TITLE: The spinal ependymal zone as a source of endogenous repair cells

across vertebrates

AUTHORS:

Catherina G. Becker*1, Thomas Becker*1, Jean-Philippe Hugnot*2,3

ADDRESSES :

1-Centre for Discovery Brain Sciences, University of Edinburgh, Biomedical

Sciences, The Chancellor’s Building, 49 Little France Crescent, Edinburgh

EH16 4SB, UK

2-INSERM U1051, INM, Hopital Saint Eloi, 80 avenue Augustin Fliche, 34091

Montpellier, France

3-Université de Montpellier, Place Eugène Bataillon, 34095 Montpellier,

France

* equal contributions, listed alphabetically; correspondence to

catherina.becker@ed.ac.uk, thomas.becker@ed.ac.uk, jean-

philippe.hugnot@umontpellier.fr

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ABSTRACT:

Spinal cord injury results in the loss of neurons and axonal

connections. In mammals, including humans, this loss is permanent, but is

repaired in other vertebrates, such as salamanders and fishes. Cells in the

ependymal niche play a pivotal role for the outcome after injury. These cells

initiate proliferation and generate new neurons of different types in

regenerating species, but only glial cells, contributing to the glial scar, in

mammals. Here we compare the cellular and molecular properties of

ependymal zone cells and their environment across vertebrate classes. We

point out communalities and differences between vertebrates capable of

neuronal regeneration and those that are not. Comparisons like these may

ultimately lead to the identification of factors that tip the balance for

ependymal zone cells in mammals to produce appropriate neural cells for

endogenous repair after spinal cord injury.

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1. INTRODUCTION:

After spinal injury or in neurodegenerative diseases, lost neurons are

not replaced in mammals, including humans. In anamniotes, particularly fishes

and salamanders, this is not the case. These species readily regenerate

neurons (Grandel and Brand, 2013; Becker and Becker, 2015; Alunni and

Bally-Cuif, 2016; Cardozo et al., 2017; Tazaki et al., 2017). Newly generated

neurons may replace those that are lost after an injury. This is significant,

because secondary neuron loss after spinal injury can be quite extensive in

mammals (Park et al., 2004). Moreover, in anamniotes, new neurons can

contribute to an axonal bridge that reconnects the spinal cord, potentially

acting as relay neurons for the injured spinal cord (Goldshmit et al., 2012). In

mammals, embryonic or induced neural progenitor cells transplanted into the

lesioned spinal cord can generate relay neurons. This leads to promising axon

growth from these cells and some functional recovery (Lu et al., 2012; Lu et

al., 2014). However, network integration of these cells is limited and immune

reactions to transplants, as well as tumour formation by transplanted cells

present additional problems to overcome (Sharp et al., 2014). Therefore,

finding and reprogramming endogenous spinal stem cells for neurogenesis

could be of great benefit to repairing spinal cord lesions. Endogenous spinal

stem cells exist in the adult mammalian spinal cord around the central canal

(Weiss et al., 1996). These cells start to proliferate after a lesion, but in situ

environmental factors prevent them from generating neurons and

oligodendrogliogenesis is rare. Instead, these cells generate mainly

astrocytes that contribute to a scar in the lesion site that prevents axons from

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crossing (Stenudd et al., 2015). In anamniotes, progenitor cells are likewise

located at the central canal, proliferate after a lesion, express similar genes as

in mammals, but contribute to a very different regenerative outcome, namely

neurogenesis and functional spinal cord repair (Becker and Becker, 2015).

For these reasons, it is worthwhile to undertake a comparative description of

the importance of cells in the ependymal zone between regenerating and non-

regenerating vertebrate species. However, other cell types in the

parenchyma, such as oligodendrocyte progenitor cells and astrocytes that

react to a lesion with proliferation may also have stem cell potential, which is

reviewed elsewhere (Almad et al., 2011; Dimou and Gotz, 2014; Gotz et al.,

2015; Magnusson and Frisen, 2016).

Here we discuss mainly the zebrafish as a regenerating species, and

compare it to different mammalian species, namely rodents, monkeys, and

humans. We present evidence that ventricular cells across vertebrates are a

major source of new cells after injury and we describe some interesting

similarities between the likely spinal stem cells in species capable and not

capable of lesion-induced neurogenesis, in terms of cell morphology,

molecular signals, and transcription factors that are activated in these cells

after a lesion. We believe that from comparing the cellular and molecular

reactions of the spinal ventricular zone between anamniotes and mammals,

important principles of regenerative neurogenesis may be gleaned that could

be used in future experiments to boost neurogenesis from endogenous neural

stem cells in the injured spinal cord of mammals.

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2. The spinal ependymal zone (EZ) in zebrafish and other species

showing regenerative neurogenesis in successful spinal cord regeneration

The spinal cord of species showing regenerative neurogenesis, such

as fishes and salamanders, consists of peripheral white matter tracts of large

diameter myelinated axons, ascending sensory axons that project dorsally

and descending axons that are located ventrally. Dorsal and ventral grey

matter areas are equivalent to dorsal and ventral horns in mammals and the

spinal cord contains motor neurons and a high diversity of interneurons,

including central canal contacting neurons, such as GABAergic Kolmer-

Agduhr cells that are located in the ependymal layer (Djenoune and Wyart,

2017). Other resident cell types are oligodendroglial cells, microglial cells, and

blood vessel-associated cells. A similar layout is found in all vertebrate

classes.

However, whereas the mammalian spinal cord contains ependymal

cells around the ventricle and free astrocytes in the parenchyma, fish and

salamander spinal cords contain only one astroglia-like cell type, sometimes

called “radial glia” or “ependymoglia”. In zebrafish, we call this cell type

ependymo-radial glia (ERG) (Becker and Becker, 2015), as a way to describe

that these cells have their soma at the central canal, thereby forming the

ependymal layer, and have radial processes that span the entire thickness of

the spinal cord to form endfeet at the pia. ERGs possess 1 to 2 cilia, as shown

by light and electron microscopy (Hui et al., 2015). These are likely to be

motile, as there is a constant flow of cerebrospinal fluid (Grimes et al., 2016)

and most ERGs express the transcription factor foxj1, which is involved in

motile cilia generation (Ribeiro et al., 2017). Hence, ERGs must fulfil a

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number of homeostatic functions of ependymal cells and astrocytes. Much like

those of astrocytes, most ERG processes are positive for the glial fibrillary

acidic protein (GFAP), the glutamate transporter GLAST, brain lipid binding-

protein (BLBP), are highly branched and thus in a position to share the

homeostatic functions of astrocytes (Hui et al., 2015). Their ciliated

ependymal somata may sense signals from the central canal milieu. It has

been proposed that ERGs may not be a physically sustainable cell type in

species with thicker parenchyma, such as mammals (Reichenbach and

Wolburg, 2013). This correlates with the emergence of free astrocytes from

radial glial cells, which are only present during development in mammals.

