Ptch1 is essential for cochlear marginal cell differentiation and stria vascularis formation.

A common cause of deafness in humans is dysregulation of the endocochlear potential generated by the stria vascularis (SV). Thus, proper formation of the SV is critical for hearing. Using single-cell transcriptomics and a series of Shh signaling mutants, we discovered that the Shh receptor Patched1 (Ptch1) is essential for marginal cell (MC) differentiation and SV formation. Single-cell RNA sequencing analyses revealed that the cochlear roof epithelium is already specified into discrete domains with distinctive gene expression profiles at embryonic day 14, with Gsc as a marker gene of the MC lineage. Ptch1 deficiency leads to defective specification of MC precursors along the cochlear basal-apical regions. We demonstrated that elevated Gli2 levels impede MC differentiation through sustaining Otx2 expression and maintaining the progenitor state of MC precursors. Our results uncover an early specification of cochlear non-sensory epithelial cells and establish a crucial role of the Ptch1-Gli2 axis in regulating the development of SV.

Broad-spectrum kinome profiling identifies CDK6 upregulation as a driver of lenvatinib resistance in hepatocellular carcinoma.

Increasing evidence has demonstrated that drug resistance can be acquired in cancer cells by kinase rewiring, which is an obstacle for efficient cancer therapy. However, it is technically challenging to measure the expression of protein kinases on large scale due to their dynamic range in human proteome. We employ a lysine-targeted sulfonyl fluoride probe, named XO44, which binds to 133 endogenous kinases in intact lenvatinib-resistant hepatocellular carcinoma (HCC) cells. This analysis reveals cyclin-dependent kinase 6 (CDK6) upregulation, which is mediated by ERK/YAP1 signaling cascade. Functional analyses show that CDK6 is crucial in regulation of acquired lenvatinib resistance in HCC via augmentation of liver cancer stem cells with clinical significance. We identify a noncanonical pathway of CDK6 in which it binds and regulates the activity of GSK3ß, leading to activation of Wnt/ß-catenin signaling. Consistently, CDK6 inhibition by palbociclib or degradation by proteolysis targeting chimeras (PROTACs) is highly synergistic with lenvatinib in vitro. Interestingly, palbociclib not only exerts maximal growth suppressive effect with lenvatinib in lenvatinib-resistant HCC models but also reshapes the tumor immune microenvironment. Together, we unveil CDK6 as a druggable target in lenvatinib-resistant HCC and highlight the use of a chemical biology approach to understand nongenetic resistance mechanisms in cancer.

seRNA PAM controls skeletal muscle satellite cell proliferation and aging through trans regulation of Timp2 expression synergistically with Ddx5.

Muscle satellite cells (SCs) are responsible for muscle homeostasis and regeneration and lncRNAs play important roles in regulating SC activities. Here, in this study, we identify PAM (Pax7 Associated Muscle lncRNA) that is induced in activated/proliferating SCs upon injury to promote SC proliferation as myoblast cells. PAM is generated from a myoblast-specific super-enhancer (SE); as a seRNA it binds with a number of target genomic loci predominantly in trans. Further studies demonstrate that it interacts with Ddx5 to tether PAM SE to its inter-chromosomal targets Timp2 and Vim to activate the gene expression. Lastly, we show that PAM expression is increased in aging SCs, which leads to enhanced inter-chromosomal interaction and target genes upregulation. Altogether, our findings identify PAM as a previously unknown lncRNA that regulates both SC proliferation and aging through its trans gene regulatory activity.

Hoxb3 Regulates Jag1 Expression in Pharyngeal Epithelium and Affects Interaction With Neural Crest Cells.

Craniofacial morphogenesis depends on proper migration of neural crest cells and their interactions with placodes and other cell types. Hox genes provide positional information and are important in patterning the neural crest and pharyngeal arches (PAs) for coordinated formation of craniofacial structures. Hox genes are expressed in the surface ectoderm and epibranchial placodes, their roles in the pharyngeal epithelium and their downstream targets in regulating PA morphogenesis have not been established. We altered the Hox code in the pharyngeal region of the Hoxb3 Tg/+ mutant, in which Hoxb3 is driven to ectopically expressed in Hoxb2 domain in the second pharyngeal arch (PA2). In the transgenic mutant, ectopic Hoxb3 expression was restricted to the surface ectoderm, including the proximal epibranchial placodal region and the distal pharyngeal epithelium. The Hoxb3 Tg/+ mutants displayed hypoplasia of PA2, multiple neural crest-derived facial skeletal and nerve defects. Interestingly, we found that in the Hoxb3 Tg/+ mutant, expression of the Notch ligand Jag1 was specifically up-regulated in the ectodermal pharyngeal epithelial cells of PA2. By molecular experiments, we demonstrated that Hoxb3 could bind to an upstream genomic site S2 and directly regulate Jag1 expression. In the Hoxb3 Tg/+ mutant, elevated expression of Jag1 in the pharyngeal epithelium led to abnormal cellular interaction and deficiency of neural crest cells migrating into PA2. In summary, we showed that Hoxb3 regulates Jag1 expression and proposed a model of pharyngeal epithelium and neural crest interaction during pharyngeal arch development.