Global Gene Expression Analysis Reveals Complex Cuticle Organization of the Tribolium Compound Eye.

The red flour beetle Tribolium castaneum is a resource-rich model for genomic and developmental studies. To extend previous studies on Tribolium eye development, we produced transcriptomes for normal-eyed and eye-depleted heads of pupae and adults to identify differentially transcript-enriched (DE) genes in the visual system. Unexpectedly, cuticle-related genes were the largest functional class in the pupal compound eye DE gene population, indicating differential enrichment in three distinct cuticle components: clear lens facet cuticle, highly melanized cuticle of the ocular diaphragm, which surrounds the Tribolium compound eye for internal fortification, and newly identified facet margins of the tanned cuticle, possibly enhancing external fortification. Phylogenetic, linkage, and high-throughput gene knockdown data suggest that most cuticle proteins (CPs) expressed in the Tribolium compound eye stem from the deployment of ancient CP genes. Consistent with this, TcasCPR15, which we identified as the major lens CP gene in Tribolium, is a beetle-specific but pleiotropic paralog of the ancient CPR RR-2 CP gene family. The less abundant yet most likely even more lens-specific TcasCP63 is a member of a sprawling family of noncanonical CP genes, documenting a role of local gene family expansions in the emergence of the Tribolium compound eye CP repertoire. Comparisons with Drosophila and the mosquito Anopheles gambiae reveal a steady turnover of lens-enriched CP genes during insect evolution.

Functional Profiling of CFTR-Directed Therapeutics Using Pediatric Patient-Derived Nasal Epithelial Cell Models.

Functional profiling of CFTR-directed therapeutics offers the potential to provide significant benefits to young people with cystic fibrosis (CF). However, the development of 2D airway epithelial cell models for individual response tests in CF children remains a central task. The objective of this study was to determine the utility of EpiXTM technology for expansion of nasal epithelial cells for use in electrophysiological CFTR function measurements. An initial harvest of as few as 20,000 cells was sufficient to expand up to 50 million cells that were used to generate air-liquid interface (ALI) cultures for ion transport studies with the Ussing assay. CFTR function was assessed by measuring responses to forskolin and the CFTR potentiator VX-770 (ivacaftor) in ALI cultures generated from passage 3 and 4 cells. Short-circuit current (Isc) measurements of blocked CFTR currents (ΔICFTRinh) discriminated CFTR function between healthy control (wild type, WT) and patients with intermediate (F508del/R117H-7T: 56% WT) and severe (F508del/F508del: 12% WT) CF disease. For the mixed genotypes, CFTR activity for F508del/c.850dupA was 12% WT, R334W/406-1G>A was 24% WT, and CFTRdele2,3(21 kb)/CFTRdele2,3(21 kb) was 9% WT. The CFTR correctors VX-809 (lumacaftor) and VX-661 (tezacaftor) significantly increased CFTR currents for F508del/R117H to 73 and 67% WT, respectively. Cultures with the large deletion mutation CFTRdele2,3(21 kb) unexpectedly responded to VX-661 treatment (20% WT). Amiloride-sensitive sodium currents were robust and ranged between 20-80 µA/cm2 depending on the subject. In addition to characterizing the electrophysiological profile of mutant CFTR activity in cultures for five genotypes, our study exemplifies the promising paradigm of bed-to-bench side cooperation and personalized medicine.

Long-Term In Vitro Expansion of Epithelial Stem Cells Enabled by Pharmacological Inhibition of PAK1-ROCK-Myosin II and TGF-ß Signaling.

Despite substantial self-renewal capability in vivo, epithelial stem and progenitor cells located in various tissues expand for a few passages in vitro in feeder-free condition before they succumb to growth arrest. Here, we describe the EpiX method, which utilizes small molecules that inhibit PAK1-ROCK-Myosin II and TGF-ß signaling to achieve over one trillion-fold expansion of human epithelial stem and progenitor cells from skin, airway, mammary, and prostate glands in the absence of feeder cells. Transcriptomic and epigenomic studies show that this condition helps epithelial cells to overcome stresses for continuous proliferation. EpiX-expanded basal epithelial cells differentiate into mature epithelial cells consistent with their tissue origins. Whole-genome sequencing reveals that the cells retain remarkable genome integrity after extensive in vitro expansion without acquiring tumorigenicity. EpiX technology provides a solution to exploit the potential of tissue-resident epithelial stem and progenitor cells for regenerative medicine.