the limb bud. Our findings suggest that the cholesterol moiety regulates the range and shape of the Shh morphogen gradient by restricting rather than promoting the postrelease spread of Shh across the limb bud during early development.

Sonic hedgehog signaling regulates Gli3 processing, mesenchymal proliferation, and differentiation during mouse lung organogenesis.

Lack of Sonic hedgehog (Shh) signaling, mediated by the Gli proteins, leads to severe pulmonary hypoplasia. However, the precise role of Gli genes in lung development is not well established. We show Shh signaling prevents Gli3 proteolysis to generate its repressor forms (Gli3R) in the developing murine lung. In Shh(-/-) or cyclopamine-treated wild-type (WT) lung, we found that Gli3R level is elevated, and this upregulation appears to contribute to defects in proliferation and differentiation observed in the Shh(-/-) mesenchyme, where Gli3 is normally expressed. In agreement, we found Shh(-/-);Gli3(-/-) lungs exhibit enhanced growth potential. Vasculogenesis is also enhanced; in contrast, bronchial myogenesis remains absent in Shh(-/-);Gli3(-/-) compared with Shh(-/-) lungs. Genes upregulated in Shh(-/-);Gli3(-/-) relative to Shh(-/-) lung include Wnt2 and, surprisingly, Foxf1 whose expression has been reported to be Shh-dependent. Cyclins D1, D2, and D3 antibody labelings also reveal distinct expression patterns in the normal and mutant lungs. We found significant repression of Tbx2 and Tbx3, both linked to inhibition of cellular senescence, in Shh(-/-) and partial derepression in Shh(-/-); Gli3(-/-) lungs, while Tbx4 and Tbx5 expressions are less affected in the mutants. Our findings shed light on the role of Shh signaling on Gli3 processing in lung growth and differentiation by regulating several critical genes.

Shh and Gli3 are dispensable for limb skeleton formation but regulate digit number and identity.

Most current models propose Sonic hedgehog (Shh) as the primary determinant of anteroposterior development of amniote limbs. Shh protein is said to be required to direct the formation of skeletal elements and to specify digit identity through dose-dependent activation of target gene expression. However, the identity of genes targeted by Shh, and the regulatory mechanisms controlling their expression, remain poorly understood. Gli3 (the gene implicated in human Greig cephalopolysyndactyly syndrome) is proposed to negatively regulate Shh by restricting its expression and influence to the posterior mesoderm. Here we report genetic analyses in mice showing that Shh and Gli3 are dispensable for formation of limb skeletal elements: Shh(-/-) Gli3(-/-) limbs are distally complete and polydactylous, but completely lack wild-type digit identities. We show that the effects of Shh signalling on skeletal patterning and ridge maintenance are necessarily mediated through Gli3. We propose that the function of Shh and Gli3 in limb skeletal patterning is limited to refining autopodial morphology, imposing pentadactyl constraint on the limb's polydactyl potential, and organizing digit identity specification, by regulating the relative balance of Gli3 transcriptional activator and repressor activities.