Characterization of Human Pancreatic Islet Cells using a Single-Cell Western Blot Platform.

OBJECTIVE: Islet transplantation is an effective treatment for type 1 diabetes. However, transplant success depends on quick islet assessment because islets deteriorate 2-3 days after isolation. A new tool, single-cell Western blot (scWestern), offers results within one day. In this study, we aimed to test the suitability of scWestern to detect protein markers for beta (insulin), alpha (glucagon), and delta (somatostatin) cells, the three major endocrine cell types in islets. METHODS: We characterized the antibody specificity, signal intensity, and cell identification on the scWestern platform, and then compared the islet cell composition analysis between scWestern and immunohistochemistry performed by the Integrated Islet Distribution Program (IIDP). RESULTS: Islet cell composition is comparable for alpha and beta cells, but not delta cells. Protein expression levels of insulin, glucagon, and somatostatin in individual islet cells varied greatly, highlighting cell type heterogeneity. Surprisingly, scWestern revealed double-hormonal cells (~1%), co-expressing insulin and somatostatin or insulin and glucagon, in non-diabetic and non-obese adult human islets, which was confirmed by confocal immunofluorescence microscopy. CONCLUSIONS: These results demonstrate that each alpha, beta, and delta cells express varying levels of peptide hormones, and a small subpopulation co-expresses double hormones in normal human islets. The scWestern platform will enable timely assessment of beta cell mass in isolated islets before clinical transplantation.

Trefoil Factor 2 Expressed by the Murine Pancreatic Acinar Cells Is Required for the Development of Islets and for ß-Cell Function During Aging.

Exocrine-to-endocrine cross talk in the pancreas is crucial to maintain ß-cell function. However, the molecular mechanisms underlying this cross talk are largely undefined. Trefoil factor 2 (Tff2) is a secreted factor known to promote the proliferation of ß-cells in vitro, but its physiological role in vivo in the pancreas is unknown. Also, it remains unclear which pancreatic cell type expresses Tff2 protein. We therefore created a mouse model with a conditional knockout of Tff2 in the murine pancreas. We find that the Tff2 protein is preferentially expressed in acinar but not ductal or endocrine cells. Tff2 deficiency in the pancreas reduces ß-cell mass on embryonic day 16.5. However, homozygous mutant mice are born without a reduction of ß-cells and with acinar Tff3 compensation by day 7. When mice are aged to 1 year, both male and female homozygous and male heterozygous mutants develop impaired glucose tolerance without affected insulin sensitivity. Perifusion analysis reveals that the second phase of glucose-stimulated insulin secretion from islets is reduced in aged homozygous mutant compared with controls. Collectively, these results demonstrate a previously unknown role of Tff2 as an exocrine acinar cell-derived protein required for maintaining functional endocrine ß-cells in mice.

Biological hypoxia in pre-transplant human pancreatic islets induces transplant failure in diabetic mice.

Evaluating the quality of isolated human islets before transplantation is crucial for predicting the success in treating Type 1 diabetes. The current gold standard involves time-intensive in vivo transplantation into diabetic immunodeficient mice. Given the susceptibility of isolated islets to hypoxia, we hypothesized that hypoxia present in islets before transplantation could indicate compromised islet quality, potentially leading to unfavorable outcomes. To test this hypothesis, we analyzed expression of 39 hypoxia-related genes in human islets from 85 deceased donors. We correlated gene expression profiles with transplantation outcomes in 327 diabetic mice, each receiving 1200 islet equivalents grafted into the kidney capsule. Transplantation outcome was post-transplant glycemic control based on area under the curve of blood glucose over 4 weeks. In linear regression analysis, DDIT4 (R = 0.4971, P < 0.0001), SLC2A8 (R = 0.3531, P = 0.0009) and HK1 (R = 0.3444, P = 0.0012) had the highest correlation with transplantation outcome. A multiple regression model of 11 genes increased the correlation (R = 0.6117, P < 0.0001). We conclude that assessing pre-transplant hypoxia in human islets via gene expression analysis is a rapid, viable alternative to conventional in vivo assessments. This approach also underscores the importance of mitigating pre-transplant hypoxia in isolated islets to improve the success rate of islet transplantation.