2.1 Ependymo-radial glial cells as promoters of axonal regeneration

ERGs represent a unique cell type that also has multiple roles after

injury. An injury to the spinal cord in zebrafish leads to proliferation of ERGs in

the ependymal layer (Reimer et al., 2008; Ogai et al., 2014; Ribeiro et al., 2017),

again similar to mammals (Meletis et al., 2008). One role of new glial cells near

the lesion site is presumably to seal the blood-brain barrier, as it has been

shown that this proliferation of astrocytes (astrogliosis) is beneficial for lesion

outcome also in mammals (Sabelström et al., 2013; Anderson et al., 2016). In

mammals, astrogliosis is often considered inhibitory for the regeneration of

axons over the lesion site (Lang et al., 2014). In contrast, in zebrafish,

processes of GFAP+ astrocyte-like cells, derived from ERGs, elongate across

the lesion site and fasciculate with regenerating axons to reconnect the spinal

cord (Goldshmit et al., 2012; Mokalled et al., 2016; Wehner et al., 2017). Hence,

ERG-derived glia in zebrafish is permissive to axon regeneration by not

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forming a barrier as in mammals. In addition, glial processes that connect the

two ends of the severed spinal cord, termed “glial bridge” (Goldshmit et al.,

2012) could provide a scaffold for regenerating axons. A number of

experimental studies show a correlation between disruption of the glial bridge,

axon crossing and functional recovery in zebrafish. For example, ERGs and

lesion site cells produce connective tissue growth factor a (ctgfa). Disrupting

ctgfa expression in a mutant impaired glial bridging and axonal regeneration,

which could both be restored by global over-expression of ctgfa or applying

recombinant human CTFG (Mokalled et al., 2016). ERGs/lesion site glia also

produce and are responsive to fibroblast growth factor (Fgf). Inhibition of Fgf

signalling disrupted both glial bridging and axon regrowth. Moreover,

experimentally increasing FGF pathway activity accelerated glial and axonal

regeneration (Goldshmit et al., 2012). Finally, activity of the Wnt pathway has

been reported in ERGs of lesioned larval and adult zebrafish and inhibition of

the pathway inhibited both glial bridging and axon regrowth (Briona et al.,

2015; Strand et al., 2016).

However, in larval zebrafish, live observations indicate dynamic growth

of axons into the lesion site, mostly independently of glial processes (Wehner

et al., 2017). Moreover, “axonal bridging” (continuity of axon labelling between

the spinal cord ends) is established earlier than glial bridging. Importantly,

when GFAP+ glia, and therefore most of the glial bridge, was conditionally

ablated, axonal bridging was unimpaired (Wehner et al., 2017). Similarly, spinal

cord transection in the adult eel led to bridging of the gap by glial processes

and axons, with axons “always the most rostral component in the bridge”

(Dervan and Roberts, 2003b; Dervan and Roberts, 2003a). These observations

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suggest that the relative importance of the glial scaffold function may depend

on species and developmental stage of fishes. The glial scaffold function

could be particularly important in adult fish, in which distances that axons

have to bridge are larger than in larvae. In addition, ERG-derived growth

factors, such as Ctgfa and Fgf could promote axon regeneration directly. To

further elucidate the interdependency of axonal and glial bridging in the future,

there-dimensional whole-mount analyses of the adult lesion site using tissue

clearing methods, in combination with cell type-specific manipulations of the

above pathways will be informative.

It has to be kept in mind that a spinal lesion site shows high cellular

complexity. For example, cell type specific manipulation of the Wnt pathway

using the TetON system indicated that Wnt activity is most important in lesion-

site fibroblasts to induce deposition of regeneration-promoting Col XII.

(Wehner et al., 2017). How ERGs interact with important non-neural lesion

site cells remains to be elucidated.

2.3 Ependymo-radial glial cells as progenitor cells

Besides roles in re-establishing spinal cord integrity and continuity,

ERGs function as progenitor cells for neurons after a lesion. In mammals,

progeny of ependymal cells is restricted mostly to glial fates (Barnabe-Heider

et al., 2010). In contrast, early observations of histological preparations

indicated that the injured spinal cord of fishes is capable of regenerating

neurons from the ependymal zone (Kirsche, 1950). After a lesion of the spinal

cord in adult zebrafish, birth of new motor neurons, serotonergic interneurons,

V2-like interneurons, and other interneuron cell types has been observed

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(Reimer et al., 2008; Kuscha et al., 2012a; Kuscha et al., 2012b). These cell

types are likely derived from ERGs based on the following evidence:

Proliferating cells at the central canal vastly outnumber parenchymal

proliferating cells and different types of neurons show wedge-shaped

distributions with the tip of the wedge in specific dorso-ventral positions of the

ependymal layer (Reimer et al., 2008). For example in the ventral spinal cord,

new serotonergic neurons are generated and serotonin-positive neurons can

be found within the ependymal zone during regeneration, but never in

unlesioned spinal cords (Kuscha et al., 2012a). In the case of motor neurons,

specific ventricular ERGs are labelled for both GFP, driven by the regulatory

sequences of the motor neuron progenitor gene olig2, and the transcription

factor mnx1 (also known as Hb9) during regeneration, but never in the

unlesioned spinal cord. A likely explanation for this double-labeling is that

differentiating motor neurons express the early marker mnx1, while they retain

the relatively stable GFP protein from the time the ventricular cell was still a

progenitor (Reimer et al., 2008). In this case, GFP protein acts as a short-term

lineage tracer. In injured larval zebrafish, genetic lineage tracing using

inducible Cre recombinase under the GFAP promoter, an ERG marker, was

used to demonstrate that HuC positive neurons are generated from spinal

ERGs (Briona et al., 2015). This matches similar experiments from other CNS

areas (Kroehne et al., 2011). In summary, ERGs are the only known source

for neurons after injury to the spinal cord so far.

