Overgrowth Phenotype Research Articles

Drosophila Ribosomal Protein Mutants Control Tissue Growth Non-Autonomously via Effects on the Prothoracic Gland and Ecdysone

The ribosome is critical for all aspects of cell growth due to its essential role in protein synthesis. Paradoxically, many Ribosomal proteins (Rps) act as tumour suppressors in Drosophila and vertebrates. To examine how reductions in Rps could lead to tissue overgrowth, we took advantage of the observation that an RpS6 mutant dominantly suppresses the small rough eye phenotype in a cyclin E hypomorphic mutant (cycEJP). We demonstrated that the suppression of cycEJP by the RpS6 mutant is not a consequence of restoring CycE protein levels or activity in the eye imaginal tissue. Rather, the use of UAS-RpS6 RNAi transgenics revealed that the suppression of cycEJP is exerted via a mechanism extrinsic to the eye, whereby reduced Rp levels in the prothoracic gland decreases the activity of ecdysone, the steroid hormone, delaying developmental timing and hence allowing time for tissue and organ overgrowth. These data provide for the first time a rationale to explain the counter-intuitive organ overgrowth phenotypes observed for certain members of the Minute class of Drosophila Rp mutants. They also demonstrate how Rp mutants can affect growth and development cell non-autonomously.

Oxidative stress induces overgrowth of theDrosophilaneuromuscular junction

Synaptic terminals are known to expand and contract throughout an animal's life. The physiological constraints and demands that regulate appropriate synaptic growth and connectivity are currently poorly understood. In previous work, we identified a Drosophila model of lysosomal storage disease (LSD), spinster (spin), with larval neuromuscular synapse overgrowth. Here we identify a reactive oxygen species (ROS) burden in spin that may be attributable to previously identified lipofuscin deposition and lysosomal dysfunction, a cellular hallmark of LSD. Reducing ROS in spin mutants rescues synaptic overgrowth and electrophysiological deficits. Synapse overgrowth was also observed in mutants defective for protection from ROS and animals subjected to excessive ROS. ROS are known to stimulate JNK and fos signaling. Furthermore, JNK and fos in turn are known potent activators of synapse growth and function. Inhibiting JNK and fos activity in spin rescues synapse overgrowth and electrophysiological deficits. Similarly, inhibiting JNK, fos, and jun activity in animals with excessive oxidative stress rescues the overgrowth phenotype. These data suggest that ROS, via activation of the JNK signaling pathway, are a major regulator of synapse overgrowth. In LSD, increased autophagy contributes to lysosomal storage and, presumably, elevated levels of oxidative stress. In support of this suggestion, we report here that impaired autophagy function reverses synaptic overgrowth in spin. Our data describe a previously unexplored link between oxidative stress and synapse overgrowth via the JNK signaling pathway.

Role of Epigenetics in the Placenta: Alterations in DNA Promoter Mmethylation and Limprinted Genes

Many researchers are focusing on epigenetic regulation in the placenta. Because the proper development and function of the placenta are crucial for the normal healthy growth and survival of the developing fetus, the study of epigenetic alterations is providing critical insights into the biology of fetal development and the pathogenesis of complications in pregnancy. One epigenetic alteration is genomic imprinting. Alleles from both the father and mother are necessary for normal human and placental development. For example, the paternally expressed IGF2 gene and maternally expressed H19 gene are located in 11p15.5 and they are coordinately regulated by differentially methylated regions (DMRs). The main phenotype of Silver-Russell syndrome is severe fetal growth retardation (FGR) that is caused by a reduction in IGF2 transcription as a result of a loss of methylation at the H19 DMR. Hypermethylation at the H19 DMR is found in 30% of cases of Beckwith-Wiedemann syndrome and the overgrowth phenotype. The other main mechanism is a promoter DNA methylation. Hypomethylation of a promoter of the SERPINA3 gene in the placenta is associated with FGR and preeclampsia. These clues to the epigenetic phenomena involved in the processes of human development and disease in pregnancy have been found in recent years.

