Dose-dependent responses to canonical Wnt transcriptional complexes in the regulation of mammalian nephron progenitors.

In vivo and in vitro studies argue that concentration dependent Wnt signaling regulates mammalian nephron progenitor cell (NPC) programs. Canonical Wnt signaling is regulated through the stabilization of ß-catenin, a transcriptional co-activator when complexed with Lef/Tcf DNA binding partners. Utilizing the GSK3ß inhibitor CHIR99021 (CHIR), to block GSK3ß-dependent destruction of ß-catenin, we examined dose-dependent responses to ß-catenin in NPCs, using mRNA transduction to modify gene expression. Low CHIR-dependent proliferation of NPCs was blocked on ß-catenin removal with evidence of NPCs arresting at the G2-M transition. While NPC identity was maintained following ß-catenin removal, mRNA-seq identified low CHIR and ß-catenin dependent genes. High CHIR activated nephrogenesis. Nephrogenic programming was dependent on Lef/Tcf factors and ß-catenin transcriptional activity. Molecular and cellular features of early nephrogenesis were driven in the absence of CHIR by a mutated, stabilized form of ß-catenin. Chromatin association studies indicate low and high CHIR response genes are likely direct targets of canonical Wnt transcriptional complexes. Together these studies provide evidence for concentration dependent Wnt-signaling in the regulation of NPCs and provide new insight into Wnt targets initiating mammalian nephrogenesis.

Worth a Double Take? An In-Depth Review of Lung Retransplantation.

Provided advancements in Lung Transplantation (LT) survival, the efficacy of Lung Retransplantation (LRT) has often been debated. Decades of retrospective analyses on thousands of LRT cases provide insight enabling predictive patient criteria for retransplantation. This review used the Preferred Reporting Items for Systematic Reviews and Meta Analyses (PRISMA) guidelines. The PubMed search engine was utilized for articles relating to LRT published through August 2023, and a systematic review was performed using Covidence software version 2.0 (Veritas Health Innovation, Australia). Careful patient selection is vital for successful LRT, and the benefit leans in favor of those in optimal health following their initial transplant. However, the lack of a standardized approach remains apparent. Through an in-depth review, we will address considerations such as chronic lung allograft dysfunction, timing to LRT, surgical and perioperative complexity, and critical ethical concerns that guide the current practice as it relates to this subset of patients for whom LRT is the only therapeutic option available.

Canonical Wnt transcriptional complexes are essential for induction of nephrogenesis but not maintenance or proliferation of nephron progenitors.

Wnt regulated transcriptional programs are associated with both the maintenance of mammalian nephron progenitor cells (NPC) and their induction, initiating the process of nephrogenesis. How opposing transcriptional roles are regulated remain unclear. Using an in vitro model replicating in vivo events, we examined the requirement for canonical Wnt transcriptional complexes in NPC regulation. In canonical transcription, Lef/Tcf DNA binding proteins associate the transcriptional co-activator ß-catenin. Wnt signaling is readily substituted by CHIR99021, a small molecule antagonist of glycogen synthase kinase-3ß (GSK3ß). GSK3ß inhibition blocks Gskß-dependent turnover of ß-catenin, enabling formation of Lef/Tcf/ß-catenin transcriptional complexes, and enhancer-mediated transcriptional activation. Removal of ß-catenin activity from NPCs under cell expansion conditions (low CHIR) demonstrated a non-transcriptional role for ß-catenin in the CHIR-dependent proliferation of NPCs. In contrast, CHIR-mediated induction of nephrogenesis, on switching from low to high CHIR, was dependent on Lef/Tcf and ß-catenin transcriptional activity. These studies point to a non-transcriptional mechanism for ß-catenin in regulation of NPCs, and potentially other stem progenitor cell types. Further, analysis of the ß-catenin-directed transcriptional response provides new insight into induction of nephrogenesis. Summary Statement: The study provides a mechanistic understanding of Wnt/ ß-catenin activity in self-renewal and differentiation of mammalian nephron progenitors.

Cadherin Adhesion Complexes Direct Cell Aggregation in the Epithelial Transition of Wnt-Induced Nephron Progenitor Cells.

