Matthew Kofron

Global analysis of the transcriptional network controlling Xenopus endoderm formation

A conserved molecular pathway has emerged controlling endoderm formation in Xenopus zebrafish and mice. Key genes in this pathway include Nodal ligands and transcription factors of the Mix-like paired homeodomain class, Gata4-6 zinc-finger factors and Sox17 HMG domain proteins. Although a linear epistatic pathway has been proposed, the precise hierarchical relationships between these factors and their downstream targets are largely unresolved. Here, we have used a combination of microarray analysis and loss-of-function experiments to examine the global regulatory network controlling Xenopus endoderm formation. We identified over 300 transcripts enriched in the gastrula endoderm, including most of the known endoderm regulators and over a hundred uncharacterized genes. Surprisingly only 10% of the endoderm transcriptome is regulated as predicted by the current linear model. We find that Nodal genes, Mixer and Sox17 have both shared and distinct sets of downstream targets, and that a number of unexpected autoregulatory loops exist between Sox17 and Gata4-6, between Sox17 and Bix1/Bix2/Bix4, and between Sox17 and Xnr4. Furthermore, we find that Mixer does not function primarily via Sox17 as previously proposed. These data provides new insight into the complexity of endoderm formation and will serve as valuable resource for establishing a complete endoderm gene regulatory network.

Jun NH2-terminal kinase (JNK) prevents nuclear β-catenin accumulation and regulates axis formation in Xenopus embryos

Jun NH2-terminal kinases (JNKs) regulate convergent extension movements in Xenopus embryos through the noncanonical Wnt/planar cell polarity pathway. In addition, there is a high level of maternal JNK activity spanning from oocyte maturation until the onset of gastrulation that has no defined functions. Here, we show that maternal JNK activation requires Dishevelled and JNK is enriched in the nucleus of Xenopus embryos. Although JNK activity is not required for the glycogen synthase kinase-3-mediated degradation of β-catenin, inhibition of the maternal JNK signaling by morpholino-antisense oligos causes hyperdorsalization of Xenopus embryos and ectopic expression of the Wnt/β-catenin target genes. These effects are associated with an increased level of nuclear and nonmembrane-bound β-catenin. Moreover, ventral injection of the constitutive-active Jnk mRNA blocks β-catenin-induced axis duplication, and dorsal injection of active Jnk mRNA into Xenopus embryos decreases the dorsal marker gene expression. In mammalian cells, activation of JNK signaling reduces Wnt3A-induced and β-catenin-mediated gene expression. Furthermore, activation of JNK signaling rapidly induces the nuclear export of β-catenin. Taken together, these results suggest that JNK antagonizes the canonical Wnt pathway by regulating the nucleocytoplasmic transport of β-catenin rather than its cytoplasmic stability. Thus, the high level of sustained maternal JNK activity in early Xenopus embryos may provide a timing mechanism for controlling the dorsal axis formation.

Targeting the Human Papillomavirus E6 and E7 Oncogenes through Expression of the Bovine Papillomavirus Type 1 E2 Protein Stimulates Cellular Motility

ABSTRACT Expression of the high-risk human papillomavirus (HPV) E6 and E7 oncogenes is essential for the initiation and maintenance of cervical cancer. The repression of both was previously shown to result in activation of their respective tumor suppressor targets, p53 and pRb, and subsequent senescence induction in cervical cancer cells. Consequently, viral oncogene suppression is a promising approach for the treatment of HPV-positive tumors. One well-established method of E6/E7 repression involves the reexpression of the viral E2 protein which is usually deleted in HPV-positive cancer cells. Here, we show that, surprisingly, bovine papillomavirus type 1 (BPV1) E2 but not RNA interference-mediated E6/E7 repression in HPV-positive cervical cancer cells stimulates cellular motility and invasion. Migration correlated with the dynamic formation of cellular protrusions and was dependent upon cell-to-cell contact. While E2-expressing migratory cells were senescent, migration was not a general feature of cellular senescence or cell cycle arrest and was specifically observed in HPV-positive cervical cancer cells. Interestingly, E2-expressing cells not only were themselves motile but also conferred increased motility to admixed HeLa cervical cancer cells. Together, our data suggest that repression of the viral oncogenes by E2 stimulates the motility of E6/E7-targeted cells as well as adjacent nontargeted cancer cells, thus raising the possibility that E2 expression may unfavorably increase the local invasiveness of HPV-positive tumors.