4S-fluorination of ProB29 in insulin lispro slows fibril formation.

Recombinant insulin is a life-saving therapeutic for millions of patients affected by diabetes mellitus. Standard mutagenesis has led to insulin variants with improved control of blood glucose; for instance, the fast-acting insulin lispro contains two point mutations that suppress dimer formation and expedite absorption. However, insulins undergo irreversible denaturation, a process accelerated for the insulin monomer. Here we replace ProB29 of insulin lispro with 4R-fluoroproline, 4S-fluoroproline, and 4,4-difluoroproline. All three fluorinated lispro variants reduce blood glucose in diabetic mice, exhibit similar secondary structure as measured by CD, and rapidly dissociate from the zinc- and resorcinol-bound hexamer upon dilution. Notably, however, we find that 4S-fluorination of ProB29 delays the formation of undesired insulin fibrils that can accumulate at the injection site in vivo and can complicate insulin production and storage. These results demonstrate how subtle molecular changes achieved through non-canonical amino acid mutagenesis can improve the stability of protein therapeutics.

Physiomimetic Fluidic Culture Platform on Microwell-Patterned Porous Collagen Scaffold for Human Pancreatic Islets.

Pancreatic islet transplantation is one of the clinical options for certain types of diabetes. However, difficulty in maintaining islets prior to transplantation limits the clinical expansion of islet transplantations. Our study introduces a dynamic culture platform developed specifically for primary human islets by mimicking the physiological microenvironment, including tissue fluidics and extracellular matrix support. We engineered the dynamic culture system by incorporating our distinctive microwell-patterned porous collagen scaffolds for loading isolated human islets, enabling vertical medium flow through the scaffolds. The dynamic culture system featured four 12 mm diameter islet culture chambers, each capable of accommodating 500 islet equivalents (IEQ) per chamber. This configuration calculates > five-fold higher seeding density than the conventional islet culture in flasks prior to the clinical transplantations (442 vs 86 IEQ/cm2). We tested our culture platform with three separate batches of human islets isolated from deceased donors for an extended period of 2 weeks, exceeding the limits of conventional culture methods for preserving islet quality. Static cultures served as controls. The computational simulation revealed that the dynamic culture reduced the islet volume exposed to the lethal hypoxia (< 10 mmHg) to ~1/3 of the static culture. Dynamic culture ameliorated the morphological islet degradation in long-term culture and maintained islet viability, with reduced expressions of hypoxia markers. Furthermore, dynamic culture maintained the islet metabolism and insulin-secreting function over static culture in a long-term culture. Collectively, the physiological microenvironment-mimetic culture platform supported the viability and quality of isolated human islets at high-seeding density. Such a platform has a high potential for broad applications in cell therapies and tissue engineering, including extended islet culture prior to clinical islet transplantations and extended culture of stem cell-derived islets for maturation.

Activation of ductal progenitor-like cells from adult human pancreas requires extracellular matrix protein signaling.

Ductal progenitor-like cells are a sub-population of ductal cells in the adult human pancreas that have the potential to contribute to regenerative medicine. However, the microenvironmental cues that regulate their activation are poorly understood. Here, we establish a 3-dimensional suspension culture system containing six defined soluble factors in which primary human ductal progenitor-like and ductal non-progenitor cells survive but do not proliferate. Expansion and polarization occur when suspension cells are provided with a low concentration (5% v/v) of Matrigel, a sarcoma cell product enriched in many extracellular matrix (ECM) proteins. Screening of ECM proteins identified that collagen IV can partially recapitulate the effects of Matrigel. Inhibition of integrin α1ß1, a major collagen IV receptor, negates collagen IV- and Matrigel-stimulated effects. These results demonstrate that collagen IV is a key ECM protein that stimulates the expansion and polarization of human ductal progenitor-like and ductal non-progenitor cells via integrin α1ß1 receptor signaling.