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2.4 Ependymo-radial glial cells retain their neural tube identities from

development

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The observation that different cell types appear to derive from different

dorso-ventral positions of the ependymal layer is reminiscent of the

developing spinal cord, in which ventral and dorsal morphogen gradients set

up discrete progenitor domains that express different combinations of

transcription factors in the neural tube (Ferg et al., 2014). Indeed, though

apparently of homogeneous morphology, adult ERGs express low levels of

domain-defining transcription factor combinations along the dorso-ventral axis

in the intact late larval and adult spinal cord (Reimer et al., 2009; Briona and

Dorsky, 2014) (Fig. 1). The ERGs at the ventral midline express sonic

hedgehog (shh), a ventralising morphogen derived from the embryonic

floorplate. Adjacent domains express other transcription factors, similar to

those expressed during development of the ventral spinal cord, e.g. nkx6.1,

dbx1, as well as olig2 as the single defining transcription factor of the motor

neuron progenitor domain (Fig. 1). pax6 is not expressed in the very ventral

spinal cord, but is expressed around the rest of the central canal. This is a

difference to the developing neural tube, where pax6 is also not present in the

most dorsal region (Wilson and Maden, 2005). A likely explanation for this

discrepancy between the dorsal aspects of the developing and adult spinal

cord is that during conversion of the primitive neural tube lumen to the central

canal, dorsal regions of the neural tube are not included in the central canal

ependyma (Kondrychyn et al., 2013). Consistent with this idea, we failed to

detect markers for dorsal progenitor identity, such as pax7 and the dorsal

morphogens bmp 2 and 4 in the adult zebrafish spinal cord (Kuscha et al.,

2012b). In contrast, ERGs in the dorsal spinal cord of salamanders are

immuno-positive for Pax7 (Schnapp et al., 2005). Adult ERGs appear to

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display a degree of lineage restriction, which depends on their developmental

dorso-ventral position of origin (Fig. 1). Therefore, the possibility exists that in

adult zebrafish, neuronal cell types that during development are derived from

very dorsal ventricular cells are not regenerated. Dorsal neuronal cell types

are mostly sensory and lack of regeneration of these could contribute to the

previously reported failure of re-innervation of sensory axon targets after

injury (Becker et al., 1997; Becker et al., 2005). Of note, after tail amputation,

salamanders regenerate the entire tail, including a fully reformed spinal cord

(Schnapp et al., 2005). This is also the case in the weakly electric fish

Sternarchus albifrons (Anderson and Waxman, 1981). Lineage analysis in

axolotls by grafting genetically labelled tissue early in development has shown

that the regenerated spinal cord is formed by cells of the pre-existing cord,

with clonal analysis showing plasticity of progenitors between dorsal and

ventral fates (McHedlishvili et al., 2007). However, it is not known whether

regenerated salamander or fish spinal cords contain the full complement of

(dorsal) neurons.

After a transection lesion in zebrafish, the spinal cord fuses at the

lesion site in an incomplete physical structure consisting mostly of myelinated

and unmyelinated axons crossing the lesion site (Becker et al., 1997).

However, the central canal adjacent to the lesion site widens considerably, a

feature shared with other regenerating and non-regenerating species

(Cardozo et al., 2017). Concomitant with this widening, progenitor domains

expand. At the same time, expression levels of signals, e.g. shh mRNA, and

transcription factors (nkx6.1, olig2, pax6), found at low levels in ERGs of the

unlesioned spinal cord, increase (Reimer et al., 2008). However, the spatial

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relationships between expression domains of these factors are comparable in

the developing neural tube, the adult EZ, and the lesioned EZ. In other words,

adult ERGs are likely the direct progeny of the early neuroepithelial cells in

the same relative positions. Whether ERGs intrinsically retain their positional

identity and potential to generate specific neuronal cell types or whether this

has to be maintained by constant low-level homeostatic signalling, for

example by Shh (see below), remains to be determined.

All ERGs could represent spinal neural stem cells. Alternatively, true

stem cells may generate specific ERGs that act as transit amplifying cells and

are then lineage-restricted. ERGs express the neural stem cell marker sox2

and other stem cell-related markers, such as pou5f1 (also known as oct4),

which is essential for their lesion-induced proliferation (Ogai et al., 2014; Hui

et al., 2015). Evidence for the presence of distinct stem cells in the ventricular

zone comes from label-retaining experiments with the base-analog BrdU.

These experiments indicated that the ventricular zone of the lesioned spinal

cord contains a subpopulation of cells that retain BrdU labelling for a long time

after injury and are therefore slow-proliferating cells (Reimer et al., 2008).

Slow proliferation is one defining criterion for neural stem cells (Johansson et

al., 1999). Whether these potential stem cells are also lineage-restricted is

unknown.

ERGs may show some flexibility in their potential to generate different

cell types. For example, the pMN-like progenitor domain starts to generate

motor neurons during early development, followed by oligodendrogliogenesis

during later development (Ravanelli and Appel, 2015). Upon a spinal lesion in

adult zebrafish, this domain reverts back to generating motor neurons (Reimer

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et al., 2008). In fact, we could demonstrate that at late larval stages, a spinal

lesion leads to re-initiation of motor neuron generation at the expense of

developmentally on-going oligodendrogliogenesis from the pMN-like domain

(Ohnmacht et al., 2016).

2.5 Signals in regenerative neurogenesis

A mechanical lesion of the spinal cord elicits regenerative proliferation

in ERGs (Reimer et al., 2008; Ogai et al., 2014; Hui et al., 2015; Ribeiro et al.,

2017). Genetically ablating motor neurons in the absence of a mechanical

lesion has the same effect. Both types of injury induce an immune response,

indicated by increased presence of microglial cells and macrophages (Becker

and Becker, 2001; Ohnmacht et al., 2016), important mediators of the

regenerative response. Indeed, inhibiting the innate immune response using

the drug dexamethasone, reduced microglia activation in larval zebrafish and

reduced numbers of regenerated motor neurons after injury (Ohnmacht et al.,

2016). It has been shown that in the stab-lesioned telencephalon of zebrafish,

the immune response is necessary and sufficient to induce regenerative

neurogenesis by a mechanism that involves Leukotriene C4 and its cognate

receptor Cysteinyl leukotriene receptor 1, which is present on progenitor cells

(Kyritsis et al., 2012). It is not known whether spinal ERGs receive the same

or different signals from the immune system.