An Ana2/Ctp/Mud Complex Regulates Spindle Orientation in Drosophila Neuroblasts

Drosophila neural stem cells, larval brain neuroblasts (NBs), align their mitotic spindles along the apical/basal axis during asymmetric cell division (ACD) to maintain the balance of self-renewal and differentiation. Here, we identified a protein complex composed of the tumor suppressor anastral spindle 2 (Ana2), a dynein light-chain protein Cut up (Ctp), and Mushroom body defect (Mud), which regulates mitotic spindle orientation. We isolated two ana2 alleles that displayed spindle misorientation and NB overgrowth phenotypes in larval brains. The centriolar protein Ana2 anchors Ctp to centrioles during ACD. The centriolar localization of Ctp is important for spindle orientation. Ana2 and Ctp localize Mud to the centrosomes and cell cortex and facilitate/maintain the association of Mud with Pins at the apical cortex. Our findings reveal that the centrosomal proteins Ana2 and Ctp regulate Mud function toorient the mitotic spindle during NB asymmetric division.

Hippo pathway inhibits Wnt signaling to restrain cardiomyocyte proliferation and heart size.

Genetic regulation of mammalian heart size is poorly understood. Hippo signaling represents an organ-size control pathway in Drosophila, where it also inhibits cell proliferation and promotes apoptosis in imaginal discs. To determine whether Hippo signaling controls mammalian heart size, we inactivated Hippo pathway components in the developing mouse heart. Hippo-deficient embryos had overgrown hearts with elevated cardiomyocyte proliferation. Gene expression profiling and chromatin immunoprecipitation revealed that Hippo signaling negatively regulates a subset of Wnt target genes. Genetic interaction studies indicated that β-catenin heterozygosity suppressed the Hippo cardiomyocyte overgrowth phenotype. Furthermore, the Hippo effector Yap interacts with β-catenin on Sox2 and Snai2 genes. These data uncover a nuclear interaction between Hippo and Wnt signaling that restricts cardiomyocyte proliferation and controls heart size.

The NAB-Brk Signal Bifurcates at JNK to Independently Induce Apoptosis and Compensatory Proliferation

Apoptosis operates to eliminate damaged or potentially dangerous cells. This loss is often compensated by extra proliferation of neighboring cells. Studies in Drosophila imaginal discs suggest that the signal for the additional growth emanates from the dying cells. In particular, it was suggested that the initiator caspase Dronc mediates compensatory proliferation (CP) through Dp53 in wing discs. However, the exact mechanism that governs this CP remained poorly understood. We have previously shown that elimination of misspecified cells due to reduced Dpp signaling is achieved by the interaction of the co-repressor NAB with the transcriptional repressor Brk, which in turn induces Jun N-terminal kinase-dependent apoptosis. Here, we performed a systematic in vivo loss- and gain-of-function analysis to study NAB-induced death and CP. Our findings indicate that the NAB primary signal activates JNK, which in turn transmits two independent signals. One triggers apoptosis through the pro-apoptotic proteins Reaper and Hid, which in turn promote activation of caspases by the apoptosome components Ark and Dronc. The other signal induces CP in a manner that is independent of the death signal, Dronc, or Dp53. Once induced, the apoptotic pathway further activates a CP response. Our data suggest that JNK is the candidate factor that differentiates between apoptosis that involves CP and apoptosis that does not.

Cartilage ultrastructure in proteoglycan‐deficient zebrafish mutants brings to light new candidate genes for human skeletal disorders

