ZNRF3 and RNF43 are closely related transmembrane E3 ubiquitin ligases with significant roles in development and cancer. Conventionally, their biological functions have been associated with regulating WNT signaling receptor ubiquitination and degradation. However, our proteogenomic studies have revealed EGFR as the protein most negatively correlated with ZNRF3/RNF43 mRNA levels in multiple human cancers. Through biochemical investigations, we demonstrate that ZNRF3/RNF43 interact with EGFR via their extracellular domains, leading to EGFR ubiquitination and subsequent degradation facilitated by the E3 ligase RING domain. Overexpression of ZNRF3 reduces EGFR levels and suppresses cancer cell growth in vitro and in vivo, whereas knockout of ZNRF3/RNF43 stimulates cell growth and tumorigenesis through upregulated EGFR signaling. Together, these data suggest ZNRF3 and RNF43 as novel E3 ubiquitin ligases of EGFR and establish the inactivation of ZNRF3/RNF43 as a driver of increased EGFR signaling, ultimately promoting cancer progression. This discovery establishes a connection between two fundamental signaling pathways, EGFR and WNT, at the level of cytoplasmic membrane receptors, uncovering a novel mechanism underlying the frequent co-activation of EGFR and WNT signaling in development and cancer.

Wnt receptors of the Frizzled (Fzd) family are widely considered to exhibit substantial functional redundancy, complicating efforts to therapeutically target individual receptors. Fzd2 was believed to be functionally redundant with Fzd1 and Fzd7, based on previously published global knockout mouse studies. By contrast, homozygosity for a Fzd2 global knockout mouse allele developed by the International Mouse Phenotype Consortium (IMPC) has been reported to cause embryonic lethality, suggesting that Fzd2 is critical for early embryonic development. If global deletion of Fzd2 leads to early lethality, conditional deletion models are necessary to identify tissue-specific phenotypes. We found that a previously published Fzd2 conditional deletion model does not eliminate Fzd2. We have generated a new conditional model to address the contradictory previous studies and allow tissue-specific studies of Fzd2. We successfully inserted two loxP sites around the Fzd2 gene and confirmed that subsequent Cre-mediated recombination creates a Fzd2 null allele. Global deletion of Fzd2 in this model does not cause embryonic lethality while limb-specific deletion causes limb shortening. This work supports the hypothesis that Fzd2 regulates limb development and emphasizes the importance of thoroughly validating newly generated mouse models.

RNF43 and ZNRF3 are transmembrane E3 ubiquitin ligases that negatively regulate Wnt signaling by promoting ubiquitination and degradation of Frizzled receptors. Loss of either gene enhances Wnt/β-catenin signaling and has been linked to tumorigenesis. Wnt signaling is a key regulator of skeletal development and bone homeostasis, and pharmacologic activation of this pathway is an established therapy for osteoporosis. In Xenopus laevis, simultaneous disruption of rnf43 and znrf3 results in supernumerary limb formation; however, their roles in mammalian limb development and skeletal maintenance remain unclear. We demonstrate that mice homozygous for null alleles of both Rnf43 and Znrf3 do not develop supernumerary limbs. Because activation of Wnt/β-catenin signaling in osteoblasts increases bone mass, we hypothesized that osteoblast-specific deletion of Rnf43 and/or Znrf3 would produce a high-bone-mass phenotype. Instead, osteoblast-specific loss of Znrf3 resulted in age- and sex-dependent reductions in trabecular bone mass, characterized by decreased bone mineral density and bone volume fraction, reduced trabecular number, and increased trabecular separation. Cortical bone exhibited increased cross-sectional size with reduced cortical area fraction and altered structural properties, while tissue mineral density was unchanged. In contrast, deletion of Rnf43 had minimal skeletal effects, and combined deletion of both genes did not exacerbate the phenotype observed with loss of Znrf3 alone. These findings support the conclusion that deletion of Znrf3 produces skeletal outcomes distinct from constitutive β-catenin activation and identify ZNRF3 as the predominant RNF43/ZNRF3 family member regulating bone architecture in mature osteoblasts.

