Notch Signaling Controls Generation of Motor Neurons in the Lesioned Spinal Cord of Adult Zebrafish

Abstract

In mammals, increased Notch signaling is held partly responsible for a lack of neurogenesis after a spinal injury. However, this is difficult to test in an essentially nonregenerating system. We show that in adult zebrafish, which exhibit lesion-induced neurogenesis, e.g., of motor neurons, the Notch pathway is also reactivated. Although apparently compatible with neuronal regeneration in zebrafish, forced activity of the pathway significantly decreased progenitor proliferation and motor neuron generation. Conversely, pharmacological inhibition of the pathway increased proliferation and motor neuron numbers. This demonstrates that Notch is a negative signal for regenerative neurogenesis, and, importantly, that spinal motor neuron regeneration can be augmented in an adult vertebrate by inhibiting Notch signaling.

Upregulation of anti-apoptotic factors in upper motor neurons after spinal cord injury in adult zebrafish

“…Our data were in good agreement with previous findings in the adult zebrafish brain, reporting that the number of newly-generated neurons is extremely low in the region where descending neurons exist (Zupanc et al, 2005;Hinsch and Zupanc, 2007). In contrast to the upper motor neurons in the brainstem, motor neurons in the spinal cord become apoptotic after SCI, and neurogenesis from the ependymal zone around the central canal is activated via sonic hedgehog and/or Notch signaling (Reimer et al, 2008;Reimer et al, 2009;Dias et al, 2012).…”

Function of Sox2 in ependymal cells of lesioned spinal cords in adult zebrafish

“…In contrast to such tragic events, adult zebrafish can regenerate motor neurons in the spinal cord and protect upper motor neurons in the brainstem against cell death via several regenerative mechanisms (Becker and Becker, 2007;Dias et al, 2012;Ogai et al, 2012).…”

“…Immunohistochemistry against Sox2 (1:500; GeneTex, CA, USA) and/or PCNA (1:1000; Sigma-Aldrich) was carried out as described previously (Dias et al, 2012) using appropriate Alexa Fluor 488-or 594-conjugated secondary antibodies (1:200; Life Technologies, CA, USA). In situ hybridization was also performed using a method described previously (Dias et al, 2012) with a zebrafish sox2-specific antisense cRNA probe.…”

“…In situ hybridization was also performed using a method described previously (Dias et al, 2012) with a zebrafish sox2-specific antisense cRNA probe. Immunoreaction and in situ hybridization images were acquired using a confocal microscope (LSM510; Carl Zeiss, Oberkochen, Germany).…”

“…The number of PCNA-positive (PCNA + ) cells was counted as described previously (Dias et al, 2012) and was calculated for the entire 1.5 mm surrounding the lesion site.…”