Top Notch Level 2

[Athenasby Regalado]

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Genetic studies have implicated Notch signaling in the maintenance of pancreatic progenitors. However, how Notch signaling regulates the quiescent, proliferative or differentiation behaviors of pancreatic progenitors at the single-cell level remains unclear. Here, using single-cell genetic analyses and a new transgenic system that allows dynamic assessment of Notch signaling, we address how discrete levels of Notch signaling regulate the behavior of endocrine progenitors in the zebrafish intrapancreatic duct. We find that these progenitors experience different levels of Notch signaling, which in turn regulate distinct cellular outcomes. High levels of Notch signaling induce quiescence, whereas lower levels promote progenitor amplification. The sustained downregulation of Notch signaling triggers a multistep process that includes cell cycle entry and progenitor amplification prior to endocrine differentiation. Importantly, progenitor amplification and differentiation can be uncoupled by modulating the duration and/or extent of Notch signaling downregulation, indicating that these processes are triggered by distinct levels of Notch signaling. These data show that different levels of Notch signaling drive distinct behaviors in a progenitor population.

In this retrospective study, we used anteroposterior plain radiographs of the neck to analyze sternal notch level in relation to the upper thoracic spine and to assess the usefulness of this relation in deciding how to approach the upper thoracic spine. We reviewed 53 patients' anteroposterior plain radiographs of the cervicothoracic spine and thoracic magnetic resonance imaging (MRI) scans. On the plain radiographs, we drew a horizontal line joining the lower-fifth edge of the medial end of the 2 clavicles; on the midsagittal thoracic MRI scans, we drew a tangential line to the sternal notch. Then we noted the vertebral level of the 2 lines. In all cases, the horizontal line on the plain radiographs and the tangential line on the MRI scans corresponded to each other without discrepancy. We evaluated this method in a patient with a fractured T3 vertebral body, in whom a satisfactory procedure was performed using low anterior cervical spine approach. As the level of sternal notch is found to be present below the level of T2 and T3 radiologically in most cases, a low cervical approach can be contemplated in most patients with upper thoracic spine pathology depending on their sternal level as determined by preoperative radiographs. MRI scans are not needed to decide the approach, as it can be assessed with plain radiographs alone, as shown in this study.

The Notch pathway controls proliferation during development and in adulthood, and is frequently affected in many disorders. However, the genetic sensitivity and multi-layered transcriptional properties of the Notch pathway has made its molecular decoding challenging. Here, we address the complexity of Notch signaling with respect to proliferation, using the developing Drosophila CNS as model. We find that a Notch/Su(H)/E(spl)-HLH cascade specifically controls daughter, but not progenitor proliferation. Additionally, we find that different E(spl)-HLH genes are required in different neuroblast lineages. The Notch/Su(H)/E(spl)-HLH cascade alters daughter proliferation by regulating four key cell cycle factors: Cyclin E, String/Cdc25, E2f and Dacapo (mammalian p21CIP1/p27KIP1/p57Kip2). ChIP and DamID analysis of Su(H) and E(spl)-HLH indicates direct transcriptional regulation of the cell cycle genes, and of the Notch pathway itself. These results point to a multi-level signaling model and may help shed light on the dichotomous proliferative role of Notch signaling in many other systems.

Copyright: 2016 Bivik et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

The Notch signal transduction pathway plays a central role during animal development, and is also critical for tissue homeostasis during adulthood [1]. Notch signaling typically acts as a short-range, cell-cell communication system, which can trigger a multitude of cellular responses, including proliferation, differentiation and programmed cell death. The outcome of Notch activation is highly context-dependent, and with respect to e.g., proliferation, Notch can act both as an anti- and pro-proliferative regulator [2].