Rare, Tightly-Bound, Multi-Cellular Clusters in the Pancreatic Ducts of Adult Mice Function Like Progenitor Cells and Survive and Proliferate After Acinar Cell Injury.

Pancreatic ductal progenitor cells have been proposed to contribute to adult tissue maintenance and regeneration after injury, but the identity of such ductal cells remains elusive. Here, from adult mice, we identify a near homogenous population of ductal progenitor-like clusters, with an average of 8 cells per cluster. They are a rare subpopulation, about 0.1% of the total pancreatic cells, and can be sorted using a fluorescence-activated cell sorter with the CD133highCD71lowFSCmid-high phenotype. They exhibit properties in self-renewal and tri-lineage differentiation (including endocrine-like cells) in a unique 3-dimensional colony assay system. An in vitro lineage tracing experiment, using a novel HprtDsRed/+ mouse model, demonstrates that a single cell from a cluster clonally gives rise to a colony. Droplet RNAseq analysis demonstrates that these ductal clusters express embryonic multipotent progenitor cell markers Sox9, Pdx1, and Nkx6-1, and genes involved in actin cytoskeleton regulation, inflammation responses, organ development, and cancer. Surprisingly, these ductal clusters resist prolonged trypsin digestion in vitro, preferentially survive in vivo after a severe acinar cell injury and become proliferative within 14 days post-injury. Thus, the ductal clusters are the fundamental units of progenitor-like cells in the adult murine pancreas with implications in diabetes treatment and tumorigenicity.

Incorporation of Aliphatic Proline Residues into Recombinantly Produced Insulin.

Analogs of proline can be used to expand the chemical space about the residue while maintaining its uniquely restricted conformational space. Here, we demonstrate the incorporation of 4R-methylproline, 4S-methylproline, and 4-methyleneproline into recombinant insulin expressed in Escherichia coli. These modified proline residues, introduced at position B28, change the biophysical properties of insulin: Incorporation of 4-methyleneproline at B28 accelerates fibril formation, while 4-methylation speeds dissociation from the pharmaceutically formulated hexamer. This work expands the scope of proline analogs amenable to incorporation into recombinant proteins and demonstrates how noncanonical amino acid mutagenesis can be used to engineer the therapeutically relevant properties of protein drugs.

Changes in the gut microbiota of NOD mice in response to an oral Salmonella-based vaccine against type 1 diabetes.

We developed an oral Salmonella-based vaccine that prevents and reverses diabetes in non-obese diabetic (NOD) mice. Related to this, the gastrointestinal tract harbors a complex dynamic population of microorganisms, the gut microbiome, that influences host homeostasis and metabolism. Changes in the gut microbiome are associated with insulin dysfunction and type 1 diabetes (T1D). Oral administration of diabetic autoantigens as a vaccine can restore immune balance. However, it was not known if a Salmonella-based vaccine would impact the gut microbiome. We administered a Salmonella-based vaccine to prediabetic NOD mice. Changes in the gut microbiota and associated metabolome were assessed using next-generation sequencing and gas chromatography-mass spectrometry (GC-MS). The Salmonella-based vaccine did not cause significant changes in the gut microbiota composition immediately after vaccination although at 30 days post-vaccination changes were seen. Additionally, no changes were noted in the fecal mycobiome between vaccine- and control/vehicle-treated mice. Significant changes in metabolic pathways related to inflammation and proliferation were found after vaccine administration. The results from this study suggest that an oral Salmonella-based vaccine alters the gut microbiome and metabolome towards a more tolerant composition. These results support the use of orally administered Salmonella-based vaccines that induced tolerance after administration.

Methylcellulose colony assay and single-cell micro-manipulation reveal progenitor-like cells in adult human pancreatic ducts.

