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The Hippo signaling pathway is widely considered a master regulator of organ growth because of the prominent overgrowth phenotypes caused by experimental manipulation of its activity. Contrary to this model, we show here that removing Hippo transcriptional output did not impair the ability of the mouse liver and Drosophila eyes to grow to their normal size. Moreover, the transcriptional activity of the Hippo pathway effectors Yap/Taz/Yki did not correlate with cell proliferation, and hyperactivation of these effectors induced gene expression programs that did not recapitulate normal development. Concordantly, a functional screen in Drosophila identified several Hippo pathway target genes that were required for ectopic overgrowth but not normal growth. Thus, Hippo signaling does not instruct normal growth, and the Hippo-induced overgrowth phenotypes are caused by the activation of abnormal genetic programs. Description Rethinking Hippo signaling Mutations that cause dysregulation of the Hippo signaling pathway are known to cause excessive growth of organs, which has led many researchers to think of this pathway as a master regulator of organ growth. Studying fruit fly eye discs and mouse livers, Kowalczyk et al. found instead that Hippo signaling does not instruct normal growth. The Hippo-induced overgrowth phenotypes appear to be caused by Hippo signaling activating abnormal genetic programs. These findings challenge a long-standing idea about the role of Hippo signaling in organ growth and suggest the need to re-evaluate our understanding of its function in other contexts, such as in cancer and regeneration. —SMH and BAP The function of Hippo signaling during organ growth was re-evaluated in Drosophila imaginal discs and mouse livers. INTRODUCTION Accurate growth control is fundamental to ensure proper organ size and animal health. Many signaling pathways can regulate cell proliferation and apoptosis, which in turn determine the size of an organ. However, mechanisms that can sense when a developing organ has reached its proper size and instruct the cessation of further growth remain elusive. Such mechanisms may also orchestrate regeneration and induce tumorigenesis when defective. In this context, the Hippo signaling pathway has attracted much attention, because experimental hyperactivation of its downstream effectors, the transcriptional coactivators Yap/Taz/Yki, can drive cell proliferation and cause substantial organ overgrowth and tumor development. RATIONALE The Hippo pathway integrates diverse biochemical and mechanical cell-cell and cell–extracellular matrix signals to regulate gene expression through Yap/Taz/Yki. In a current model of growth control, the activity of Yap/Taz/Yki directs the right amount of cell proliferation during development such that they are active when organs grow and inactivated when they stop growing. We reasoned that a signaling pathway functioning as a central regulator of organ growth should fulfil three requirements: (i) normal growth does not start or terminate properly without its input, (ii) its activity correlates with the spatial and temporal pattern of cell proliferation, and (iii) when hyperactivated, it produces larger but otherwise normal organs. We thus investigated whether the Hippo pathway fits these criteria for an organ growth control mechanism. RESULTS To address these hypotheses, we studied two commonly used models for organ growth control, the Drosophila eye and the mouse liver, in which we examined the effects of loss of Hippo signaling output on growth, its pattern of activity during normal development, and the genetic program induced by the experimental hyperactivation of Yap/Taz/Yki. Our data show that Hippo pathway output is not required for the developmental proliferation of liver precursor and Drosophila retinal cells. Furthermore, the transcriptional activity of Yap/Taz/Yki did not correlate with cell proliferation during development, and their experimental hyperactivation in hepatocytes and Drosophila retinal cells, which caused overgrowth, did not reactivate progenitor programs. Rather, Yap/Taz/Yki were active and required in specific cell types, namely Drosophila squamous peripodial epithelial cells and mouse cholangiocytes, and their hyperactivation induced aberrant gene expression profiles that partly resembled the normal programs of these cells. Finally, a functional screen in Drosophila identified several Hippo pathway target genes that were required for ectopic overgrowth but not for normal growth. CONCLUSION Our data show that Hippo signaling does not instruct normal organ growth, and that the classic Hippo mutant overgrowth phenotypes represent Yap/Taz/Yki gain-of-function situations that involve ectopic expression of genes that are normally expressed in Hippo-dependent cell types. Cells that require endogenous Hippo pathway output, such as squamous cells, have in common being mechanically strained. Thus, Hippo signaling is involved in cellular responses to mechanical strain and in the differentiation and homeostasis of morphologically challenged cells. These findings correct a long-standing misconception about the role of Hippo signaling in organ growth and reveal the need to re-evaluate our understanding of its function in other contexts, such as in cancer and regeneration. Functional analysis of the Hippo pathway in organ growth control. The regulation of the Hippo signaling pathway has been thought to determine the amount of organ growth during development. However, analysis of the endogenous activity and effects of genetic manipulations of the pathway in the developing mouse liver and Drosophila eye show that Hippo signaling does not instruct normal organ size. In the Hippo pathway schematic (top left), Drosophila proteins are shown on the left and mouse proteins on the right, separated by a hyphen.