Recently, a role for the adaptive immune system has been described

for zebrafish spinal cord regeneration. Regulatory T cells (Tregs) accumulate in

the adult spinal cord after injury and show secretion of Neurotrophin 3 (Nt3).

This is an organ-specific reaction, as Tregs secrete other factors in the injured

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retina or heart. Lack of Tregs impaired ERG proliferation, neurogenesis, axon

regrowth and behavioural recovery, whereas application of recombinant Nt3

rescued proliferation and neurogenesis (Hui et al., 2017). This demonstrates a

direct promoting influence of Tregs on regenerative neurogenesis.

Spinal ERGs also react to classical developmental signals during

regeneration. For example, Shh, derived from ventral ERGs, stimulates

generation of serotonergic and motor neurons. This has been shown by

blocking Shh signalling with the plant-derived small molecule inhibitor

cyclopamine (Reimer et al., 2009; Kuscha et al., 2012a). In the future it will be

interesting to determine how this ventrally derived signal reaches the more

dorsal progenitor cells in the adult spinal cord, which is larger and more

complex than the developing neural tube, in which Shh acts in a comparable

fashion.

Notch signalling is a developmental short-range signal involved in

determining fate of adult stem cells and maintenance of “stemness” in

zebrafish (Alunni and Bally-Cuif, 2016). Ligands (deltaC, jagged1b), receptors

(notch1a/b) and down-stream effectors (her4.1, her4.5, her9) are strongly

upregulated from undetectable levels after an injury of the adult spinal cord.

These genes are expressed in partially non-overlapping domains of ERGs

and may reflect different notch activities in different progenitor domains.

Functional analyses using the gamma-secretase inhibitor DAPT to inhibit

notch signalling and heat-shock inducible over-expression of the active

intracellular domain of the notch receptor have shown that notch activity

attenuates lesion-induced neurogenesis of motor neurons (Dias et al., 2012).

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In the unlesioned spinal cord, manipulations of notch had no effect, as

opposed to different brain regions, where notch inhibition leads to ERG

proliferation (Alunni et al., 2013; de Oliveira-Carlos et al., 2013). Thus, the

notch pathway is probably not constitutively active in the zebrafish spinal cord.

This indicates that adult ERGs in the spinal cord are not only different from

constitutively active ERGs in other CNS regions, but also from other quiescent

ERGs, inasmuch as quiescence in spinal ERGs is not dependent on notch

signalling.

Wnt signalling regulates proliferation of spinal ERGs after injury, as the

pathway is active in some ERGs and pharmacological or genetic global

interference with the Wnt pathway inhibited neurogenesis in injured larvae

(Briona et al., 2015). The effect on some ERGs could be indirect, as only a

minority of ERGs show pathway activity after injury in larval zebrafish (Wehner

et al., 2017). Future research will have to elucidate source and target cells of

Wnt signalling.

FGF signalling is upregulated in ERGs of the lesioned spinal cord, as

indicated by increased expression of the FGF receptor 2 and target genes

pea3, erm and spry4 in these cells. FGF controls proliferation of ERG-derived

glia, as shown by global pharmacological inhibition and over-expression of a

dominant-negative FGF receptor variant. Conversely, in a mutant for the FGF

downstream gene and inhibitor spry4, proliferation was enhanced. The source

for FGF ligands are ERGs themselves and neurons (Goldshmit et al., 2012). It

has not been analysed whether neurogenesis was altered after FGF

manipulations, but given the influence on proliferation this seems likely. Other

well-known developmental pathways, such as retinoic acid signalling,

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components of which are upregulated after a lesion (Reimer et al., 2009), or

BMP signalling have not been functionally analysed.

Descending dopaminergic and serotonergic axons promote

regeneration of motor neurons, as shown by a reduction in motor neuron

regeneration following pharmacological ablation of serotonergic (by 5,7-

dihydroxytryptamine) or dopaminergic axons (by 6-hydroxydopamine). Under

these conditions, ERG proliferation and motor neuron regeneration was

impaired. ERGs express dopamine (drd4a) and serotonin receptors (hrt1aa

and hrt3a) (Reimer et al., 2013; Barreiro-Iglesias et al., 2015). Due to the fact

that almost all dopaminergic and serotonergic innervation of the spinal cord is

derived from the brain, this effect is only seen rostral to a lesion site. That is

because caudal to the lesion site, dopaminergic and serotonergic axons

degenerate by Wallerian degeneration even without toxin mediated ablation.

Conversely, addition of dopamine agonists or serotonin augments motor

neuron regeneration caudal to a lesion site that is normally deprived of these

molecules (Reimer et al., 2013; Barreiro-Iglesias et al., 2015). Both signals

also enhance developmental motor neuron generation, such that sensitivity to

dopamine and serotonin during regeneration can be considered a

recapitulation of a developmental mechanism. Interestingly, neither serotonin

nor dopamine injections into unlesioned animals led to any motor neuron

generation. Hence, while these signals modulate the regenerative

neurogenesis programme, they are insufficient to elicit regenerative

neurogenesis from quiescent spinal ERGs.

2.6 Gene expression changes in Ependymo-radial glial cells

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The transitions from quiescence to proliferation and to neurogenesis

necessarily lead to changes in the gene expression programmes in ERGs.

Expression profiles of a spinal lesion site have revealed a number of

transcription factors and other genes that change expression after injury, such

as those involved in the inflammatory response, cell proliferation,

neurogenesis, patterning, and axon outgrowth-related genes (Hui et al.,

2014). Other genes found to be upregulated in spinal ERGs after injury are

HMGB1, syntenin-a, contactin-2, major vault protein, sox11b, mcam, and L1.2

(Guo, Y. et al., 2011; Lin et al., 2012; Pan et al., 2013; Yu and Schachner,

2013; Fang et al., 2014; Chen et al., 2016; Liu et al., 2016). Functional

studies, relying on anti-sense morpholino application to a spinal lesion site,

have found beneficial roles for regeneration of these genes, which might have

been due to morpholino uptake into axotomised neurons, ERGs or both.