In this study, we find that Notch/E(spl)-HLH signaling is globally required to regulate the Type I>0 switch. To dissect the Notch downstream events and the role of the different E(spl)-HLH genes, we utilized TILLING and CRISPR/Cas9 mutagenesis, as well as BAC recombineering, to generate novel individual mutants for all seven E(spl)-HLH genes. Strikingly, in spite of their reported genetic redundancy, we find that, when placed over a genomic deletion removing all seven genes, individual E(spl)-HLH mutations can significantly affect the Type I>0 daughter proliferation switch. Intriguingly, different E(spl)-HLH genes affect the switch in different NB lineages. With respect to cell cycle components, Notch signaling regulates several key cell cycle proteins, including CycE, E2f, Stg and Dap. Moreover, ChIP-seq and DamID-seq demonstrates binding of Su(H), E(spl)m5-HLH and E(spl)m8-HLH to E(spl)-C, CycE, stg, E2f and dap. These results help resolve the Notch pathway with respect to the Type I>0 switch, by identifying the main Notch components, the critical downstream targets, as well as the molecular and genetic interactions involved. We propose an intriguing multi-levelNotch signaling cassette involved in the Type I>0 daughter proliferation switch, where primary-level Notch signaling results in activation of E(spl)-HLH and cell cycle genes, and second-level Notch signaling results in E(spl)-HLH repressing a partly overlapping set of cell cycle genes. This multi-levelmode of Notch signaling may help ensure precise timing and fidelity of the Type I>0 switch, and may shed light upon the sensitivity and dynamics of Notch signaling, as well as its dichotomous nature with respect to proliferation control, in many other systems.

These studies demonstrate that in spite of redundancy between the E(spl) genes in relation to other Notch functions, with respect to the Type I>0 switch we observe weak but significant effects in single gene mutants for six of the seven E(spl)-HLH genes, revealed when placed over a deficiency removing the entire E(spl) region. Strikingly, we furthermore find evidence for selective utilization of different E(spl) genes in different NBs, with m3 and m8 only acting in NB3-3A and m7 in NB5-6T.

A number of gain-of-function studies have demonstrated strong effects when expressing the Notch-Intracellular Domain truncation (NICD) [37]. To address the sufficiency of Notch signaling to trigger the Type I>0 switch, we therefore misexpressed NICD using the insc-Gal4 driver, a driver expressed by most if not all NBs from St11 and onwards. We analyzed NB and daughter proliferation in both the thorax and abdomen, at two different stages. In line with the selective role for Notch signaling in controlling daughter but not NB proliferation, we did not observe any effect on NB proliferation at any stage, neither in thorax nor abdomen (S5B Fig). We did however observe significant reduction of daughter proliferation, evident in both thorax and abdomen at St12 (S5B Fig). We thus find that NICD can trigger the Type I>0 switch.

We conclude that ectopic E(spl)-HLH expression can trigger a premature Type I>0 switch, without strongly affecting NB proliferation, and that triggering a premature switch logically leads to reduction of cell numbers generated in a lineage.

We recently found that the Type I>0 daughter proliferation switch is under control also of the late temporal gene castor (cas) and the Hox gene Antennapedia (Antp)[23]. Cas is part of the temporal cascade of transcription factors (Hb>Kr>Pdm>Cas>Grh) playing out in most, if not all, NBs [42]. Antp is gradually expressed in NBs over time, and hence also shows a temporal expression profile [23, 43].

Previous studies did not reveal cross-regulation in NBs between cas, Antp or Notch signaling [23, 27]. We therefore addressed if m8 can act combinatorially with cas and Antp. First, looking at pros>cas-Antp co-misexpression, as anticipated from previous studies [23], we noted reduction of daughter proliferation in both thorax and abdomen (Fig 5E and 5F). In contrast to Notch signaling, both cas and Antp are also involved in the control of NB proliferation exit at the end of lineage development [23]. Indeed, pros>cas-Antp co-misexpression resulted in reduction also of NB proliferation, in both thorax and abdomen, at both St12 and St14 (Fig 5E and 5F). Next, we co-misexpressed m8CK2 with cas-Antp, and observed striking combinatorial reduction of daughter proliferation, in both thorax and abdomen, at both St12 and St14 (Fig 5C and 5D and 5E and 5F). Similar to cas-Antp co-misexpression, we also noted reduced NB proliferation in m8CK2-cas-Antp co-misexpression, but this was not significantly increased from that observed in cas-Antp co-misexpression (Fig 5E and 5F).

We conclude that stabilized m8 can act strongly combinatorially with cas and Antp to trigger a premature Type I>0 switch. In addition, misexpression of all three genes can to a lower degree reduce NB proliferation, but does not act combinatorially in this regard.

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