Forced Notch Signaling Inhibits Commissural Axon Outgrowth in the Developing Chick Central Nerve System

Abstract

BackgroundA collection of in vitro evidence has demonstrated that Notch signaling plays a key role in the growth of neurites in differentiated neurons. However, the effects of Notch signaling on axon outgrowth in an in vivo condition remain largely unknown.Methodology/Principal FindingsIn this study, the neural tubes of HH10-11 chick embryos were in ovo electroporated with various Notch transgenes of activating or inhibiting Notch signaling, and then their effects on commissural axon outgrowth across the floor plate midline in the chick developing central nerve system were investigated. Our results showed that forced expression of Notch intracellular domain, constitutively active form of RBPJ, or full-length Hes1 in the rostral hindbrain, diencephalon and spinal cord at stage HH10-11 significantly inhibited commissural axon outgrowth. On the other hand, inhibition of Notch signaling by ectopically expressing a dominant-negative form of RBPJ promoted commissural axonal growth along the circumferential axis. Further results revealed that these Notch signaling-mediated axon outgrowth defects may be not due to the alteration of axon guidance since commissural axon marker TAG1 was present in the axons in floor plate midline, and also not result from the changes in cell fate determination of commissural neurons since the expression of postmitotic neuron marker Tuj1 and specific commissural markers TAG1 and Pax7 was unchanged.Conclusions/SignificanceWe first used an in vivo system to provide evidence that forced Notch signaling negatively regulates commissural axon outgrowth.

Notch signaling and neural connectivity

“…Consistent with in vitro studies, Notch and its ligands are present on both axons and dendrites of mammalian neurons in vivo [22, 23]. Notch activation acutely limits the number and/or length of neurites [24], and in Drosophila , absence of Notch cell-autonomously decreases the number of filopodia on identified CNS pioneer growth cones [25]. …”

In silico analyses and global transcriptional profiling reveal novel putative targets for Pea3 transcription factor related to its function in neurons

“…Although Wnt signaling was long known for its role in early embryonic development, their role in growth cone and axon guidance have been identified only a decade ago [ 50 , 51 ]. Notch signaling is involved in the early development of many systems, nervous system being one—it was shown to be important for axonal outgrowth as well as dendritic patterning in various model systems [ 52 – 54 ]. Hippo pathway, which is known to be a common regulator of organ size in development, was recently shown to mediate ephrinB/EphB signaling in peripheral nerve regeneration [ 55 ].…”

Notch-Rbpj signaling is required for the development of noradrenergic neurons in mouse locus coeruleus

“…Plasmid preparation, dual-luciferase reporter assay and in vitro gene expression assay Expression plasmids encoding Hes1, NICD, and Rbpj-VP16 (a constitutively active form of Rbpj) and an siRNA expression plasmid targeting Hes1 (Hes1 siRNA) were prepared as described previously (Shi et al, 2011). Ascl1-and Phox2a-luciferase reporter plasmids were constructed by subcloning the 59-upsteam elements of mouse Ascl1 (Arvidsson et al, 2005) or Phox2a (Benjanirut et al, 2006) into the pGL3-enhancer vector (Promega, Madison, WI, USA).…”

“…In ovo electroporation was performed as previously described (Shi et al, 2011). In brief, after fertilized chicken eggs were incubated to Hamburger and Hamilton (HH) stage HH10-11, 0.5 ml of expression plasmid (1.0 mg/ml in sterile PBS) or Hes1 siRNA plasmid was injected into the midbrain aqueduct of chicken embryo with glass capillaries (see schematic in Fig.…”