Specific gene manipulation in ERGs will be needed to discern a role for these

genes in ERG function. We anticipate that expression profiling of highly

purified progenitor cell populations in combination with cell type specific

manipulations, e.g. using the TetON system (Wehner et al., 2017) or

conditional CRISPR interference (Yin et al., 2016) will clarify the contribution

of specific genes and pathways in ERGs to functional spinal cord regeneration

in the future.

3. The spinal ependymal zone in mammals

3.1 Rodents

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3.1.1 Ependymal zone composition and regionalisation

The rodent ependymal zone (EZ) is a pseudo-epithelium originating

mainly from the ventral part of the developing neuroepithelium, as the dorsal

walls of the neuroepithelium fuse at approximately E15 in the mouse (Fu et

al., 2003). Cells from the roof plate in the developing spinal cord may also

contribute to dorsal EZ cells (Hugnot, unpublished) (Sevc et al., 2009). The

formation of the EZ zone is controlled by Shh signalling, as indicated by the

observation that in the absence of late Shh production it is not formed (Yu et

al., 2013). In the caudal part of the spinal cord, the EZ might be derived from

a secondary neurulation process through a mesenchymal-neuroepithelial

conversion (Lowery and Sive, 2004). In adults, the cervical EZ has a wide

lumen while in the thoracic and lumbar regions, the lumen is reduced to a slit

(Sturrock, 1981). The EZ contains different types of highly-polarized

ependymocytes which can be distinguished both morphologically and using

marker expression. As in zebrafish, dorsal and ventral midline cells are

characterized by a very long radial morphology. These cells maintain long

filament-rich processes extending to the pia and are sometimes referred as

tanycytes (Seitz et al., 1981; Sturrock, 1981; Mothe and Tator, 2005;

Rodriguez et al., 2005). At the lateral level, ependymocytes are either cuboid

or show a radial morphology with a process ending on a vessel. Compared to

the ependymocytes in the subventricular zone, in the spinal cord, these cells

are not multiciliated but mostly feature two motile cilia (occasionally one, three

or four cilia can be found) associated with large basal bodies but without

daughter centriole (Alfaro‐Cervello et al., 2012).

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Two other non-ependymocyte cell types are also encountered in the EZ

region. 1/ cerebral-fluid contacting neurons (CSF-N) are cells equivalent to

Kolmer-Agduhr cells in zebrafish. These neurons have been observed in

many species, including lamprey and macaques. In rodents they are present

at the different levels of the spinal cord (Vigh et al., 2004; Djenoune et al.,

2014). Two types of CSF-N, lateral and ventral CSF-N, with different

developmental origin and properties have been described (Petracca et al.,

2016). These neurons have immature electrophysiological properties and

express Dcx, a marker expressed by immature neurons during development

and in adult neurogenesis (Shechter et al., 2007; Marichal et al., 2009;

Sabourin et al., 2009; Orts-Del’Immagine et al., 2014; Orts-Del'Immagine et

al., 2017). They also maintain expression of transcription factors found in the

developing spinal cord, such as Nkx6.1, Nkx2.2, FoxA2, Gata2/3 (Sabourin et

al., 2009; Petracca et al., 2016). However, CSF-N are not produced by

ongoing adult neurogenesis, but instead are produced late during

development from two ventral domains of the spinal cord (Kútna et al., 2014;

Petracca et al., 2016). These neurons express functional P2X2 ATP channels,

as well as high levels of the polycystic kidney disease-like channels (PKD2L1

and PKD1L2), which are very good markers for these cells (Huang et al.,

2006). CSF-N are mostly GABAergic and send axons toward the caudal part

of the spinal cord (Stoeckel et al., 2003). Their role in mammals remains

enigmatic, but they express ion channels responsive to pH, osmolarity, and

mechanical stimulation. This suggests that they are involved in the monitoring

of CSF composition and movement. These cells send a process into the

lumen and this process is terminated by a large vesicle-containing structure

20

called budge (several µm in size). Presence of this structure indicates the

potential for an intense secretory activity into the CSF. The role of these cells

in the control of cell proliferation in the adult ependyma remains unexplored.

In other CNS niches, GABA signalling controls neural stem cell quiescence

and proliferation (Alfonso et al., 2012; Pontes et al., 2013). Thus GABAergic

CSF-N may serve a similar function in the spinal cord. 2/ the second type of

non-ependymocyte cell found in the EZ region are central-canal contacting

astrocytes (Accs) which can be observed using GFAP-GFP reporter mice

(Sabourin et al., 2009), electron microscopy (Alfaro‐Cervello et al., 2012) and

marker expression (Sabourin et al., 2009; Fiorelli et al., 2013). These

infrequent cells, are mostly situated in the dorsal and ventral parts of the EZ

(80% of cells) and unlike ependymocytes, they have a single cilium with a

daughter centriole. In some cases, Accs send long processes along the

ventral and dorsal midline fissure (i.e. a fold of the pia mater into the

parenchyma) and appear to reach the pia or midline-located pial-lined

interstitial perivascular spaces that are fluid-filled (also known as Virchow-

Robin spaces). Accs represent a heterogeneous population, as only some of

these cells contain Nestin and Vimentin-containing intermediate filaments

(Alfaro‐Cervello et al., 2012). Accs do not proliferate (Alfaro‐Cervello et al.,

2012) or at very low levels (Fiorelli et al., 2013). However, Nestin+ proliferating

cells have been reported in the dorsal region of the EZ (Hamilton et al., 2009),

suggesting cellular heterogeneity in this part of the EZ.

With regard to marker expression, most ependymocytes have a Nestin-

Vimentin+, S100b+, CD24+, EphrinB1+, Sox2+, Sox9+, and CD133+ phenotype

(Sabourin et al., 2009; Pfenninger et al., 2011) and weakly express GFAP

21

(Alfaro‐Cervello et al., 2012). In comparison, radial midline glia cells located at

the roof and floor of the EZ, show lower Vimentin/CD24 expression and higher

levels of GFAP expression (Alfaro‐Cervello et al., 2012). Further marker

analysis has revealed that the adult EZ is in fact a mosaic of cells that show

different developmental gene expression, notably along the dorsal-ventral and

rostro-caudal axes. Almost all EZ cells express the ventral transcription factor

Nkx6.1 (Fu et al., 2003; Sabourin et al., 2009), but Pax6 is expressed only in

the dorsal part of the EZ (Yu et al., 2013) (Hugnot unpublished) and Nato3 is

expressed only in the ventral region (Khazanov et al., 2016). Nato3 is a

transcription factor found in the floor plate during development. Data from the

gene atlas and transgenic mice confirm that genes that during development

are specifically expressed in the dorsal EZ, e.g. MDK (Hugnot and Franzen,

2011), or its ventral part, e.g. Nkx2.2 (Yu et al., 2013) or Shh (Hugnot and

Franzen, 2011), are maintained at the adult stage. Radial cells found in the

roof of the EZ express the Ret receptor tyrosine kinase (Pfenninger et al.,

2011), as well as Zeb1, a transcription factor regulated by TGF-β signalling

(Sabourin et al., 2009). These cells form the dorsal median septum and may

have specific functions, which could also be shared by radial cells found in the

ventral part of the EZ (Alfaro‐Cervello et al., 2012).