Progenitor cells capable of self-renewal and differentiation in the adult human pancreas are an under-explored resource for regenerative medicine. Using micro-manipulation and three-dimensional colony assays we identify cells within the adult human exocrine pancreas that resemble progenitor cells. Exocrine tissues were dissociated into single cells and plated into a colony assay containing methylcellulose and 5% Matrigel. A subpopulation of ductal cells formed colonies containing differentiated ductal, acinar, and endocrine lineage cells, and expanded up to 300-fold with a ROCK inhibitor. When transplanted into diabetic mice, colonies pre-treated with a NOTCH inhibitor gave rise to insulin-expressing cells. Both colonies and primary human ducts contained cells that simultaneously express progenitor transcription factors SOX9, NKX6.1, and PDX1. In addition, in silico analysis identified progenitor-like cells within ductal clusters in a single-cell RNA sequencing dataset. Therefore, progenitor-like cells capable of self-renewal and tri-lineage differentiation either pre-exist in the adult human exocrine pancreas, or readily adapt in culture.

Microwell culture platform maintains viability and mass of human pancreatic islets.

Background: Transplantation of the human pancreatic islets is a promising approach for specific types of diabetes to improve glycemic control. Although effective, there are several issues that limit the clinical expansion of this treatment, including difficulty in maintaining the quality and quantity of isolated human islets prior to transplantation. During the culture, we frequently observe the multiple islets fusing together into large constructs, in which hypoxia-induced cell damage significantly reduces their viability and mass. In this study, we introduce the microwell platform optimized for the human islets to prevent unsolicited fusion, thus maintaining their viability and mass in long-term cultures. Method: Human islets are heterogeneous in size; therefore, two different-sized microwells were prepared in a 35 mm-dish format: 140 µm × 300 µm-microwells for <160 µm-islets and 200 µm × 370 µm-microwells for >160 µm-islets. Human islets (2,000 islet equivalent) were filtered through a 160 µm-mesh to prepare two size categories for subsequent two week-cultures in each microwell dish. Conventional flat-bottomed 35 mm-dishes were used for non-filtered islets (2,000 islet equivalent/2 dishes). Post-cultured islets are collected to combine in each condition (microwells and flat) for the comparisons in viability, islet mass, morphology, function and metabolism. Islets from three donors were independently tested. Results: The microwell platform prevented islet fusion during culture compared to conventional flat bottom dishes, which improved human islet viability and mass. Islet viability and mass on the microwells were well-maintained and comparable to those in pre-culture, while flat bottom dishes significantly reduced islet viability and mass in two weeks. Morphology assessed by histology, insulin-secreting function and metabolism by oxygen consumption did not exhibit the statistical significance among the three different conditions. Conclusion: Microwell-bottomed dishes maintained viability and mass of human islets for two weeks, which is significantly improved when compared to the conventional flat-bottomed dishes.

Isolation and Characterization of Colony-Forming Progenitor Cells from Adult Pancreas.

Obtaining, growing, and analysis of pancreatic progenitor cells. Adult stem and progenitor cells have been successfully used for cell-based therapies such as transplantation of hematopoietic stem cells for various diseases. Whether stem and progenitor cells in the adult pancreas can be identified and used for replacement therapy has been a highly controversial topic. To address this controversy, our laboratory has developed in vitro colony assays to detect and characterize individual pancreatic stem and progenitor-like cells. We found that a subpopulation of ductal cells in the adult murine pancreas has the abilities to self-renew and differentiate into multiple pancreatic lineages in three-dimensional space in methylcellulose-containing semisolid media. This protocol details the techniques used for culturing and characterizing these pancreatic stem and progenitor-like cells, which we have named pancreatic colony-forming units (PCFUs), as well as their progenies (colonies). The techniques presented here include dissociation of pancreases, sorting antibody-stained cells with a fluorescence-activated cell sorter, viral transduction of dissociated pancreatic cells, growth of PCFUs in semi-solid media, whole-mount immunostaining and Western blot analysis for proteins expressed in colonies, and kidney capsule transplantation of colonies for in vivo functional analysis.

