Sonic hedgehog is a polarized signal for motor neuron regeneration in adult zebrafish

Abstract

In contrast to mammals, the spinal cord of adult zebrafish has the capacity to reinitiate generation of motor neurons after a lesion. Here we show that genes involved in motor neuron development, i.e., the ventral morphogen sonic hedgehog a (shha), as well as the transcription factors nkx6.1 and pax6, together with a Tg(olig2:egfp) transgene, are expressed in the unlesioned spinal cord of adult zebrafish. Expression is found in ependymoradial glial cells lining the central canal in ventrodorsal positions that match expression domains of these genes in the developing neural tube. Specifically, Tg(olig2:egfp)(+) ependymoradial glial cells, the adult motor neuron progenitors (pMNs), coexpress Nkx6.1 and Pax6, thus defining an adult pMN-like zone. shha is expressed in distinct ventral ependymoradial glial cells. After a lesion, expression of all these genes is strongly increased, while relative spatial expression domains are maintained. In addition, expression of the hedgehog (hh) receptors patched1 and smoothened becomes detectable in ependymoradial glial cells including those of the pMN-like zone. Cyclopamine-induced knock down of hh signaling significantly reduces ventricular proliferation and motor neuron regeneration. Expression of indicator genes for the FGF and retinoic acid signaling pathways was also increased in the lesioned spinal cord. This suggests that a subclass of ependymoradial glial cells retain their identity as motor neuron progenitors into adulthood and are capable of reacting to a sonic hedgehog signal and potentially other developmental signals with motor neuron regeneration after a spinal lesion.

Figure 1.

Expression of olig2 mRNA is increased in the lesioned adult spinal cord close to the lesion site at 2 weeks postlesion. A, Schematic presentation of transcription factor domains set up in the developing vertebrate neural tube by a ventrodorsal gradient of Shh (modified after Vallstedt and Kullander, 2007). Different progenitor zones (p0–3) give rise to distinct classes of interneurons (V0–3). The pMN zone gives rise to motor neurons (MN). B, Cross-sections through the adult spinal cord are shown at the level of the central canal (dorsal is up). Ventricular cells in ventrolateral positions around the central canal express detectable levels of olig2 mRNA in the lesioned spinal cord. Scale bar, 25 μm.

Figure 2.

Evidence for a pMN-equivalent zone in the ependymal layer of the adult spinal cord. Spinal cross-sections at the level of the central canal (asterisks) are shown. In the unlesioned (arrows) and lesioned spinal cord (brackets) Pax6/Tg(olig2:egfp)/Nkx6.1 coexpressing ependymoradial glial cells are present. Expression of all markers is increased close to a spinal lesion site at 2 weeks postlesion. Arrowheads, Oligodendrocytes. Scale bar, 20 μm.

Figure 3.

Increased expression of shha and proliferation of Tg(shha:gfp)⁺ ependymoradial glial cells in the ventricular zone of the lesioned spinal cord. Cross-sections are shown of whole spinal cross-sections (Tg(shha:gfp) in A only) or just for the area around in the ventricle at high magnification (all other images). Asterisks indicate the position of the ventricle. A, Expression of shha mRNA is increased in the ventral-most position of the ventricle and Tg(shha:gfp)⁺ ventral ependymoradial glial cells, which form part of the glial limitans (arrows), are more numerous at 2 weeks postlesion. B, Tg(shha:gfp)⁺ ependymoradial glial cells actively proliferate at 2 weeks postlesion, as indicated by double labeling with PCNA antibodies (arrows). Scale bars, 25 μm.

Figure 4.

Tg(shha:gfp)⁺ ependymoradial glial cells coexpress Nkx6.1, but not Pax6. Spinal cross-sections at the level of the central canal (outlined by dots) are shown. Arrows point to an individual Tg(shha:gfp)⁺/Nkx6.1⁺/Pax6⁻ cell. Scale bar, 15 μm.

Figure 5.

hh pathway genes are upregulated in ependymal cells of the lesioned spinal cord. Cross-sections of the adult spinal cord at the level of the central canal (asterisk) are shown. A, Expression of patched1 and smoothened mRNA is increased after a lesion. Note polarized (stronger ventral) expression of patched1 mRNA, whereas smoothened mRNA is detectable along the dorsoventral extent of the central canal of the lesioned spinal cord. B, In the lesioned, but not the unlesioned spinal cord, Tg(olig2:egfp)⁺ ependymoradial glial cells express detectable levels of mRNA for patched1 (arrow), an indicator of hh pathway activity. Scale bars: A, 25 μm; B, 15 μm.

Figure 6.

Expression of patched1 and olig2 mRNA in the adult lesioned spinal cord is specifically reduced by intraperitoneal cyclopamine injection. PCR amplification of patched1 and olig2, using GAPDH as a standard, is shown. Tomatidine injection is used as a negative control.

Figure 7.

Cyclopamine treatment impairs motor neuron regeneration. A, Spinal cross-sections are shown. Asterisks indicate the central canal. Cyclopamine injection reduces the number of newly generated motor neurons (Tg(hb9:gfp)⁺; arrows) and proliferating cells in the ventricular zone (PCNA⁺). Arrowheads indicate large motor neurons in unlesioned animals. B, Quantification of reduced numbers of small, newly generated Tg(hb9:gfp)⁺ motor neurons and PCNA⁺ profiles depicted in A. Values for unlesioned animals are given for reference (Reimer et al., 2008). Control, Injected with tomatidine. Scale bar, 50 μm.

Figure 8.

FGF and retinoic acid signaling pathway related genes show increased expression in the lesioned adult spinal cord. PCR analyses of unlesioned and lesioned (14 d postlesion) spinal cord, using GAPDH levels as a standard, are shown. All indicated genes show robust upregulation in lesioned tissue. Patched1 (ptc1) as an indicator for Shh pathway activity is included as a positive control.

Figure 9.

Summary of known events associated with motor neuron regeneration in adult zebrafish. Schematic cross-sections through the spinal cord are shown (the central canal is represented by a gray oval). Different ependymoradial glial cells in the unlesioned spinal cord express low levels (light shading) of different transcription factor combinations found in similar dorsoventral positions in the embryonic neural tube. After a lesion, the central canal widens close to the lesion site, concomitant with an increase in numbers of ependymoradial glial cells that also express higher levels of transcription factors (darker shading). A ventrodorsal hh signal, evidenced by a ventrodorsal patched1 (ptc1) mRNA expression gradient in ependymoradial glial cells, contributes to production of Hb9⁺/islet-1/-2⁺/ChAT⁺ motor neurons (MNs) from pMN-like ependymoradial glial cells (green) after a lesion. Increased expression of genes related to FGF and retinoic acid signaling suggests involvement of these signals in motor neuron regeneration.