Wan-Jin Lu

p53 ancestry: gazing through an evolutionary lens

Evolutionary patterns indicate that primordial p53 genes predated the appearance of cancer. Therefore, wild-type tumour suppressive functions and mutant oncogenic functions that give celebrity status to this gene family were probably co-opted from unrelated primordial activities. Is it possible to deduce what these early functions might have been? And might this knowledge provide a platform for therapeutic opportunities?

Meiotic recombination provokes functional activation of the p53 regulatory network.

Germline Quality Control The p53 tumor suppressor protein plays a key role in protecting organisms from aberrant cancer cells. But during evolution, animals would rarely, if ever, have lived long enough to develop cancer and so need such a function of p53. What, then, were the original functions for which p53 was selected? Lu et al. (p. 1278) observed a pulse of p53 activation during Drosophila development in the female germ line. In cancer, p53 is activated in response to DNA damage. Similarly, in this study, breaks in DNA that occur normally during meiosis also caused p53 activation. In animals in which resolution of DNA breaks during crossing over was inhibited, activation of p53 was prolonged; furthermore, when p53 was also lacking oogenesis was abnormal. Exactly how activation of p53 contributes to the process of chromosome recombination during meiosis remains unclear, but it may provide quality control, only allowing survival of gametes that possess intact DNA. A tumor suppressor appears to protect against aberrant chromosome breakage during meiosis. The evolutionary appearance of p53 protein probably preceded its role in tumor suppression, suggesting that there may be unappreciated functions for this protein. Using genetic reporters as proxies to follow in vivo activation of the p53 network in Drosophila, we discovered that the process of meiotic recombination instigates programmed activation of p53 in the germ line. Specifically, double-stranded breaks in DNA generated by the topoisomerase Spo11 provoked functional p53 activity, which was prolonged in cells defective for meiotic DNA repair. This intrinsic stimulus for the p53 regulatory network is highly conserved because Spo11-dependent activation of p53 also occurs in mice. Our findings establish a physiological role for p53 in meiosis and suggest that tumor-suppressive functions may have been co-opted from primordial activities linked to recombination.

The Bax/Bak ortholog in Drosophila, Debcl, exerts limited control over programmed cell death

Bcl-2 family members are pivotal regulators of programmed cell death (PCD). In mammals, pro-apoptotic Bcl-2 family members initiate early apoptotic signals by causing the release of cytochrome c from the mitochondria, a step necessary for the initiation of the caspase cascade. Worms and flies do not show a requirement for cytochrome c during apoptosis, but both model systems express pro- and anti-apoptotic Bcl-2 family members. Drosophila encodes two Bcl-2 family members, Debcl (pro-apoptotic) and Buffy (anti-apoptotic). To understand the role of Debcl in Drosophila apoptosis, we produced authentic null alleles at this locus. Although gross development and lifespans were unaffected, we found that Debcl was required for pruning cells in the developing central nervous system. debcl genetically interacted with the ced-4/Apaf1 counterpart dark, but was not required for killing by RHG (Reaper, Hid, Grim) proteins. We found that debclKO mutants were unaffected for mitochondrial density or volume but, surprisingly, in a model of caspase-independent cell death, heterologous killing by murine Bax required debcl to exert its pro-apoptotic activity. Therefore, although debcl functions as a limited effector of PCD during normal Drosophila development, it can be effectively recruited for killing by mammalian members of the Bcl-2 gene family.

Lessons from p53 in non-mammalian models

p53 is a fundamental determinant of oncogenesis, aging physiology and neurodegenerative pathologies. The complexity of the p53 regulatory network can hinder attempts to fully understand how this oncogenic protein operates within/ between cells to constrain growth potential. Orthologs of p53 in non-mammalian models, such as zebrafish, Drosophila and Caenorhabditis elegans, afford simpler models that illuminate core properties of this ancient network. The existence of p53 in short-lived organisms, where cancers do not occur, argues that tumor suppression per se was not the evolutionary pressure shaping p53. Instead, p53 as a constraint against tumor growth was probably co-opted from more ancestral, nonautonomous functions that are entirely unknown. Describing these functional properties will be essential for a comprehensive picture of the p53 regulatory network in normal and disease states. The tractable systems described here offer sophisticated genetic tools and fundamental insights that will continue to be indispensable models for achieving this goal.