Adult Murine Pancreatic Progenitors Require Epidermal Growth Factor and Nicotinamide for Self-Renewal and Differentiation in a Serum- and Conditioned Medium-Free Culture.

Adult pancreatic stem and progenitor cells may serve as an alternative source of insulin-secreting endocrine cells in cell replacement therapy for type 1 diabetes, but much remained unknown about these cells. We previously identified adult murine pancreatic progenitor-like cells that displayed in vitro self-renewal and tri-lineage differentiation activities in a three-dimensional colony/organoid assay containing 1% methylcellulose and 5% Matrigel. However, the presence of other undefined culture components, such as serum and conditioned medium, has prevented a complete understanding of the signals required for progenitor cell growth. Here, we have established a serum-free, conditioned medium-free colony assay with the inclusion of seven defined factors: epidermal growth factor (EGF), R-Spondin 1 (RSPO1), Noggin, nicotinamide, exendin-4, activin B, and vascular endothelial growth factor (VEGF)-A. The requirements for colony growth were characterized and we found that EGF and nicotinamide were necessary and sufficient for the colony growth and long-term self-renewal of these progenitors. However, the seven factor (7F) culture medium better induced colony size and self-renewal in long-term culture than EGF plus nicotinamide alone. Individual 3-week-old colonies grown in the 7F culture medium expressed ductal, acinar, and endocrine lineage markers, suggesting that tri-lineage differentiation of the tri-potent progenitors was occurring without genetic manipulation. A delayed inhibition of Notch signaling using small molecules in 2-week-old cultures enhanced endocrine gene expression in 3-week-old colonies. This better-defined colony assay system will enable our and other laboratories for in-depth mechanistic studies on the biology of these progenitor cells.

lolal Is an Evolutionarily New Epigenetic Regulator of dpp Transcription during Dorsal-Ventral Axis Formation.

Secreted ligands in the Dpp/BMP family drive dorsal-ventral (D/V) axis formation in all Bilaterian species. However, maternal factors regulating Dpp/BMP transcription in this process are largely unknown. We identified the BTB domain protein longitudinals lacking-like (lolal) as a modifier of decapentaplegic (dpp) mutations. We show that Lolal is evolutionarily related to the Trithorax group of chromatin regulators and that lolal interacts genetically with the epigenetic factor Trithorax-like during Dpp D/V signaling. Maternally driven Lolal(HA) is found in oocytes and translocates to zygotic nuclei prior to the point at which dpp transcription begins. lolal maternal and zygotic mutant embryos display significant reductions in dpp, pMad, and zerknullt expression, but they are never absent. The data suggest that lolal is required to maintain dpp transcription during D/V patterning. Phylogenetic data revealed that lolal is an evolutionarily new gene present only in insects and crustaceans. We conclude that Lolal is the first maternal protein identified with a role in dpp D/V transcriptional maintenance, that Lolal and the epigenetic protein Trithorax-like are essential for Dpp D/V signaling and that the architecture of the Dpp D/V pathway evolved in the arthropod lineage after the separation from vertebrates via the incorporation of new genes such as lolal.

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas.

