Ngn3 is recognized as a regulator of pancreatic endocrine formation, and Notch signaling as an important negative regulator Ngn3 gene expression. By conditionally controlling expression of Ngn3 in the pancreas, we find that these two signaling components are dynamically linked. This connection involves transcriptional repression as previously shown, but also incorporates a novel post-translational mechanism. In addition to its ability to promote endocrine fate, we provide evidence of a competing ability of Ngn3 in the patterning of multipotent progenitor cells in turn controlling the formation of ducts. On one hand, Ngn3 cell-intrinsically activates endocrine target genes; on the other, Ngn3 cell-extrinsically promotes lateral signaling via the Dll1>Notch>Hes1 pathway which substantially limits its ability to sustain endocrine formation. Prior to endocrine commitment, the Ngn3-mediated activation of the Notch>Hes1 pathway impacts formation of the trunk domain in the pancreas causing multipotent progenitors to lose acinar, while gaining endocrine and ductal, competence. The subsequent selection of fate from such bipotential progenitors is then governed by lateral inhibition, where Notch>Hes1-mediated Ngn3 protein destabilization serves to limit endocrine differentiation by reducing cellular levels of Ngn3. This system thus allows for rapid dynamic changes between opposing bHLH proteins in cells approaching a terminal differentiation event. Inhibition of Notch signaling leads to Ngn3 protein stabilization in the normal mouse pancreas explants. We conclude that the mutually exclusive expression pattern of Ngn3/Hes1 proteins in the mammalian pancreas is partially controlled through Notch-mediated post-translational regulation and we demonstrate that the formation of insulin-producing beta-cells can be significantly enhanced upon induction of a pro-endocrine drive combined with the inhibition of Notch processing.

Background & AimsThe genetic specification of the compartmentalized pancreatic acinar/centroacinar unit is poorly understood. Growth factor independence-1 (Gfi1) is a zinc finger transcriptional repressor that regulates hematopoietic stem cell maintenance, pre-T-cell differentiation, formation of granulocytes, inner ear hair cells, and the development of secretory cell types in the intestine. As GFI1/Gfi1 is expressed in human and rodent pancreas, we characterized the potential function of Gfi1 in mouse pancreatic development.MethodsGfi1 knockout mice were analyzed at histological and molecular levels, including qRT-PCR, in situ hybridization, immunohistochemistry, and electron microscopy.ResultsLoss of Gfi1 impacted formation and structure of the pancreatic acinar/centroacinar unit. Histologic and ultrastructural analysis of Gfi1-null pancreas revealed specific defects at the level of pancreatic acinar cells as well as the centroacinar cells (CACs) in Gfi1−/− mice when compared with wild-type littermates. Pancreatic endocrine differentiation, islet architecture, and function were unaffected. Organ domain patterning and the formation of ductal cells occurred normally during the murine secondary transition (E13.5–E14.5) in the Gfi1−/− pancreas. However, at later gestational time points (E18.5), expression of cellular markers for CACs was substantially reduced in Gfi1−/− mice, corroborated by electron microscopy imaging of the acinar/centroacinar unit. The reduction in CACs was correlated with an exocrine organ defect. Postnatally, Gfi1 deficiency resulted in severe pancreatic acinar dysplasia, including loss of granulation, autolytic vacuolation, and a proliferative and apoptotic response.ConclusionsGfi1 plays an important role in regulating the development of pancreatic CACs and the function of pancreatic acinar cells.

Wnt proteins are secreted hydrophobic glycoproteins that act over long distances through poorly understood mechanisms. We discovered that Wnt7a is secreted on extracellular vesicles (EVs) following muscle injury. Structural analysis identified the motif responsible for Wnt7a secretion on EVs that we term the Exosome Binding Peptide (EBP). Addition of the EBP to an unrelated protein directed secretion on EVs. Disruption of palmitoylation, knockdown of WLS, or deletion of the N-terminal signal peptide did not affect Wnt7a secretion on purified EVs. Bio-ID analysis identified Coatomer proteins as candidates responsible for loading Wnt7a onto EVs. The crystal structure of EBP bound to the COPB2 coatomer subunit, the binding thermodynamics, and mutagenesis experiments, together demonstrate that a dilysine motif in the EBP mediates binding to COPB2. Other Wnts contain functionally analogous structural motifs. Mutation of the EBP results in a significant impairment in the ability of Wnt7a to stimulate regeneration, indicating that secretion of Wnt7a on exosomes is critical for normal regeneration in vivo. Our studies have defined the structural mechanism that mediates binding of Wnt7a to exosomes and elucidated the singularity of long-range Wnt signalling.

Objective. To develop a novel deep learning-based 3D in vivo dose reconstruction framework with an electronic portal imaging device (EPID) for magnetic resonance-linear accelerators (MR-LINACs). Approach. The proposed method directly back-projected 2D portal dose into 3D patient coarse dose, which bypassed the complicated patient-to-EPID scatter estimation step used in conventional methods. A pre-trained convolutional neural network (CNN) was then employed to map the coarse dose to the final accurate dose. The electron return effect caused by the magnetic field was captured with the CNN model. Patient dose and portal dose datasets were synchronously generated with Monte Carlo simulation for 96 patients (78 cases for training and validation and 18 cases for testing) treated with fixed-beam intensity-modulated radiotherapy in four different tumor sites, including the brain, nasopharynx, lung, and rectum. Beam angles from the training dataset were further rotated 2–3 times, and doses were recalculated to augment the datasets. Results. The comparison between reconstructed doses and MC ground truth doses showed mean absolute errors <0.88% for all tumor sites. The averaged 3D γ-passing rates (3%, 2 mm) were 97.42%±2.66% (brain), 98.53%±0.95% (nasopharynx), 99.41%±0.46% (lung), and 98.63%±1.01% (rectum). The dose volume histograms and indices also showed good consistency. The average dose reconstruction time, including back projection and CNN dose mapping, was less than 3 s for each individual beam. Significance. The proposed method can be potentially used for accurate and fast 3D dosimetric verification for online adaptive radiotherapy using MR-LINACs.