A collective form of cell death requires homeodomain interacting protein kinase

We examined post-eclosion elimination of the Drosophila wing epithelium in vivo where collective “suicide waves” promote sudden, coordinated death of epithelial sheets without a final engulfment step. Like apoptosis in earlier developmental stages, this unique communal form of cell death is controlled through the apoptosome proteins, Dronc and Dark, together with the IAP antagonists, Reaper, Grim, and Hid. Genetic lesions in these pathways caused intervein epithelial cells to persist, prompting a characteristic late-onset blemishing phenotype throughout the wing blade. We leveraged this phenotype in mosaic animals to discover relevant genes and establish here that homeodomain interacting protein kinase (HIPK) is required for collective death of the wing epithelium. Extra cells also persisted in other tissues, establishing a more generalized requirement for HIPK in the regulation of cell death and cell numbers.

p53 activity is selectively licensed in the Drosophila stem cell compartment

Oncogenic stress provokes tumor suppression by p53 but the extent to which this regulatory axis is conserved remains unknown. Using a biosensor to visualize p53 action, we find that Drosophila p53 is selectively active in gonadal stem cells after exposure to stressors that destabilize the genome. Similar p53 activity occurred in hyperplastic growths that were triggered either by the RasV12 oncoprotein or by failed differentiation programs. In a model of transient sterility, p53 was required for the recovery of fertility after stress, and entry into the cell cycle was delayed in p53- stem cells. Together, these observations establish that the stem cell compartment of the Drosophila germline is selectively licensed for stress-induced activation of the p53 regulatory network. Furthermore, the findings uncover ancestral links between p53 and aberrant proliferation that are independent of DNA breaks and predate evolution of the ARF/Mdm2 axis. DOI: http://dx.doi.org/10.7554/eLife.01530.001

Stromal Gli2 activity coordinates a niche signaling program for mammary epithelial stem cells