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. However, it is still unknown whether stem and progenitor cells exist in the adult pancreas. Research strategies using cre-lox lineage-tracing in adult mice have yielded results that either support or refute the idea that beta cells can be generated from the ducts, the presumed location where adult pancreatic progenitors may reside. These in vivo cre-lox lineage-tracing methods, however, cannot answer the questions of self-renewal and multi-lineage differentiation-two criteria necessary to define a stem cell. To begin addressing this technical gap, we devised 3-dimensional colony assays for pancreatic progenitors. Soon after our initial publication, other laboratories independently developed a similar, but not identical, method called the organoid assay. Compared to the organoid assay, our method employs methylcellulose, which forms viscous solutions that allow the inclusion of extracellular matrix proteins at low concentrations. The methylcellulose-containing assays permit easier detection and analyses of progenitor cells at the single-cell level, which are critical when progenitors constitute a small sub-population, as is the case for many adult organ stem cells. Together, results from several laboratories demonstrate in vitro self-renewal and multi-lineage differentiation of pancreatic progenitor-like cells from mice. The current protocols describe two methylcellulose-based colony assays to characterize mouse pancreatic progenitors; one contains a commercial preparation of murine extracellular matrix proteins and the other an artificial extracellular matrix protein known as a laminin hydrogel. The techniques shown here are 1) dissociation of the pancreas and sorting of CD133(+)Sox9/EGFP(+) ductal cells from adult mice, 2) single cell manipulation of the sorted cells, 3) single colony analyses using microfluidic qRT-PCR and whole-mount immunostaining, and 4) dissociation of primary colonies into single-cell suspensions and re-plating into secondary colony assays to assess self-renewal or differentiation.

Cells with surface expression of CD133highCD71low are enriched for tripotent colony-forming progenitor cells in the adult murine pancreas.

Progenitor cells in the adult pancreas are potential sources of endocrine beta cells for treating type 1 diabetes. Previously, we identified tri-potent progenitor cells in the adult (2-4month-old) murine pancreas that were capable of self-renewal and differentiation into duct, acinar, and endocrine cells in vitro. These progenitor cells were named pancreatic colony-forming units (PCFUs). However, because PCFUs are a minor population in the pancreas (~1%) they are difficult to study. To enrich PCFUs, strategies using cell-surface marker analyses and fluorescence-activated cell sorting were developed. We found that CD133(high)CD71(low) cells, but not other cell populations, enriched PCFUs by up to 30 fold compared to the unsorted cells. CD133(high)CD71(low) cells generated primary, secondary, and subsequent colonies when serially re-plated in Matrigel-containing cultures, suggesting self-renewal abilities. In the presence of a laminin hydrogel, CD133(high)CD71(low) cells gave rise to colonies that contained duct, acinar, and Insulin(+)Glucagon(+) double-hormonal endocrine cells. Colonies from the laminin hydrogel culture were implanted into diabetic mice, and five weeks later duct, acinar, and Insulin(+)Glucagon(-) cells were detected in the grafts, demonstrating tri-lineage differentiation potential of CD133(high)CD71(low) cells. These CD133(high)CD71(low) cells will enable future studies of putative adult pancreas stem cells in vivo.

New gene evolution in the bonus-TIF1-γ/TRIM33 family impacted the architecture of the vertebrate dorsal-ventral patterning network.

Uncovering how a new gene acquires its function and understanding how the function of a new gene influences existing genetic networks are important topics in evolutionary biology. Here, we demonstrate nonconservation for the embryonic functions of Drosophila Bonus and its newest vertebrate relative TIF1-γ/TRIM33. We showed previously that TIF1-γ/TRIM33 functions as an ubiquitin ligase for the Smad4 signal transducer and antagonizes the Bone Morphogenetic Protein (BMP) signaling network underlying vertebrate dorsal-ventral axis formation. Here, we show that Bonus functions as an agonist of the Decapentaplegic (Dpp) signaling network underlying dorsal-ventral axis formation in flies. The absence of conservation for the roles of Bonus and TIF1-γ/TRIM33 reveals a shift in the dorsal-ventral patterning networks of flies and mice, systems that were previously considered wholly conserved. The shift occurred when the new gene TIF1-γ/TRIM33 replaced the function of the ubiquitin ligase Nedd4L in the lineage leading to vertebrates. Evidence of this replacement is our demonstration that Nedd4 performs the function of TIF1-γ/TRIM33 in flies during dorsal-ventral axis formation. The replacement allowed vertebrate Nedd4L to acquire novel functions as a ubiquitin ligase of vertebrate-specific Smad proteins. Overall our data reveal that the architecture of the Dpp/BMP dorsal-ventral patterning network continued to evolve in the vertebrate lineage, after separation from flies, via the incorporation of new genes.