CRIM1 Complexes with ß-catenin and Cadherins, Stabilizes Cell-Cell Junctions and Is Critical for Neural Morphogenesis

In multicellular organisms, morphogenesis is a highly coordinated process that requires dynamically regulated adhesion between cells. An excellent example of cellular morphogenesis is the formation of the neural tube from the flattened epithelium of the neural plate. Cysteine-rich motor neuron protein 1 (CRIM1) is a single-pass (type 1) transmembrane protein that is expressed in neural structures beginning at the neural plate stage. In the frog Xenopus laevis, loss of function studies using CRIM1 antisense morpholino oligonucleotides resulted in a failure of neural development. The CRIM1 knockdown phenotype was, in some cases, mild and resulted in perturbed neural fold morphogenesis. In severely affected embryos there was a dramatic failure of cell adhesion in the neural plate and complete absence of neural structures subsequently. Investigation of the mechanism of CRIM1 function revealed that it can form complexes with ß-catenin and cadherins, albeit indirectly, via the cytosolic domain. Consistent with this, CRIM1 knockdown resulted in diminished levels of cadherins and ß-catenin in junctional complexes in the neural plate. We conclude that CRIM1 is critical for cell-cell adhesion during neural development because it is required for the function of cadherin-dependent junctions.

Foxi2 Is an Animally Localized Maternal mRNA in Xenopus , and an Activator of the Zygotic Ectoderm Activator Foxi1e

Foxi1e is a zygotic transcription factor that is essential for the expression of early ectodermal genes. It is expressed in a highly specific pattern, only in the deep cell layers of the animal hemisphere, and in a mosaic pattern in which expressing cells are interspersed with non-expressing cells. Previous work has shown that several signals in the blastula control this expression pattern, including nodals, the TGFβ family member Vg1, and Notch. However, these are all inhibitory, which raises the question of what activates Foxi1e. In this work, we show that a related Forkhead family protein, Foxi2, is a maternal activator of Foxi1e. Foxi2 mRNA is maternally encoded, and highly enriched in animal hemisphere cells of the blastula. ChIP assays show that it acts directly on upstream regulatory elements of Foxi1e. Its effect is specific, since animal cells depleted of Foxi2 are able to respond normally to mesoderm inducing signals from vegetal cells. Foxi2 thus acts as a link between the oocyte and the early pathway to ectoderm, in a similar fashion to the vegetally localized VegT acts to initiate endoderm and mesoderm formation.

Localization and stretch-dependence of lung elastase activity in development and compensatory growth

Synthesis and remodeling of the lung matrix is necessary for primary and compensatory lung growth. Because cyclic negative force is applied to developing lung tissue during the respiratory cycle, we hypothesized that stretch is a critical regulator of lung matrix remodeling. By using quantitative image analysis of whole-lung and whole-lobe elastin in situ zymography images, we demonstrated that elastase activity increased twofold during the alveolar stage of postnatal lung morphogenesis in the mouse. Remodeling was restricted to alveolar walls and ducts and was nearly absent in dense elastin band structures. In the mouse pneumonectomy model of compensatory lung growth, elastase activity increased threefold, peaking at 14 days postpneumonectomy and was higher in the accessory lobe compared with other lobes. Remodeling during normal development and during compensatory lung growth was different with increased major airway and pulmonary arterial remodeling during development but not regeneration, and with homogenous remodeling throughout the parenchyma during development, but increased remodeling only in subpleural regions during compensatory lung growth. Left lung wax plombage prevented increased lung elastin during compensatory lung growth. To test whether the adult lung retains an innate capacity to remodel elastin, we developed a confocal microscope-compatible stretching device. In ex vivo adult mouse lung sections, lung elastase activity increased exponentially with strain and in peripheral regions of lung more than in central regions. Our study demonstrates that lung elastase activity is stretch-dependent and supports a model in which externally applied forces influence the composition, structure, and function of the matrix during periods of alveolar septation.

Long- and short-range signals control the dynamic expression of an animal hemisphere-specific gene in Xenopus

Little is known of the control of gene expression in the animal hemisphere of the Xenopus embryo. Here we show that expression of FoxI1e, a gene essential for normal ectoderm formation, is expressed regionally within the animal hemisphere, in a highly dynamic fashion. In situ hybridization shows that FoxI1e is expressed in a wave-like fashion that is initiated on the dorsal side of the animal hemisphere, extends across to the ventral side by the mid-gastrula stage, and is then turned off in the dorsal ectoderm, the neural plate, at the neurula stage. It is confined to the inner layers of cells in the animal cap, and is expressed in a mosaic fashion throughout. We show that this dynamic pattern of expression is controlled by both short- and long-range signals. Notch signaling controls both the mosaic, and dorsal/ventral changes in expression, and is controlled, in turn, by Vg1 signaling from the vegetal mass. FoxI1e expression is also regulated by nodal signaling downstream of VegT. Canonical Wnt signaling contributes only to late changes in the FoxI1e expression pattern. These results provide new insights into the roles of vegetally localized mRNAs in controlling zygotic genes expressed in the animal hemisphere by long-range signaling. They also provide novel insights into the role of Notch signaling at the earliest stages of vertebrate development.