Double duty for mammary stem cell niche The stem cell niche is a complex local signaling microenvironment that regulates stem cell activity for tissue and organ maintenance and regeneration. As well as responding locally, during puberty, the mammary gland stem cell niche also responds to systemic hormonal signals. Zhao et al. have found that Gli2, a transcriptional effector of Hedgehog signaling, coordinates the niche-signaling program and activates expression of receptors for the mammatrophic hormones estrogen and growth hormone throughout the mammary gland (see the Perspective by Robertson). Disease may result not only from stem cell defects, but also from dysregulation of the microenvironment. Science, this issue p. eaal3485; see also p. 250 The GLI2 transcription factor regulates stromal cell expression of signals controlling breast epithelial stem cell activity. INTRODUCTION The stem cell niche is a complex signaling microenvironment that acts locally to sustain stem cell activity in tissue maintenance and regeneration. Although the cellular constitution and signaling activity of the stem cell niche is coming into focus in a variety of tissues, genetic regulatory factors that specify the niche are less clear. The activity of such factors is particularly intriguing in organs such as the breast, where the niche provides local signals for tissue homeostasis but also must be entrained by circulating hormones that induce the dramatic changes of puberty. RATIONALE The epithelial stem cells of a variety of organs respond to secreted signals generated in subjacent stromal cells, in a manner often dependent on activity of the Hedgehog (Hh) signaling pathway. Gli2, which encodes the major transcriptional effector of Hh signaling, is expressed in a subset of stromal cells adjacent to the ductal epithelium of the mouse mammary gland and is highly expressed at terminal end buds and end structures of the pubertal and adult mammary gland, respectively, which represent the sites of mammary epithelial stem cells. RESULTS Stromal ablation of a conditional Gli2fl allele with Fsp1Cre, a stromally expressed recombinase allele (producing Gli2∆S mice), caused a delay in mammary ductal development, reduced the number of mammary gland stromal cells and volume of extracellular matrix, and caused abnormal mammary duct distension. Stromal Gli2 ablation did not alter development of the ovary or pituitary, nor their production of mammatrophic hormones such as estrogen or growth hormone, but did affect ductal regeneration, as indicated by a fivefold decrease in outgrowth efficiency of mammary stem cells (MaSCs) transplanted into Gli2∆S mammary glands. These findings suggest that Gli2 specifies a stromal niche signaling program that critically regulates MaSC activity. FACS-isolated mammary stromal cells showed Gli2-dependent expression of factors that stimulate epithelial stem cell renewal, ductal outgrowth, and morphogenesis, including specific members of the IGF, WNT, FGF, and HGF families of secreted peptides. Single-cell analysis showed expression of these factors in a subset of stromal cells in a manner dependent on Gli2 function. Receptors for the mammatrophic hormones estrogen and growth hormone were also expressed in a Gli2-dependent manner, and estrogen and growth hormone treatment of cultured stromal cells induced these secreted peptide factors, suggesting that a critical role of Gli2 in mammary stromal niche cells is to render these cells responsive to systemic mammatrophic hormones. Supporting this conclusion, mammary gland implants of polymer fragments releasing IGF1 and WNT2 rescued Gli2∆S ductal growth phenotypes, whereas growth hormone–releasing polymer did not. CONCLUSION We find that the Hedgehog pathway transcriptional effector GLI2 specifies a stromal cell niche signaling program that supports mammary epithelial stem cells in pubertal and virgin adult mice. This program includes expression of factors such as IGF and WNT, and GLI2 acts in part by causing expression of receptors for hormones such as estrogen and growth hormone, thus entraining local stem cell niche activity to systemic mammatrophic hormones. Our work illustrates the central role of the stromal niche in controlling epithelial stem cell activity and suggests that niche failure may underlie pathogenesis of certain diseases, including the deficient breast development and hormonal insensitivity associated with the human disorder, combined pituitary hormone deficiency. Stromal GLI2 coordinates mammary stem cell niche signaling program. Transcriptional regulation by Hedgehog pathway effector GLI2 in stromal cells of the mammary gland coordinates a hormone-responsive niche signaling program that directs epithelial stem cell activity during the changes of puberty (schematic, left). Gli2-deficient stromal cells in mouse mammary are unable to support normal ductal outgrowth and morphogenesis of transplanted, green fluorescent protein–labeled stem cells (right). The stem cell niche is a complex local signaling microenvironment that sustains stem cell activity during organ maintenanceorgan maintenance and regeneration. The mammary gland niche must support its associated stem cells while also responding to systemic hormonal regulation that triggers pubertal changes. We find that Gli2, the major Hedgehog pathway transcriptional effector, acts within mouse mammary stromal cells to direct a hormone-responsive niche signaling program by activating expression of factors that regulate epithelial stem cells as well as receptors for the mammatrophic hormones estrogen and growth hormone. Whereas prior studies implicate stem cell defects in human disease, this work shows that niche dysfunction may also cause disease, with possible relevance for human disorders and in particular the breast growth pathogenesis associated with combined pituitary hormone deficiency.

Control of inflammation by stromal Hedgehog pathway activation restrains colitis

Significance Inflammatory bowel disease (IBD) is a debilitating disorder with limited treatment options. Here, we report that manipulation of Hedgehog (Hh) pathway signaling affects disease severity in the well-established dextran sulfate mouse model of colitis. Genetic and pharmacologic manipulations that decrease Hh pathway signaling in the colon worsen colitis. Conversely, manipulations that increase Hh pathway signaling ameliorate colitis. We find that Hh pathway stimulation exerts its effects partially through increased expression of the antiinflammatory cytokine IL-10 in Hh pathway-responsive stromal cells and concomitant increases in CD4+Foxp3+ regulatory T cells in the colon. Our studies suggest that pharmacologic Hh pathway stimulation in colonic stromal cells may be a strategy to treat IBD. Inflammation disrupts tissue architecture and function, thereby contributing to the pathogenesis of diverse diseases; the signals that promote or restrict tissue inflammation thus represent potential targets for therapeutic intervention. Here, we report that genetic or pharmacologic Hedgehog pathway inhibition intensifies colon inflammation (colitis) in mice. Conversely, genetic augmentation of Hedgehog response and systemic small-molecule Hedgehog pathway activation potently ameliorate colitis and restrain initiation and progression of colitis-induced adenocarcinoma. Within the colon, the Hedgehog protein signal does not act directly on the epithelium itself, but on underlying stromal cells to induce expression of IL-10, an immune-modulatory cytokine long known to suppress inflammatory intestinal damage. IL-10 function is required for the full protective effect of small-molecule Hedgehog pathway activation in colitis; this pharmacologic augmentation of Hedgehog pathway activity and stromal IL-10 expression are associated with increased presence of CD4+Foxp3+ regulatory T cells. We thus identify stromal cells as cellular coordinators of colon inflammation and suggest their pharmacologic manipulation as a potential means to treat colitis.

