A functional genetic screen identifies aurora kinase b as an essential regulator of Sox9-positive mouse embryonic lung progenitor cells.

Development of a branching tree in the embryonic lung is crucial for the formation of a fully mature functional lung at birth. Sox9+ cells present at the tip of the primary embryonic lung endoderm are multipotent cells responsible for branch formation and elongation. We performed a genetic screen in murine primary cells and identified aurora kinase b (Aurkb) as an essential regulator of Sox9+ cells ex vivo. In vivo conditional knockout studies confirmed that Aurkb was required for lung development but was not necessary for postnatal growth and the repair of the adult lung after injury. Deletion of Aurkb in embryonic Sox9+ cells led to the formation of a stunted lung that retained the expression of Sox2 in the proximal airways, as well as Sox9 in the distal tips. Although we found no change in cell polarity, we showed that loss of Aurkb or chemical inhibition of Aurkb caused Sox9+ cells to arrest at G2/M, likely responsible for the lack of branch bifurcation. This work demonstrates the power of genetic screens in identifying novel regulators of Sox9+ progenitor cells and lung branching morphogenesis.

A serine in the first transmembrane domain of the human E3 ubiquitin ligase MARCH9 is critical for down-regulation of its protein substrates.

The membrane-associated RING-CH (MARCH) family of membrane-bound E3 ubiquitin ligases regulates the levels of cell-surface membrane proteins, many of which are involved in immune responses. Although their role in ubiquitin-dependent endocytosis and degradation of cell-surface proteins is extensively documented, the features of MARCH proteins and their substrates that drive the molecular recognition events leading to ubiquitin transfer remain poorly defined. In this study, we sought to determine the features of human MARCH9 that are required for regulating the surface levels of its substrate proteins. Consistent with previous studies of other MARCH proteins, we found that susceptibility to MARCH9 activity is encoded in the transmembrane (TM) domains of its substrates. Accordingly, substitutions at specific residues and motifs within MARCH9's TM domains resulted in varying degrees of functional impairment. Most notably, a single serine-to-alanine substitution in the first of its two TM domains rendered MARCH9 completely unable to alter the surface levels of two different substrates: the major histocompatibility class I molecule HLA-A2 and the T-cell co-receptor CD4. Solution NMR analysis of a MARCH9 fragment encompassing the two TM domains and extracellular connecting loop revealed that the residues contributing most to MARCH9 activity are located in the α-helical portions of TM1 and TM2 that are closest to the extracellular face of the lipid bilayer. This observation defines a key region required for substrate regulation. In summary, our biochemical and structural findings demonstrate that specific sequences in the α-helical MARCH9 TM domains make crucial contributions to its ability to down-regulate its protein substrates.

Lung morphogenesis is orchestrated through Grainyhead-like 2 (Grhl2) transcriptional programs.

The highly conserved transcription factor Grainyhead-like 2 (Grhl2) exhibits a dynamic expression pattern in lung epithelium throughout embryonic development. Using a conditional gene targeting approach to delete Grhl2 in the developing lung epithelium, our results demonstrate that Grhl2 plays multiple roles in lung morphogenesis that are essential for respiratory function. Loss of Grhl2 leads to impaired ciliated cell differentiation and perturbed formation of terminal saccules. Critically, a substantial increase in Sox9-positive distal tip progenitor cells was observed following loss of Grhl2, suggesting that Grhl2 plays an important role in branching morphogenesis. Gene transcription profiling of Grhl2-deficient lung epithelial cells revealed a significant down regulation of Elf5, a member of the Ets family of transcription factors. Furthermore, ChIP and comparative genomic analyzes confirmed that Elf5 is a direct transcriptional target of Grhl2. Taken together, these results support the hypothesis that Grhl2 controls normal lung morphogenesis by tightly regulating the activity of distal tip progenitor cells.

Glimma: interactive graphics for gene expression analysis.