In the developing mammalian kidney, nephron formation is initiated by a subset of nephron progenitor cells (NPCs). Wnt input activates a ß-catenin ( Ctnnb1 )-driven, transcriptional nephrogenic program. In conjunction, induced mesenchymal NPCs transition through a pre-tubular aggregate to an epithelial renal vesicle, the precursor for each nephron. How this critical mesenchymal-to-epithelial transition (MET) is regulated is unclear. In an in vitro mouse NPC culture model, activation of the Wnt pathway results in the aggregation of induced NPCs into closely-packed, cell clusters. Genetic removal of ß-catenin resulted in a failure of both Wnt pathway-directed transcriptional activation and the formation of aggregated cell clusters. Modulating extracellular Ca 2+ levels showed cell-cell contacts were Ca 2+ -dependent, suggesting a role for cadherin (Cdh)-directed cell adhesion. Molecular analysis identified Cdh2 , Cdh4 and Cdh11 in uninduced NPCs and the up-regulation of Cdh3 and Cdh4 accompanying the Wnt pathway-induced MET. Genetic removal of all four cadherins, and independent removal of α-catenin, which couples Cdh-ß-catenin membrane complexes to the actin cytoskeleton, abolished cell aggregation in response to Wnt pathway activation. However, the ß-catenin driven inductive transcriptional program was unaltered. Together with the accompanying paper (Bugacov et al ., submitted), these data demonstrate that distinct cellular activities of ß-catenin - transcriptional regulation and cell adhesion - combine in the mammalian kidney programs generating differentiated epithelial nephron precursors from mesenchymal nephron progenitors. Summary statement: Our study highlights the role of Wnt-ß-catenin pathway regulation of cadherin-mediated cell adhesion in the mesenchymal to epithelial transition of induced nephron progenitor cells.

The developing mouse coronal suture at single-cell resolution.

Sutures separate the flat bones of the skull and enable coordinated growth of the brain and overlying cranium. The coronal suture is most commonly fused in monogenic craniosynostosis, yet the unique aspects of its development remain incompletely understood. To uncover the cellular diversity within the murine embryonic coronal suture, we generated single-cell transcriptomes and performed extensive expression validation. We find distinct pre-osteoblast signatures between the bone fronts and periosteum, a ligament-like population above the suture that persists into adulthood, and a chondrogenic-like population in the dura mater underlying the suture. Lineage tracing reveals an embryonic Six2+ osteoprogenitor population that contributes to the postnatal suture mesenchyme, with these progenitors being preferentially affected in a Twist1+/-; Tcf12+/- mouse model of Saethre-Chotzen Syndrome. This single-cell atlas provides a resource for understanding the development of the coronal suture and the mechanisms for its loss in craniosynostosis.

A ß-catenin-driven switch in TCF/LEF transcription factor binding to DNA target sites promotes commitment of mammalian nephron progenitor cells.

The canonical Wnt pathway transcriptional co-activator ß-catenin regulates self-renewal and differentiation of mammalian nephron progenitor cells (NPCs). We modulated ß-catenin levels in NPC cultures using the GSK3 inhibitor CHIR99021 (CHIR) to examine opposing developmental actions of ß-catenin. Low CHIR-mediated maintenance and expansion of NPCs are independent of direct engagement of TCF/LEF/ß-catenin transcriptional complexes at low CHIR-dependent cell-cycle targets. In contrast, in high CHIR, TCF7/LEF1/ß-catenin complexes replaced TCF7L1/TCF7L2 binding on enhancers of differentiation-promoting target genes. Chromosome confirmation studies showed pre-established promoter-enhancer connections to these target genes in NPCs. High CHIR-associated de novo looping was observed in positive transcriptional feedback regulation to the canonical Wnt pathway. Thus, ß-catenin's direct transcriptional role is restricted to the induction of NPCs, where rising ß-catenin levels switch inhibitory TCF7L1/TCF7L2 complexes to activating LEF1/TCF7 complexes at primed gene targets poised for rapid initiation of a nephrogenic program.