Neuronal delivery of Hedgehog directs spatial patterning of taste organ regeneration

Significance The maintenance of taste sensory organs (taste buds) in the tongue has been known for 140 years to depend on sensory innervation from distant neurons by an unknown mechanism. We find that maintenance and regeneration of taste receptor cells (TRCs) within taste buds requires neuronal delivery of the Sonic hedgehog (Shh) protein signal, thus explaining loss of taste sensation associated with Hedgehog pathway antagonism in patients and illustrating the principle that spatial patterning of TRC regeneration is specified by the projection pattern of Shh-expressing sensory neurons. We also find that pharmacologic Hedgehog pathway activation accelerates TRC recovery, suggesting a means to ameliorate the loss of taste sensation and appetite and the associated delay in recovery in cancer patients undergoing chemotherapy. How organs maintain and restore functional integrity during ordinary tissue turnover or following injury represents a central biological problem. The maintenance of taste sensory organs in the tongue was shown 140 years ago to depend on innervation from distant ganglion neurons, but the underlying mechanism has remained unknown. Here, we show that Sonic hedgehog (Shh), which encodes a secreted protein signal, is expressed in these sensory neurons, and that experimental ablation of neuronal Shh expression causes loss of taste receptor cells (TRCs). TRCs are also lost upon pharmacologic blockade of Hedgehog pathway response, accounting for the loss of taste sensation experienced by cancer patients undergoing Hedgehog inhibitor treatment. We find that TRC regeneration following such pharmacologic ablation requires neuronal expression of Shh and can be substantially enhanced by pharmacologic activation of Hedgehog response. Such pharmacologic enhancement of Hedgehog response, however, results in additional TRC formation at many ectopic sites, unlike the site-restricted regeneration specified by the projection pattern of Shh-expressing neurons. Stable regeneration of TRCs thus requires neuronal Shh, illustrating the principle that neuronal delivery of cues such as the Shh signal can pattern distant cellular responses to assure functional integrity during tissue maintenance and regeneration.

MP81-10 MOLECULAR CHARACTERIZATION OF THE MURINE URINARY BLADDER USING SINGLE CELL RNA SEQUENCING

INTRODUCTION AND OBJECTIVES: The use of decellularized natural matrices to recover a functional bladder has been shown to be the most appropriate source to reconstruct this organ. Our goal was to compare evolution of recellularized acellular matrix vs non-recelullarized matrix implanted in a rat model. METHODS: All experimental procedures were performed into consideration the ethical processes described by the animal committee of the center. Sprague-Dawley rats were used for the experiment. Here we compared the efficient and functional integration of a recellularized acellular matrix, previously seeded in vitro with human adipose tissue derived mesenchymal stem cells with a non-recelullarized matrix in a rat model of partial cystectomy and bladder substitution. RESULTS: Relevant and significant anatomical differences were found few days after neo bladders implantation. The human ADSC containing matrix showed a significant recovery of mature urothelium measured as the positive reactivity to p63 at the transitional epithelium layers. The expression of the mesoderm marker, cytokeratin 7 was also significantly induced within the regenerated connective tissue. Interestingly a higher expression of smooth muscle actin was found at both, the internal and peripheral transversal and longitudinal smooth muscle layers in the hADSC-recellularized matrix. However, no significant induction in any of the transplanted groups, in comparison with native bladder, was found for CD31 expression, endothelium marker. The presence of the hADSC, by most probably the paracrine release of proregenerative factors, favors a faster and more efficient endogenous regeneration of the ectopic bladder allowing a faster voluntary urodynamic recovery within a more specialized an mature tissue. CONCLUSIONS: Although bladder recellularization and urodynamic functional recovery have been successfully tested in small animals, and here we show the relevant benefits of the use of human ADSC on this regeneration process, more efficient procedures ex-vivo with a better understanding of the mechanisms involved of the ectopic transplanted cells is required in order to improve the tissue maturation in terms of peripheral nerve innervation and vascularization for a proper clinical translation