Drosophila CORL is required for Smad2-mediated activation of Ecdysone Receptor expression in the mushroom body.

CORL proteins (FUSSEL/SKOR proteins in humans) are related to Sno/Ski oncogenes but their developmental roles are unknown. We have cloned Drosophila CORL and show that its expression is restricted to distinct subsets of cells in the central nervous system. We generated a deletion of CORL and noted that homozygous individuals rarely survive to adulthood. Df(4)dCORL adult escapers display mushroom body (MB) defects and Df(4)dCORL larvae are lacking Ecdysone Receptor (EcR-B1) expression in MB neurons. This is phenocopied in CORL-RNAi and Smad2-RNAi clones in wild-type larvae. Furthermore, constitutively active Baboon (type I receptor upstream of Smad2) cannot stimulate EcR-B1 MB expression in Df(4)dCORL larvae, which demonstrates a formal requirement for CORL in Smad2 signaling. Studies of mouse Corl1 (Skor1) revealed that it binds specifically to Smad3. Overall, the data suggest that CORL facilitates Smad2 activity upstream of EcR-B1 in the MB. The conservation of neural expression and strong sequence homology of all CORL proteins suggests that this is a new family of Smad co-factors.

Fat facets deubiquitylation of Medea/Smad4 modulates interpretation of a Dpp morphogen gradient.

The ability of secreted Transforming Growth Factor ß (TGFß) proteins to act as morphogens dictates that their influence be strictly regulated. Here, we report that maternally contributed fat facets (faf; a homolog of USP9X/FAM) is essential for proper interpretation of the zygotic Decapentaplegic (Dpp) morphogen gradient that patterns the embryonic dorsal-ventral axis. The data suggest that the loss of faf reduces the activity of Medea (a homolog of Smad4) below the minimum necessary for adequate Dpp signaling and that this is likely due to excessive ubiquitylation on a specific lysine. This study supports the hypothesis that the control of cellular responsiveness to TGFß signals at the level of Smad4 ubiquitylation is a conserved mechanism required for proper implementation of a morphogen gradient.

Wg signaling via Zw3 and mad restricts self-renewal of sensory organ precursor cells in Drosophila.

It is well known that the Dpp signal transducer Mad is activated by phosphorylation at its carboxy-terminus. The role of phosphorylation on other regions of Mad is not as well understood. Here we report that the phosphorylation of Mad in the linker region by the Wg antagonist Zw3 (homolog of vertebrate Gsk3-ß) regulates the development of sensory organs in the anterior-dorsal quadrant of the wing. Proneural expression of Mad-RNA interference (RNAi) or a Mad transgene with its Zw3/Gsk3-ß phosphorylation sites mutated (MGM) generated wings with ectopic sensilla and chemosensory bristle duplications. Studies with pMad-Gsk (an antibody specific to Zw3/Gsk3-ß-phosphorylated Mad) in larval wing disks revealed that this phosphorylation event is Wg dependent (via an unconventional mechanism), is restricted to anterior-dorsal sensory organ precursors (SOP) expressing Senseless (Sens), and is always co-expressed with the mitotic marker phospho-histone3. Quantitative analysis in both Mad-RNAi and MGM larval wing disks revealed a significant increase in the number of Sens SOP. We conclude that the phosphorylation of Mad by Zw3 functions to prevent the self-renewal of Sens SOP, perhaps facilitating their differentiation via asymmetric division. The conservation of Zw3/Gsk3-ß phosphorylation sites in vertebrate homologs of Mad (Smads) suggests that this pathway, the first transforming growth factor ß-independent role for any Smad protein, may be widely utilized for regulating mitosis during development.