Coupling organelle inheritance with mitosis to balance growth and differentiation.

INTRODUCTION Adult tissues must balance growth and differentiation to develop and maintain homeostasis. Excessive differentiation can lead to aging and poor wound healing. Too much growth is observed in hyperproliferative disorders and cancers. How tissue imbalances arise in disease states is poorly understood. Skin is an excellent system for understanding the importance of this balance. Essential for keeping harmful microbes out and retaining body fluids, the skin barrier is maintained by an inner layer of proliferative basal progenitors, which generate a constant outward flux of terminally differentiating cells. It is known that when epidermal progenitors accumulate mutations that will give rise to malignancy, they change their program of gene expression. However, the extent to which cancer progression involves a gain of proliferation versus a loss of differentiation is unclear. A detailed molecular knowledge of how normal basal epidermal progenitors transition from a proliferative, undifferentiated state to a terminally differentiated state allows us to investigate how this process goes awry in a tumorigenic state. We use a genetic screen to identify which of the gene changes that occur in both early cell commitment and cancer are integral to maintaining the balance between growth and differentiation. RATIONALE Epithelial cancers are among the most prevalent and life-threatening cancers worldwide. Despite intensive research, the mechanisms by which these cancers evade regulatory systems working to balance differentiation and proliferation remain poorly understood. To provide new insights into how malignancies arise and how this might be exploited in advancing cancer therapeutics, we tackled this problem in the developing skin where these regulatory systems are established. RESULTS To understand how the balance between growth and differentiation is controlled, we first devised a strategy to transcriptionally profile epidermal stem cells and their terminally differentiating progeny. Using this method, we defined the earliest molecular events associated with the commitment of epidermal progenitors to their differentiation program. Of the many changes that occur, we focused on the cohort of genes that are also mutated in human epithelial cancers. To sift through which of these genes are functional drivers in cancers and how they perturb homeostasis, we conducted an in vivo epidermal RNA interference (RNAi) screen to identify candidates that are selectively enriched or depleted in proliferative progenitors relative to their differentiating progeny. We focused on PEX11b, a protein associated with peroxisomes, organelles involved in fatty acid and energy metabolism. PEX11b deficiency compromised epidermal terminal differentiation and barrier formation. Without PEX11b, peroxisomes functioned but failed to localize and therefore segregate properly during mitosis. Probing deeper, we discovered that in normal cells, peroxisomes take on stereotyped positions during mitosis. However, after depletion of PEX11b, peroxisomes failed to localize. Localization was directly coupled to mitotic progression, and when peroxisomes were mislocalized, a mitotic delay occurred. During this delay, spindles rotated uncontrollably, subsequently leading to perturbed polarized divisions and skewed daughter fates. Using a recently developed light-activated organelle repositioning technique to ectopically move peroxisomes, we found that altering peroxisomal localization in a PEX11b-independent manner also causes mitotic alterations. CONCLUSION Through transcriptional profiling and RNAi screening, we defined molecular targets associated with either increased proliferation or differentiation. One such target, the peroxisome membrane protein PEX11b, was required for epidermal development. The imbalance in epidermal differentiation that resulted from PEX11b deficiency and peroxisome mislocalization in mitosis was caused by an inability of basal stem cells to orient their spindle perpendicularly relative to the underlying basement membrane. For a stratified epithelium, where spindle orientation plays a critical role in establishing tissue architecture and homeostasis, this defect had dire consequences. Our findings unveil a role for organelle inheritance in mitosis, spindle attachment and alignment, and the choice of daughter progenitors to differentiate or remain stem-like.