During development, the spinal cord is regionalised along the rostro-

caudal axis by expression of different Hox transcription factors, the so-called

“Hox code”. Ependymocytes of the adult spinal cord maintain expression of

developmental Hox genes (Pfenninger et al., 2011). In addition,

neurospheres, derived from cervical, thoracic and lumbar spinal levels

maintain the expression of the combination of hox genes corresponding to the

22

segment they were isolated from over several passages in vitro (Sabourin et

al., 2009).

3.1.2 Proliferation in the adult spinal cord

EZ cells strongly proliferate after birth, corresponding to the elongation

of the spinal cord, which doubles its length over the first nine post-natal

weeks. During this period, proliferation is progressively reduced until the

spinal cord reaches its final size at around postnatal week 12 in mice

(Sabourin et al., 2009; Alfaro‐Cervello et al., 2012). However, EZ cells

maintain a low level of proliferation in adulthood which declines with age

(Gonzalez-Fernandez et al., 2016). The main other cell type to proliferate in

the adult spinal cord are oligodendrocyte progenitor cells in the parenchyma.

Proliferating cells in the EZ have been observed in its dorsal (Hamilton et al.,

2009) or dorsal and ventral region (Alfaro‐Cervello et al., 2012). Electron

microscopy indicates that bi-ciliated ependymocytes are the main source of

proliferative cells in the EZ (Alfaro‐Cervello et al., 2012). This low number of

cilia in spinal cord ependymocytes correlates with proliferative activity,

because ependymocytes in the brain are multiciliated and do not proliferate

(Spassky et al., 2005; Mirzadeh et al., 2008). Cilia are cellular organelles

controlling proliferation (Han and Alvarez-Buylla, 2010) and the presence of

multiple cilia might not be compatible with proliferation (Brooks and

Wallingford, 2014).

The growth factors and molecular mechanisms underlying

ependymocyte proliferation in the spinal cord remain ill-defined. Comparative

gene expression analysis revealed that in the spinal cord these cells are

23

enriched for expression of retinoic acid (RA)-responsive genes, including Hox

genes (Pfenninger et al., 2011). In contrast, ependymocytes at the lateral

ventricle of the brain show higher expression levels of Hey1, a transcription

factor downstream of Notch signalling, as well as expression of several genes

related to the TGF beta1 signalling (Pfenninger et al., 2011). Treatment of

purified spinal ependymocytes with RA increases their proliferation in vitro and

these cells also appear to respond to RA injection in vivo (Pfenninger et al.,

2011). Hox genes are controlled by RA and are involved in the proliferation of

many cell types (Foronda et al., 2009), thus the strong expression of Hox

genes in spinal cord ependymocytes may indicate the involvement of these

genes in the proliferative response to RA. Endogenous growth factors such as

FGF2 and EGF may also be involved in the proliferation of adult

ependymocytes, as infusion of these growth factors into the CSF increases

proliferation of these cells in vivo and in vitro (Weiss et al., 1996; Kojima and

Tator, 2002; Martens et al., 2002). FGF2 and EGF may be derived from the

vasculature, as some ependymocytes send radial extension to vessels. This

suggests intimate cross-talk between these cells, a situation reminiscent of

the neurovascular niche in the SVZ and hippocampus. Interestingly, exercise

increases ependymocytes proliferation (Cizkova et al., 2009) in the spinal

cord. Several growth factors have been shown to be increased in expression

after physical exercise, including FGF2 (Gómez-Pinilla et al., 1997) which

might contribute to ependymocyte proliferation. Ependymocytes also express

high levels of receptor B for endothelins (EDNRB), which are cytokines

expressed by vascular cells (Peters et al., 2003; Sabourin et al., 2009).

Endothelin 1 and 3 enhance the proliferation of embryonic spinal cord stem

24

cells in vitro, suggesting that these growth factors may also be involved in the

proliferation of EZ cells in adults (Sabourin et al., 2009). Finally, another

signal to be considered with respect to the control of adult EZ proliferation is

Wnt, as several ligands and receptors for this pathway are found to be

expressed in this region (Gonzalez-Fernandez et al., 2016) and Wnt-reporter

mice reveal Wnt pathway activity in some EZ cells (White et al., 2010; Garbe

and Ring, 2012; González-Fernández et al., 2014).

The vast majority of proliferating cells remain in the EZ and there is no

clear evidence for substantial migration into the parenchyma. Few cells

expressing oligodendrocytic markers (Olig2, Sox10) are produced during

postnatal development and adulthood in the EZ (Sevc et al., 2014) but their

contribution to formation of new oligodendrocytes in the parenchyma is still

undefined. Endogenous neurogenesis has been described in the dorsal horn

of the spinal cord (Shechter et al., 2007; Rusanescu, 2016). These new

neurons are involved in nociception (Rusanescu and Mao, 2015) and their

production is enhanced by mechanosensation (Shechter et al., 2011) and

peripheral nerve injury (Rusanescu and Mao, 2017). However, it is more likely

that these cells are produced by local progenitors rather than by EZ cells.