MOTIVATION: graphics for RNA-sequencing and microarray gene expression analyses may contain upwards of tens of thousands of points. Details about certain genes or samples of interest are easily obscured in such dense summary displays. Incorporating interactivity into summary plots would enable additional information to be displayed on demand and facilitate intuitive data exploration. RESULTS: The open-source Glimma package creates interactive graphics for exploring gene expression analysis with a few simple R commands. It extends popular plots found in the limma package, such as multi-dimensional scaling plots and mean-difference plots, to allow individual data points to be queried and additional annotation information to be displayed upon hovering or selecting particular points. It also offers links between plots so that more information can be revealed on demand. Glimma is widely applicable, supporting data analyses from a number of well-established Bioconductor workflows ( limma , edgeR and DESeq2 ) and uses D3/JavaScript to produce HTML pages with interactive displays that enable more effective data exploration by end-users. Results from Glimma can be easily shared between bioinformaticians and biologists, enhancing reporting capabilities while maintaining reproducibility. AVAILABILITY AND IMPLEMENTATION: The Glimma R package is available from http://bioconductor.org/packages/Glimma/ . CONTACT: su.s@wehi.edu.au , law@wehi.edu.au or mritchie@wehi.edu.au.

Cauli: a mouse strain with an Ift140 mutation that results in a skeletal ciliopathy modelling Jeune syndrome.

Cilia are architecturally complex organelles that protrude from the cell membrane and have signalling, sensory and motility functions that are central to normal tissue development and homeostasis. There are two broad categories of cilia; motile and non-motile, or primary, cilia. The central role of primary cilia in health and disease has become prominent in the past decade with the recognition of a number of human syndromes that result from defects in the formation or function of primary cilia. This rapidly growing class of conditions, now known as ciliopathies, impact the development of a diverse range of tissues including the neural axis, craniofacial structures, skeleton, kidneys, eyes and lungs. The broad impact of cilia dysfunction on development reflects the pivotal position of the primary cilia within a signalling nexus involving a growing number of growth factor systems including Hedgehog, Pdgf, Fgf, Hippo, Notch and both canonical Wnt and planar cell polarity. We have identified a novel ENU mutant allele of Ift140, which causes a mid-gestation embryonic lethal phenotype in homozygous mutant mice. Mutant embryos exhibit a range of phenotypes including exencephaly and spina bifida, craniofacial dysmorphism, digit anomalies, cardiac anomalies and somite patterning defects. A number of these phenotypes can be attributed to alterations in Hedgehog signalling, although additional signalling systems are also likely to be involved. We also report the identification of a homozygous recessive mutation in IFT140 in a Jeune syndrome patient. This ENU-induced Jeune syndrome model will be useful in delineating the origins of dysmorphology in human ciliopathies.

Dual inhibition of BCL-XL and MCL-1 is required to induce tumour regression in lung squamous cell carcinomas sensitive to FGFR inhibition.

Genetic alterations in the fibroblast growth factor receptors (FGFRs) have been described in multiple solid tumours including bladder cancer, head and neck and lung squamous cell carcinoma (SqCC). However, recent clinical trials showed limited efficacy of FGFR-targeted therapy in lung SqCC, suggesting combination therapy may be necessary to improve patient outcomes. Here we demonstrate that FGFR therapy primes SqCC for cell death by increasing the expression of the pro-apoptotic protein BIM. We therefore hypothesised that combining BH3-mimetics, potent inhibitors of pro-survival proteins, with FGFR-targeted therapy may enhance the killing of SqCC cells. Using patient-derived xenografts and specific inhibitors of BCL-2, BCL-XL, and MCL-1, we identified a greater reliance of lung SqCC cells on BCL-XL and MCL-1 compared to BCL-2 for survival. However, neither BCL-XL nor MCL-1 inhibitors alone provided a survival benefit in combination FGFR therapy in vivo. Only triple BCL-XL, MCL-1, and FGFR inhibition resulted in tumour volume regression and prolonged survival in vivo, demonstrating the ability of BCL-XL and MCL-1 proteins to compensate for each other in lung SqCC. Our work therefore provides a rationale for the inhibition of MCL-1, BCL-XL, and FGFR1 to maximize therapeutic response in FGFR1-expressing lung SqCC.