The dendritic cell–T helper 17–macrophage axis controls cholangiocyte injury and disease progression in murine and human biliary atresia

Biliary atresia (BA) is a fibroinflammatory obstruction of the extrahepatic biliary tree in neonates. While intrahepatic bile duct proliferation is universal at diagnosis, bile duct paucity develops later. We hypothesized that polarized T helper lymphocyte responses orchestrate progression of intrahepatic biliary injury in this disease. Interleukin 17A (IL‐17A)‐green fluorescent protein, cluster of differentiation 11c (CD11c)/diphtheria toxin receptor, and IL‐17 receptor A−/− mice were used to examine T‐lymphocyte polarization, inflammatory leukocyte recruitment, and biliary injury in rhesus rotavirus–induced BA. Multiparameter flow cytometry and automated image analysis of immunostaining were applied to liver tissue samples from infants with BA. In the mouse model, activated CD4+ lymphocytes started to emerge in the liver on day 8 after viral challenge, while innate immune responses were waning. Plasma IL‐17A levels rose concomitantly with hepatic accumulation of T helper 17 lymphocytes and myeloid dendritic cells. Targeted depletion of CD11c+ dendritic cells diminished hepatic IL‐17A production and ameliorated intrahepatic bile duct injury. Recombinant IL‐17A induced expression of chemokine (C‐C motif) ligand 2 in neonatal cholangiocytes in vitro, and blockade of the corresponding chemokine (C‐C motif) receptor 2 reduced recruitment of inflammatory macrophages to the liver in vivo. Genetic disruption of IL‐17A signaling was associated with down‐regulation of hepatic Ccl2/Ccr2 messenger RNA expression, reduced infiltration of the liver with inflammatory Ly6Chi macrophages, and improved survival. In the liver of infants with BA, cholangiocytes were found to express IL‐17 receptor A, and the prevalence of IL‐17A+ cells was positively correlated with the degree of CD68+ macrophage infiltration at diagnosis. Hepatic CD4+ lymphocytes were chief producers of IL‐17A in patients with progressive disease undergoing liver transplantation. Conclusion: These findings identify the dendritic cell–T helper 17–macrophage axis as a target for the development of strategies to block progression of intrahepatic bile duct injury in patients with BA. (Hepatology 2017;65:174‐188).

Sox17 Regulates Insulin Secretion in the Normal and Pathologic Mouse β Cell

SOX17 is a key transcriptional regulator that can act by regulating other transcription factors including HNF1β and FOXA2, which are known to regulate postnatal β cell function. Given this, we investigated the role of SOX17 in the developing and postnatal pancreas and found a novel role for SOX17 in regulating insulin secretion. Deletion of the Sox17 gene in the pancreas (Sox17-paLOF) had no observable impact on pancreas development. However, Sox17-paLOF mice had higher islet proinsulin protein content, abnormal trafficking of proinsulin, and dilated secretory organelles suggesting that Sox17-paLOF adult mice are prediabetic. Consistant with this, Sox17-paLOF mice were more susceptible to aged-related and high fat diet-induced hyperglycemia and diabetes. Overexpression of Sox17 in mature β cells using Ins2-rtTA driver mice resulted in precocious secretion of proinsulin. Transcriptionally, SOX17 appears to broadly regulate secretory networks since a 24-hour pulse of SOX17 expression resulted in global transcriptional changes in factors that regulate hormone transport and secretion. Lastly, transient SOX17 overexpression was able to reverse the insulin secretory defects observed in MODY4 animals and restored euglycemia. Together, these data demonstrate a critical new role for SOX17 in regulating insulin trafficking and secretion and that modulation of Sox17-regulated pathways might be used therapeutically to improve cell function in the context of diabetes.

AMP kinase promotes glioblastoma bioenergetics and tumour growth

Stress is integral to tumour evolution, and cancer cell survival depends on stress management. We found that cancer-associated stress chronically activates the bioenergetic sensor AMP kinase (AMPK) and, to survive, tumour cells hijack an AMPK-regulated stress response pathway conserved in normal cells. Analysis of The Cancer Genome Atlas data revealed that AMPK isoforms are highly expressed in the lethal human cancer glioblastoma (GBM). We show that AMPK inhibition reduces viability of patient-derived GBM stem cells (GSCs) and tumours. In stressed (exercised) skeletal muscle, AMPK is activated to cooperate with CREB1 (cAMP response element binding protein-1) and promote glucose metabolism. We demonstrate that oncogenic stress chronically activates AMPK in GSCs that coopt the AMPK–CREB1 pathway to coordinate tumour bioenergetics through the transcription factors HIF1α and GABPA. Finally, we show that adult mice tolerate systemic deletion of AMPK, supporting the use of AMPK pharmacological inhibitors in the treatment of GBM.Signalling by the energy sensor kinase AMPK is generally tumour suppressive, but Chhipa et al. show that AMPK is upregulated in glioblastoma, where it phosphorylates CREB1 to enhance HIF1α and GABPA transcription and to support tumour bioenergetics.