Proteoglycans are molecules consisting of protein cores onto which sugar chains, i.e., glycosaminoglycans (GAGs) such as heparan or chondroitin sulphates, are attached. Proteoglycans are produced by nearly all cells, and once secreted they become a major component of the extracellular matrix. Cartilage is particularly rich in proteoglycans, and changes in the structure and composition of GAGs have been found in osteochondromas and osteoarthritis. The zebrafish (Danio rerio) exhibits fast development, a growth plate-like organization of its craniofacial skeleton and an availability of various mutants, making it a powerful model for the study of human skeletal disorders with unknown aetiology. We analysed skeletons from five zebrafish lines with known mutations in genes involved in proteoglycan synthesis: dackel (dak/ext2), lacking heparan sulphate; hi307 (β3gat3), deficient for most GAGs; pinscher (pic/slc35b2), presenting defective sulphation of GAGs and other molecules; hi954 (uxs1), lacking Notch and most GAGs due to impaired protein xylosylation; and knypek (kny/gpc4), missing the protein core of the Glypican-4 proteoglycan. Here we show that each mutant displays different phenotypes related to: (a) cartilage morphology; (b) composition of the extracellular matrix; (c) ultrastructure of the extracellular matrix; and (d) the intracellular ultrastructure of chondrocytes, proving that sulphated GAGs orchestrate the cartilage intra- and extracellular ultrastructures. The mild phenotype of the hi307 mutant suggests that proteoglycans consisting of a protein core and a short sugar linker might suffice for proper chondrocyte stacking. Finally, knypek supports the involvement of Glypican-4 in the craniofacial phenotype of Simpson-Golabi-Behmel syndrome and suggests GPC4 as a modulator of the overgrowth phenotype that is associated with this syndrome and is primarily caused by a mutation in GPC3. Moreover, we speculate on the potential involvement of SLC35B2, β3GAT3 and UXS1 in skeletal dysplasias. This work promotes the use of zebrafish as a model of human skeletal development and associated pathologies.

Crumbs Regulates Salvador/Warts/Hippo Signaling in Drosophila via the FERM-Domain Protein Expanded

Altered expression of apicobasal polarity factors is associated with cancer in vertebrates and tissue overgrowth in invertebrates, yet the mechanisms by which these factors affect growth-regulatory pathways are not well defined. We have tested the basis of an overgrowth phenotype driven by the Drosophila protein Crumbs (Crb), which nucleates an apical membrane complex that functionally interacts with the Par6/Par3/aPKC and Scrib/Dlg/Lgl apicobasal polarity complexes. We find that Crb-driven growth is dependent upon the Salvador/Warts/Hippo (SWH) pathway and its transcriptional effector Yorkie (Yki). Expression of the Crb intracellular domain elevates Yki activity, and this correlates in tissues and cultured cells with loss of Expanded (Ex), an apically localized SWH component that inhibits Yki. Reciprocally, loss of crb elevates Ex levels, although this excess Ex does not concentrate to its normal location at apical junctions. The Ex-regulatory domain of Crb maps to the juxtamembrane FERM-binding motif (JM), a cytoskeletal interaction domain distinct from the PDZ-binding motif (PBM) through which Crb binds polarity factors. Expression of Crb-JM drives Yki activity and organ growth with little effect on tissue architecture, while Crb-PBM reciprocally produces tissue architectural defects without significant effect on Yki activity. These studies identify Crb as a novel SWH regulator via JM-dependent effects on Ex levels and localization and suggest that discrete domains within Crb may allow it to integrate junctional polarity signals with a conserved growth pathway.

Casein kinase I epsilon somatic mutations found in breast cancer cause overgrowth in Drosophila

We are using a candidate gene approach to identify genes contributing to cancer through somatic mutation. Somatic mutations were found in breast cancer samples in the human casein kinase I epsilon (CKIepsilon) gene, a homolog of the Drosophila gene dco in which certain point mutations lead to imaginal disc overgrowth. We therefore created fly genotypes in which the dco gene carried point mutations homologous to those discovered in CKIepsilon, and tested them in vivo. The results show that the most frequent mutation discovered in breast cancer, L39Q, causes a striking overgrowth phenotype in flies. Further experiments show that this mutation affects the newly recognized Fat/Warts signaling pathway, which controls organ size and shape in both flies and mammals. Another mutation, S101R, modifies the mutant phenotype so that the affected tissue disintegrates, mimicking more aggressive forms of breast cancer. Our results thus strongly support the conclusion that CKIepsilon mutations play important roles in breast carcinogenesis.