The WNT5A-mediated Wnt/Planar Cell Polarity (Wnt/PCP) pathway plays a key role in vertebrate development, particularly in limb morphogenesis. Robinow Syndrome (RS) is a rare genetic disorder characterized primarily by craniofacial malformations and limb shortening that is linked to mutations in multiple Wnt/PCP genes. The pathogenic WNT5A point mutation, Cys83Ser (C83S), is one of the most-studied RS-associated variants to date. It has been described as a loss-of-function, hypomorphic, or dominant-negative variant based on overexpression studies in vitro and in vivo. However, a mammalian model that mimics the C83S condition in human RS patients has not yet been established, and methods to distinguish between Wnt/PCP loss-of-function and gain-of-function in vivo phenotypes remain limited. In this study, we present a novel image-based method, local misalignment score (LMS), for in situ visualization and quantification of cell alignment within long bones during late embryonic development, providing a reliable and specific in vivo readout of aberrant Wnt/PCP-associated phenotypes. Using this method to assess chondrocyte orientation across mouse limb regions, we found that the heterozygous germline Wnt5a-C83S point mutation in mice induces profound chondrocyte orientation defects. This phenotype is distinct from the disrupted chondrocyte orientation with spatially patterned severity observed in homozygous Wnt5a conditional knockout (Wnt5a-cKO) limbs, which represent a Wnt5a loss-of-function model, and from those in Wnt5a-LSL knock-in limbs, where ectopic Wnt5a expression disrupts the endogenous gradient rather than producing a true Wnt5a gain-of-function effect. We further performed a comprehensive in vitro analysis of Wnt5a-C83S in C3H10T1/2 cells using a luciferase-based KIF26B reporter system along with other established Wnt signaling readouts. The results show that the C83S mutation does not exert dominant-negative effects on Wnt/PCP signaling, consistent with our in vivo findings. In summary, our work provides new insights into the putative gain-of-function or neomorphic nature of the RS-related WNT5A mutation and its impact on WNT5A gradient-dependent limb development. We highlight the reliability of LMS as an in vivo morphological measure of chondrocyte orientation that reveals defects linked to aberrant Wnt/PCP activity. When combined with other spatially resolved readouts, LMS enables location-based evaluation of pathogenic mechanisms.

Low-density lipoprotein-related receptor 5 (LRP5) is an LDLR family member with well-defined roles in mediating Wnt signaling. Its domain structure includes four LDLR class B and three LDLR class A repeats. Class B repeats mediate binding with Wnt ligands and other effectors, while the role of the LRP5 class A repeats, known to interact with apolipoproteins within the LDLR, is unclear. Complete loss of the LRP5 gene in humans causes osteoporosis pseudoglioma, a syndrome characterized by early-onset osteoporosis and changes in retinal vascularization. We and others have previously created mice and rats completely deficient in LRP5 and reported the presence of bone and retinal vascularization defects. In this study, we created an allele of Lrp5 in mice in which the entire protein except for the class A repeats is present and expressed from the endogenous locus. Unlike in vitro studies using ectopic overexpression of LRP5, our in vivo data demonstrate that the class A repeats are essential for several normal LRP5 functions, including bone homeostasis, retinal vascularization and mammary gland development - phenotypes similar to those observed in Lrp5 null mice.

One critical mechanism through which prostate cancer (PCa) adapts to treatments targeting androgen receptor (AR) signaling is the emergence of ligand-binding domain-truncated and constitutively active AR splice variants, particularly AR-V7. While AR-V7 has been intensively studied, its ability to activate distinct biological functions compared with the full-length AR (AR-FL), and its role in regulating the metastatic progression of castration-resistant PCa (CRPC), remain unclear. Our study found that, under castrated conditions, AR-V7 strongly induced osteoblastic bone lesions, a response not observed with AR-FL overexpression. Through combined ChIP-seq, ATAC-seq, and RNA-seq analyses, we demonstrated that AR-V7 uniquely accesses the androgen-responsive elements in compact chromatin regions, activating a distinct transcription program. This program was highly enriched for genes involved in epithelial-mesenchymal transition and metastasis. Notably, we discovered that SOX9, a critical metastasis driver gene, was a direct target and downstream effector of AR-V7. Its protein expression was dramatically upregulated in AR-V7-induced bone lesions. Moreover, we found that Ser81 phosphorylation enhanced AR-V7's pro-metastasis function by selectively altering its specific transcription program. Blocking this phosphorylation with CDK9 inhibitors impaired the AR-V7-mediated metastasis program. Overall, our study has provided molecular insights into the role of AR splice variants in driving the metastatic progression of CRPC.

Supplementary Figures 1-4 from Mammalian Target of Rapamycin–Dependent Acinar Cell Neoplasia after Inactivation of Apc and Pten in the Mouse Salivary Gland: Implications for Human Acinic Cell Carcinoma

Supplementary Figure Legend from Activation of the PI3K/AKT Pathway Induces Urothelial Carcinoma of the Renal Pelvis: Identification in Human Tumors and Confirmation in Animal Models

Loss of heterozygosity and promoter hypermethylation of APC is frequently observed in human endometrial cancer, which is the most common gynecological cancer in the USA, but its carcinogenic driver status in the endometrial epithelium has not been confirmed. We have identified a novel population of progenitor endometrial epithelial cells (EECs) in mice that express lysozyme M (LysM) and give rise to approximately 15% of all EECs in adult mice. LysM is a glycoside hydrolase that is encoded by Lyz2 and functions to protect cells from bacteria as part of the innate immune system. Its expression has been shown in a subset of hematopoietic stem cells and in specialized lung and small intestinal epithelial cells. Conditional deletion of Apc in LysM + EECs results in significantly more epithelial cells compared to wild-type mice. At 5 months of age, the ApccKO mice have enlarged uterine horns with pathology that is consistent with endometrial hyperplasia with cystic endometrial glands, non-villous luminal papillae and nuclear atypia. Nuclear accumulation of β-catenin and ERα, both of which are known to induce endometrial hyperplasia, was observed in the EECs of the ApccKO mice. These results confirm that loss of APC in EECs can result in a phenotype similar to endometrial hyperplasia.