3.1.3 Neural stem cell functions of the adult ependymal zone

The EZ is a pseudo-epithelium in contact with the CSF and undergoes

mechanical stress. Like in most adult epithelia in the body, cells in the EZ

probably have to self-renew, at least to some extent. Most epithelial tissues

are renewed from resident multi-potent stem cells and/or uni-potent progenitor

cells (Blanpain et al., 2007). These stem cells can exist in a dormant or

25

activated state. This is controlled by the environment, physiological cycles or

pathological conditions. Once activated, they produce transiently amplifying

cells (TA) before differentiated cells are finally generated. This is the case in

the subventricular zone in the brain, where neural stem cells underlie adult

neurogenesis (Lim and Alvarez-Buylla, 2016). Stem cells in adults are often

maintained in specialised structures called niches that are found in specific

locations in the respective organs. These niches are highly organized

structures composed of stem and non-stem cells, in which activity of canonical

signalling pathways, such as Notch, Wnt and BMP is adequately maintained

to control stem cell status and fate. It is worth noting that some tissues do not

regenerate from stem cells, but by self-duplication of differentiated cells (for

instance liver and pancreas) (Dor et al., 2004; Yanger et al., 2014).

With regard to the EZ in the spinal cord, the situation appears complex,

as on the one hand self-duplication of ependymocytes (which show some

degrees of differentiation) has been observed using electron microscopy

(Alfaro‐Cervello et al., 2012), and on the other hand, in vitro studies provide

compelling evidence for the presence of sub-populations of stem cells in the

EZ. The presence of neural stem cells in the adult spinal cord EZ has been

demonstrated in 1996 using the neurosphere assay (Weiss et al., 1996) in

which cells are seeded at low density on a non-adherent substrate and

allowed to grow in suspension in the presence of a defined medium. Weiss et

al. found that compared to lateral ventricles, stem cells from the spinal cord

required both FGF2 and EGF to generate these neurospheres, which could

be expanded for several passages and generated oligodendrocytes, neurons,

and astrocytes when placed in differentiation conditions. Several groups have

26

confirmed that spinal EZ tissue from both mice and rats form neurospheres.

The demonstration that neurospheres are derived from the EZ has been

obtained using i/ microdissection of the central canal region (Martens et al.,

2002; Sabourin et al., 2009), ii/ animals in which the EZ cells are fluorescently

labelled by transgenes that are controlled by specific promoters (FoxJ1,

Nestin) (Meletis et al., 2008; Li et al., 2016), iii/ and by purification of EZ cells

based on surface protein presence (CD24, CD133) (Pfenninger et al., 2011).

Interestingly, the capacity of EZ cells to form neurospheres is much higher in

juveniles than in older animals (Li et al., 2016; Xu et al., 2017). At least two

cell types found in the EZ are able to form multipotent neurospheres in vitro.

Using hGFAP-CreERT2 and hGFAP-GFP mice, two groups (Sabourin et al.,

2009; Fiorelli et al., 2013) have demonstrated that GFAP+ cells found in the

EZ region generate neurospheres, which differentiate into astrocytes and

neurons. The presence of GFAP-expressing cells in primary neurospheres

was established using marker expression (Sabourin et al., 2009) and with

GFAP-GFP mice (Xu et al., 2017). However, only Sabourin et al. could

demonstrate long-term propagation of these cells, while Fiorelli et al. reported

limited self-renewal properties. Different culture conditions, i.e. sorted

(Sabourin et al., 2009) vs. non-sorted GFAP cells (Fiorelli et al., 2013), used

in these studies could account for this discrepancy. In addition to GFAP+ cells,

Fiorelli et al. found a second population of neurosphere-forming EZ cells, the

identity of which remains to be specified (Fiorelli et al., 2013). These cells

generate neurospheres that can be propagated for at least 7 passages.

Moreover, it is possible that additional rare neurosphere-forming cells are

present in the EZ region, as illustrated recently by the isolation of Oct4+

27

primitive neural stem cells (Xu et al., 2017). Using genetic lineage tracing in

two transgenic mice, Nestin-CreERT and Foxj1-CreERT mice (Meletis et al.,

2008), it has been found that FoxJ1+ and Nestin+ cells generate passageable

and multipotent neurospheres. FoxJ1 is expressed by most EZ cells including

GFAP+ cells (Hugnot, 2012 ). This is reminiscent of the brain subventricular

zone, where FoxJ1 is expressed by neurogenic GFAP+ cells (Jacquet et al.,

2009). Thus, it remains to be explored whether FoxJ1+/GFAP- and

FoxJ1+/GFAP+ EZ cells have distinct abilities to form neurospheres. In the

subventricular zone (Pastrana et al., 2009), both activated stem cell

astrocytes and transit amplifying type C cells can generate neurospheres.

Thus the situation could be similar in the spinal cord EZ region where, as

mentioned previously, several cell types co-exist. As these cells have different

morphologies and express different proteins, notably developmental genes,

e.g. Pax6, Zeb1, Nato3, new lines of transgenic mice should be designed to

further explore the diversity and properties of these EZ cells in vitro and in

vivo. The recent advances to perform single cell RNA profiling should also

shed light on the diversity of EZ cells and the presence of distinct types of

stem cells. This will show whether mammalian EZ cells have a potential to

generate different neurons that is similar to that observed for ERGs in

zebrafish.

While the presence of stem cells in the adult EZ has been reported by

many groups, the type of neurons they generate in vitro and the signals

regulating the gliogenic versus neurogenic choice remain ill-defined. The

default neuronal subtype appears to be GABAergic (Weiss et al., 1996;

Sabourin et al., 2009). However, it is very interesting that by treating rat spinal

28

cord neurospheres with developmental ventralizing and caudalizing factors,

Shh and retinoic acid, respectively, a high rate of HB9+ and Map2+

motoneuron-like cells with different levels of electrophysiological maturity can

be obtained (Moreno-Manzano et al., 2009). In addition, a high rate of

oligodendrocyte production in vitro has been reported after differentiation of

rat neurospheres, which reveals the potential of these cells for myelin repair

(Kulbatski et al., 2007; Kulbatski and Tator, 2009). However, neurospheres

derived from different spinal cord regions express specific combination of Hox

genes (Sabourin et al., 2009) and have different abilities to differentiate

(Kulbatski and Tator, 2009), thus the full capacity of these cells to generate

different types of spinal neurons remains to be established.