Chronically elevated plasma C-type natriuretic peptide level stimulates skeletal growth in transgenic mice

C-type natriuretic peptide (CNP) plays a critical role in endochondral ossification through guanylyl cyclase-B (GC-B), a natriuretic peptide receptor subtype. Cartilage-specific overexpression of CNP enhances skeletal growth and rescues the dwarfism in a transgenic achondroplasia model with constitutive active mutation of fibroblast growth factor receptor-3. For future clinical application, the efficacy of CNP administration on skeletal growth must be evaluated. Due to the high clearance of CNP, maintaining a high concentration is technically difficult. However, to model high blood CNP concentration, we established a liver-targeted CNP-overexpressing transgenic mouse (SAP-CNP tgm). SAP-CNP tgm exhibited skeletal overgrowth in proportion to the blood CNP concentration and revealed phenotypes of systemic stimulation of cartilage bones, including limbs, paws, costal bones, spine, and skull. Furthermore, in SAP-CNP tgm, the size of the foramen magnum, the insufficient formation of which results in cervico-medullary compression in achondroplasia, also showed significant increase. CNP primarily activates GC-B, but under high concentrations it cross-reacts with guanylyl cyclase-A (GC-A), a natriuretic peptide receptor subtype of atrial natriuretic peptides (ANP) and brain natriuretic peptides (BNP). Although activation of GC-A could alter cardiovascular homeostasis, leading to hypotension and heart weight reduction, the skeletal overgrowth phenotype in the line of SAP-CNP tgm with mild overexpression of CNP did not accompany decrease of systolic blood pressure or heart weight. These results suggest that CNP administration stimulates skeletal growth without adverse cardiovascular effect, and thus CNP could be a promising remedy targeting achondroplasia.

Autophagy promotes synapse development in Drosophila

Autophagy, a lysosome-dependent degradation mechanism, mediates many biological processes, including cellular stress responses and neuroprotection. In this study, we demonstrate that autophagy positively regulates development of the Drosophila melanogaster larval neuromuscular junction (NMJ). Autophagy induces an NMJ overgrowth phenotype closely resembling that of highwire (hiw), an E3 ubiquitin ligase mutant. Moreover, like hiw, autophagy-induced NMJ overgrowth is suppressed by wallenda (wnd) and by a dominant-negative c-Jun NH(2)-terminal kinase (bsk(DN)). We show that autophagy promotes NMJ growth by reducing Hiw levels. Thus, autophagy and the ubiquitin-proteasome system converge in regulating synaptic development. Because autophagy is triggered in response to many environmental cues, our findings suggest that it is perfectly positioned to link environmental conditions with synaptic growth and plasticity.

H19acts as a trans regulator of the imprinted gene network controlling growth in mice

The imprinted H19 gene produces a non-coding RNA of unknown function. Mice lacking H19 show an overgrowth phenotype, due to a cis effect of the H19 locus on the adjacent Igf2 gene. To explore the function of the RNA itself, we produced transgenic mice overexpressing H19. We observed postnatal growth reduction in two independent transgenic lines and detected a decrease of Igf2 expression in embryos. An extensive analysis of several other genes from the newly described imprinted gene network (IGN) was performed in both loss- and gain-of-function animals. We found that H19 deletion leads to the upregulation of several genes of the IGN. This overexpression is restored to the wild-type level by transgenic expression of H19. We therefore propose that the H19 gene participates as a trans regulator in the fine-tuning of this IGN in the mouse embryo. This is the first in vivo evidence of a functional role for the H19 RNA. Our results also bring further experimental evidence for the existence of the IGN and open new perspectives in the comprehension of the role of genomic imprinting in embryonic growth and in human imprinting pathologies.

Phenotypic discordance upon paternal or maternal transmission of duplications of the 11p15 imprinted regions

We report on two families in which the parental origin of duplications of the BWS imprinted regions on chromosome 11p15 influences the phenotype. In family A the transmission of a t(4; 11)(q35; p15.5) translocation results in duplication of BWSIC1 and BWSIC2. If this duplication is transmitted from the father, the extra chromosomal material has the paternal imprint. This results in overexpression of IGF2 and consequently an overgrowth phenotype. If the duplication is transmitted from the mother, the extra chromosomal material has the maternal imprint, resulting in overexpression of CDKN1C and a growth retardation phenotype. In family B an interstitial duplication of BWSIC1 results in an overgrowth phenotype when inherited from the father, similar to family A. However, no change in phenotype is observed if the duplication is transmitted through the mother suggesting that increased dosage of maternally expressed genes in the duplicated region has limited effect on the phenotype.