Supplementary Table 1 from Activation of the PI3K/AKT Pathway Induces Urothelial Carcinoma of the Renal Pelvis: Identification in Human Tumors and Confirmation in Animal Models

Supplementary Methods and Materials from Rad6B Is a Positive Regulator of β-Catenin Stabilization

The requirement for LRP5 and LRP6 to prevent β-catenin degradation in the absence of the tumor suppressor APC is unclear because cell culture models have yielded conflicting results. We previously established that osteoblast-specific loss of APC causes β-catenin accumulation and increased bone mass, while loss of both LRP5 and LRP6 reduces bone mass. We report here that the simultaneous loss of APC, LRP5, and LRP6 in osteoblasts in mice phenocopies the APC osteoblast-specific knockout. Thus, β-catenin stabilization and increased bone mass after loss of APC in osteoblasts in vivo are not dependent on LRP5 and LRP6.

Frizzled 2 (FZD2) is a transmembrane Wnt receptor. We previously identified a pathogenic human FZD2 variant in individuals with FZD2-associated autosomal dominant Robinow syndrome. The variant encoded a protein with a premature stop and loss of 17 amino acids, including a region of the consensus dishevelled-binding sequence. To model this variant, we used zygote microinjection and i-GONAD-based CRISPR/Cas9-mediated genome editing to generate a mouse allelic series. Embryos mosaic for humanized Fzd2W553* knock-in exhibited cleft palate and shortened limbs, consistent with patient phenotypes. We also generated two germline mouse alleles with small deletions: Fzd2D3 and Fzd2D4. Homozygotes for each allele exhibit a highly penetrant cleft palate phenotype, shortened limbs compared with wild type and perinatal lethality. Fzd2D4 craniofacial tissues indicated decreased canonical Wnt signaling. In utero treatment with IIIC3a (a DKK inhibitor) normalized the limb lengths in Fzd2D4 homozygotes. The in vivo replication represents an approach for further investigating the mechanism of FZD2 phenotypes and demonstrates the utility of CRISPR knock-in mice as a tool for investigating the pathogenicity of human genetic variants. We also present evidence for a potential therapeutic intervention.

Supplementary Figure 5 from Mammalian Target of Rapamycin–Dependent Acinar Cell Neoplasia after Inactivation of Apc and Pten in the Mouse Salivary Gland: Implications for Human Acinic Cell Carcinoma

Supplementary Figures 1-2 from Inactivation of Apc in the Mouse Prostate Causes Prostate Carcinoma

Supplementary Table 2 from Activation of the PI3K/AKT Pathway Induces Urothelial Carcinoma of the Renal Pelvis: Identification in Human Tumors and Confirmation in Animal Models

The ability to create genetically engineered mouse models (GEMMs) has exponentially increased our understanding of many areas of biology. Musculoskeletal biology is no exception. In this review, we will first discuss the historical development of GEMMs and how these developments have influenced musculoskeletal disease research. This review will also update our 2008 review that appeared in BONEKey, a journal that is no longer readily available online. We will first review the historical development of GEMMs in general, followed by a particular emphasis on the ability to perform tissue-specific (conditional) knockouts focusing on musculoskeletal tissues. We will then discuss how the development of CRISPR/Cas-based technologies during the last decade has revolutionized the generation of GEMMs.

Proper regulation of Wnt signaling is critical for normal bone development and homeostasis. Mutations in several Wnt signaling components, which increase the activity of the pathway in the skeleton, cause high bone mass in human subjects and mouse models. Increased bone mass is often accompanied by severe headaches from increased intracranial pressure, which can lead to fatality and loss of vision or hearing due to the entrapment of cranial nerves. In addition, progressive forehead bossing and mandibular overgrowth occur in almost all subjects. Treatments that would provide symptomatic relief in these subjects are limited.Porcupine-mediated palmitoylation is necessary for Wnt secretion and binding to the frizzled receptor. Chemical inhibition of porcupine is a highly selective method of Wnt signaling inhibition. We treated three different mouse models of high bone mass caused by aberrant Wnt signaling, including homozygosity for loss-of-function in Sost, which models sclerosteosis, and two strains of mice carrying different point mutations in Lrp5（equivalent to human G171V and A214V）, at 3 months of age with porcupine inhibitors for 5–6 weeks. Treatment significantly reduced both trabecular and cortical bone mass in all three models. This demonstrates that porcupine inhibition is potentially therapeutic for symptomatic relief in subjects who suffer from these disorders and further establishes that the continued production of Wnts is necessary for sustaining high bone mass in these models.