Abi plays an opposing role to Abl inDrosophilaaxonogenesis and synaptogenesis

Abl tyrosine kinase (Abl) regulates axon guidance by modulating actin dynamics. Abelson interacting protein (Abi), originally identified as a kinase substrate of Abl, also plays a key role in actin dynamics, yet its role with respect to Abl in the developing nervous system remains unclear. Here we show that mutations in abi disrupt axonal patterning in the developing Drosophila central nervous system (CNS). However, reducing abi gene dosage by half substantially rescues Abl mutant phenotypes in pupal lethality, axonal guidance defects and locomotion deficits. Moreover, we show that mutations in Abl increase synaptic growth and spontaneous synaptic transmission frequency at the neuromuscular junction. Double heterozygosity for abi and enabled (ena) also suppresses the synaptic overgrowth phenotypes of Abl mutants, suggesting that Abi acts cooperatively with Ena to antagonize Abl function in synaptogenesis. Intriguingly, overexpressing Abi or Ena alone in cultured cells dramatically redistributed peripheral F-actin to the cytoplasm, with aggregates colocalizing with Abi and/or Ena, and resulted in a reduction in neurite extension. However, co-expressing Abl with Abi or Ena redistributed cytoplasmic F-actin back to the cell periphery and restored bipolar cell morphology. These data suggest that abi and Abl have an antagonistic interaction in Drosophila axonogenesis and synaptogenesis, which possibly occurs through the modulation of F-actin reorganization.

The Hippo tumor-suppressor pathway regulates apical-domain size in parallel to tissue growth

The Hippo tumor-suppressor pathway controls tissue growth in Drosophila and mammals by regulating cell proliferation and apoptosis. The Hippo pathway includes the Fat cadherin, a transmembrane protein, which acts upstream of several other components that form a kinase cascade that culminates in the regulation of gene expression through the transcriptional coactivator Yorkie (Yki). Our previous work in Drosophila indicated that Merlin (Mer) and Expanded (Ex) are members of the Hippo pathway and act upstream of the Hippo kinase. In contrast to this model, it was suggested that Mer and Ex primarily regulate membrane dynamics and receptor trafficking, thereby affecting Hippo pathway activity only indirectly. Here, we examined the effects of Mer, Ex and the Hippo pathway on the size of the apical membrane and on apical-basal polarity complexes. We found that mer;ex double mutant imaginal disc cells have significantly increased levels of apical membrane determinants, such as Crb, aPKC and Patj. These phenotypes were shared with mutations in other Hippo pathway components and required Yki, indicating that Mer and Ex signal through the Hippo pathway. Interestingly, however, whereas Crb was required for the accumulation of other apical proteins and for the expansion of the apical domain observed in Hippo pathway mutants, its elimination did not significantly reverse the overgrowth phenotype of warts mutant cells. Therefore, Hippo signaling regulates cell polarity complexes in addition to and independently of its growth control function in imaginal disc cells.

Positive and Negative Glypicans

Glypicans are glycophosphatidylinositol (GPI)-anchored heparan sulfate proteoglycans that regulate the signaling of morphogens, including Wnt, Hedgehog (Hh), bone morphogenetic proteins (BMPs), and fibroblast growth factors (FGFs). Two articles show that glypicans can have opposing functions in Hedgehog signaling. Capurro et al . sought to determine how loss-of-function mutations in glypican-3 (GPC3), one of six mammalian glypicans, produce a developmental overgrowth phenotype that appears to arise by excessive proliferation and is associated with Simpson-Golabi-Behmel syndrome in humans. Increased mRNA abundance of Hh targets, as determined by reverse transcription polymerase chain reaction, suggested that signaling through the Hedgehog pathway was increased in GPC3-null mouse embryos. With cultured cells, they found that activation of a Hh reporter gene by Sonic hedgehog (Shh) or Indian hedgehog (Ihh) was inhibited when GPC3 was overexpressed and that this inhibition was retained in a GPC3 mutant (GPC3ΔGAG ) that could not be glycanated, suggesting that the core protein without heparan sulfate has biological activity. Activity required the GPI anchor, and inhibition of Hh reporter gene activation was not detected if a soluble form of GPC3 was expressed or if GPC3 was anchored to the membrane through a chimeric transmembrane domain from syndecan. Shh and GPC3 or GPC3ΔGAG coimmunoprecipitated when transiently transfected into cells, Shh bound to the surface of cells expressing GPC3, and GPC3 and GPC3ΔGAG both interacted with Shh in a surface plasmon resonance assay. Overexpression of GPC3 reduced the amount of Shh that coimmunoprecipitated with the Hedgehog receptor Patched1 (Ptc1), suggesting that GPC3 may compete with Ptc1 for Shh. GPC3-expressing cells triggered the endocytosis of Shh and the two proteins colocalized with markers of endosomes and lysosomes, suggesting that GPC3 may inhibit Hedgehog signaling by removing Hh from the cell surface. Consistent with this model, GPC3-null mouse embryos showed increased abundance of Shh based on Western blotting and immunohistochemistry of tissue sections. Hh and Wingless (Wg, also known as Wnt) signaling are involved in wing morphogenesis in Drosophila . Gallet et al . found that endocytosis of Hh and Ptc was enhanced by the glypican Dally-like (Dlp) and that this was required for full activation of the Hh pathway. Flies homozygous for the dlp 20 allele, which is a functional knockout, exhibited decreased expression of Hh target genes ( engrailed , Ptc , and decapentaplegic ) in the wing imaginal disc, and the zone in which they were expressed was narrower. A green fluorescent protein (GFP)-Dlp fusion protein colocalized with Hh and Ptc in intracellular vesicles in the imaginal disc cells. The GPI anchor of Dlp was required for Dlp internalization because replacement of the GPI anchor with a transmembrane domain (GFP-Dlp-CD2) did not produce colocalization. The number of Ptc- and Hh-positive vesicles was decreased in the dlp 20 mutants or flies expressing GFP-Dlp-CD2, whereas overexpression of GFP-Dlp increased the number of vesicles containing both Ptc and Hh. GFP-Dlp, but not GFP-Dlp-CD2, rescued the dlp 20 phenotype, suggesting that endocytosis of Dlp was important for its ability to enhance Hh signaling. Dlp was detected at the apical surface of disc epithelial cells and was endocytosed and translocated to the basolateral compartment, a process that was blocked in flies expressing a dominant-negative form of dynamin, which is required for some types of endocytosis. Ptc was stabilized at both the apical and basolateral sides of cells expressing dominant-negative dynamin, and decapentaplegic expression was decreased, suggesting impairment of Hh signaling. Dlp also contributed to Wg signaling and appeared to facilitate the endocytosis and transcytosis of Wg from the apical surface to the basolateral surface to contribute to the spread of Wg. Thus, these two papers show that glypicans use their GPI anchors to mediate endocytosis and that this either can enhance Hh signaling by stimulating internalization of the ligand-bound receptor or can inhibit Hh signaling by removing the ligand from the cell surface and preventing its interaction with its receptor Ptc. M. I. Capurro, P. Xu, W. Shi, F. Li, A. Jia, J. Filmus, Glypican-3 inhibits Hedgehog signaling during development by competing with Patched for Hedgehog binding. Dev. Cell 14 , 700-711 (2008). [PubMed] A. Gallet, L. Staccini-Lavenant, P. P. Thérond, Cellular trafficking of the glypican Dally-like is required for full-strength Hedgehog signaling and Wingless transcytosis. Dev. Cell 14 , 712-725 (2008). [PubMed]

Moesin helps to restrain synaptic growth at the Drosophila neuromuscular junction

The precise role of actin and actin-binding proteins in synaptic development is unclear. In Drosophila, overexpression of a dominant-negative NSF2 construct perturbs filamentous actin, which is associated with overgrowth of the NMJ, while co-expression of moesin, which encodes an actin binding protein, suppresses this overgrowth phenotype. These data suggest that Moesin may play a role in synaptic development at the Drosophila NMJ. To further investigate this possibility, we examined the influence of loss-of-function moesin alleles on the NSF2-induced overgrowth phenotype. We found that flies carrying P-element insertions that reduce moesin expression enhanced the NMJ overgrowth phenotype, indicating a role for Moesin in normal NMJ morphology. In addition to the NMJ overgrowth phenotype, expression of dominant-negative NSF2 is known to reduce the frequency of miniature excitatory junctional potentials and the amplitude of excitatory junctional potentials. We found that moesin coexpression did not restore the physiology of the mutant NSF2 phenotype. Together, our results demonstrate a role for moesin in regulating synaptic growth in the Drosophila NMJ and suggest that the effect of dominant-negative NSF2 on NMJ morphology and physiology may have different underlying molecular origins.

The E1 ubiquitin-activating enzyme Uba1 inDrosophilacontrols apoptosis autonomously and tissue growth non-autonomously

Ubiquitination is an essential process regulating turnover of proteins for basic cellular processes such as the cell cycle and cell death (apoptosis). Ubiquitination is initiated by ubiquitin-activating enzymes (E1), which activate and transfer ubiquitin to ubiquitin-conjugating enzymes (E2). Conjugation of target proteins with ubiquitin is then mediated by ubiquitin ligases (E3). Ubiquitination has been well characterized using mammalian cell lines and yeast genetics. However, the consequences of partial or complete loss of ubiquitin conjugation in a multi-cellular organism are not well understood. Here, we report the characterization of Uba1, the only E1 in Drosophila. We found that weak and strong Uba1 alleles behave genetically differently with sometimes opposing phenotypes. Whereas weak Uba1 alleles protect cells from cell death, clones of strong Uba1 alleles are highly apoptotic. Strong Uba1 alleles cause cell cycle arrest which correlates with failure to reduce cyclin levels. Surprisingly, clones of strong Uba1 mutants stimulate neighboring wild-type tissue to undergo cell division in a non-autonomous manner giving rise to overgrowth phenotypes of the mosaic fly. We demonstrate that the non-autonomous overgrowth is caused by failure to downregulate Notch signaling in Uba1 mutant clones. In summary, the phenotypic analysis of Uba1 demonstrates that impaired ubiquitin conjugation has significant consequences for the organism, and may implicate Uba1 as a tumor suppressor gene.

YAP1 Increases Organ Size and Expands Undifferentiated Progenitor Cells

The mechanisms that regulate mammalian organ size are poorly understood. It is unclear whether the pathways that control organ size also impinge on stem/progenitor cells. A highly expressed gene in stem cells is YAP1, the ortholog of Drosophila Yorkie, a downstream component of the Hippo pathway. Mutations in components of this pathway produce tissue overgrowth phenotypes in the fly whereas mammalian orthologs, like salvador, merlin, LATS, and YAP1, have been implicated in tumorigenesis. We report here that YAP1 increases organ size and causes aberrant tissue expansion in mice. YAP1 activation reversibly increases liver size more than 4-fold. In the intestine, expression of endogenous YAP1 is restricted to the progenitor/stem cell compartment, and activation of YAP1 expands multipotent undifferentiated progenitor cells, which differentiate upon cessation of YAP1 expression. YAP1 stimulates Notch signaling, and administration of gamma-secretase inhibitors suppressed the intestinal dysplasia caused by YAP1. Human colorectal cancers expressing higher levels of YAP1 share molecular aspects with YAP1-induced dysplastic growth in the mouse. Our data show that the Hippo signaling pathway regulates organ size in mammals and can act on stem cell compartments, indicating a potential link between stem/progenitor cells, organ size, and cancer.

A cluster of translocation breakpoints in 2q37 is associated with overexpression of NPPC in patients with a similar overgrowth phenotype

Overexpression of the C-type natriuretic peptide, encoded by the NPPC gene in 2q37.1, was recently reported in a patient presenting an overgrowth phenotype and a balanced t(2;7)(q37.1;q21.3) translocation. We present clinical, cytogenetic, and molecular data from two additional patients carrying balanced translocations involving the same 2q37.1 chromosome band and chromosomes 8 and 13, respectively. The clinical phenotype of these patients is very similar to the first patient described. In addition to the overgrowth syndrome, there is evidence of generalized cartilage dysplasia. In these two new cases, we found overexpression of NPPC, confirming that this unusual overgrowth phenotype in humans is due to the overexpression of this gene. The involvement of three different chromosomes and a cluster of breakpoints around the NPPC gene suggests that the overexpression of this gene in translocation patients could be due to its separation from a negative regulatory element located on chromosome 2, which would constitute a previously undescribed mutational mechanism.