3.1.4 The ependymal zone, spinal cord injury and regeneration

The capacity of ependymal cells to rapidly react to spinal cord injury

has been observed around 30 years ago in rats and rabbits (Matthews et al.,

1979; Vaquero et al., 1981; Bruni and Anderson, 1987). Different types of

lesions (compression, contusion, dorsal funiculus incision or hemisection,

transection) have been shown to trigger intense cell proliferation within a few

days. For a review on differences observed in EZ proliferation in the different

models used for spinal cord injury, see (McDonough and Martínez-Cerdeño,

2012). Spinal cord injury appears to be accompanied by an increase in the

stem cell pool as the formation of neurospheres formed in vitro is increased

when the tissue is taken from the injured spinal cord (Xu et al., 2006; Moreno-

Manzano et al., 2009; Barnabe-Heider et al., 2010; Fiorelli et al., 2013; Li et

al., 2016; Xu et al., 2017). However, it cannot be excluded that a fraction of

29

these neurospheres were derived through a de-differentiation process of

reactive astrocytes situated in the parenchyma (Sirko et al., 2013).

Interestingly, after injury, these neurospheres generate more

oligodendrocytes suggesting that spinal cord injury affects spinal cord stem

cell fate (Li X et al, 2016). Indeed, modifications of EZ properties by spinal

cord injury are indicated by several changes in gene expression. A number of

studies have reported a significant increase in expression of the neural

progenitor cell marker Nestin in EZ cells (Frisén et al., 1995; Namiki and

Tator, 1999; Liu et al., 2002; Shibuya et al., 2002; Takahashi et al., 2003;

Orendáčová et al., 2004; Xu et al., 2006; Foret et al., 2010). In addition,

GFAP, a marker for astrocytes, but also for neural stem cells, is increased in

EZ cells (JP Hugnot, unpublished data)(Takahashi et al., 2003; Xu et al.,

2006). Expression of ICAM-1, an adhesion molecule involved in leukocyte

transmigration, is increased in EZ cells after spinal cord injury in rats

(Isaksson et al., 1999). Notably, cell fate regulators, like β1-integrin (North et

al., 2015), Bmp4, Msx2, Notch1, Numb, Pax6, and Shh show higher

expression levels after injury (Yamamoto et al., 2001; Chen et al., 2005),

suggestive of an incomplete recapitulation of a developmental program for

neurogenesis. The re-initiation of basic developmental processes after spinal

cord injury is further illustrated by the observation of asymmetric divisions of

EZ cells, involving the Notch1 receptor after injury (Johansson et al., 1999).

This is an evolutionarily conserved feature with zebrafish (Dias et al., 2012)

and indicates that notch signalling may fulfil essential functions in

regenerative neurogenesis.

30

The molecular mechanisms governing the activation of the EZ after

injury are not fully explored. EZ cells express several receptors, such as

purinergic receptors and Ednrb (Hugnot and Franzen, 2011) and may rapidly

detect molecules released by the lesion site, such as ATP (Franke and Illes,

2014) and inflammatory cytokines. In the uninjured state, some microglial cells

are located very close to the EZ (Alfaro‐Cervello et al., 2012) and could

rapidly relay lesion-induced signals to EZ cells. Apparently, ras signalling is

required, as in the “rasless” mouse, proliferation of EZ cells after spinal injury

is reduced (Sabelström et al., 2013). With regard to Wnt signalling, despite the

downstream effector beta-catenin being expressed by EZ cells, Wnt-signalling

reporter mice did not reveal increased activation of this pathway after spinal

cord injury (White et al., 2010; González-Fernández et al., 2014). This is

similar to observations in zebrafish (Wehner et al., 2017). Compared to

trauma, demyelinating lesions appear not to be able to trigger acute

proliferation in the EZ (Guo, F. et al., 2011; Lacroix et al., 2014) indicating that

a mechanical damage with disruption of the blood-spinal cord barrier (Maikos

and Shreiber, 2007) is probably needed.

In addition to proliferation, migration of EZ cells to the lesion site and

contribution to the glial scar have been reported using genetic linage-tracing

tools (Meletis et al., 2008) or EZ dye-labelling (Mothe and Tator, 2005).

Recent evidence indicates that the contribution of EZ-derived cells to the scar

depends on age, type, and severity of the injury (Li et al., 2016; Ren et al.,

2017). The migration of EZ cells to the lesion site could be analogous to an

epithelial-mesenchymal transition process, especially as these cells already

display mesenchymal traits such as the expression of the intermediate

31

filament Vimentin and the transcription factor Zeb1, both of which are involved

in migration and epithelial-mesenchymal transition (Cheng and Eriksson,

2017). EZ cells also express the polysialylated form of NCAM (Oumesmar et

al., 1995), as well as the CXCR4 receptor (Tysseling et al., 2011). These cell

surface proteins are both involved in neural precursor migration (Cremer et

al., 2000). Whether EZ-derived cells migrate freely through the parenchyma or

are associated with other cells such as vessels or astrocytes is currently

unknown. Using post-natal spinal cord sections maintained in vitro, dorsal and

ventral migration of cells from the EZ, named funicular migratory stream, has

been observed (Mladinic et al., 2014). This is associated with nuclear Atf3

expression. Whether a funicular migratory stream exists after spinal cord

injury in mice remains to be analysed.

After injury, the fate of EZ cells appears to be primarily glial. Using

genetic lineage-tracing tools and lesions of the dorsal funiculus, Meletis et al.

have shown that EZ-derived cells give rise to Sox9+/Vim+ astrocytes (80%,

only part of them express GFAP) and only few myelinating oligodendrocytes

(3%) (Meletis et al., 2008). These astrocytes, which contribute to the core of

the glial scar (for review on glial scar see (Cregg et al., 2014; Gregoire et al.,

2015)), are beneficial for recovery, as their reduction using genetic tools

provoked further axonal loss and secondary enlargement of the lesion volume

(Sabelström et al., 2013). This positive effect of ependymal cell–derived

astrocytes on the lesioned tissue could be mediated by their expression of

neurotrophic factors, such as CNTF, HGF, and IGF-1. Hence, whereas spinal

cord-derived neurospheres generate neurons in vitro, the spinal cord

environment after injury appears to restrict the fate of progenitor cells to glial

32

phenotypes. This notion is also supported by transplantation experiments

showing that multipotent progenitors of the adult spinal cord generate mostly

glial cells when grafted into the spinal cord, but generate neurons when

placed in the hippocampus, a neurogenic environment (Shihabuddin et al.,

2000). It is not clear whether this represents a true fate switch in EZ

progenitors, or instead if this is a selective effect on survival or differentiation

of a heterogeneous population of EZ progenitors. After spinal cord injury, the

strong inflammation and the presence of gliogenic cytokines such as BMPs

(Setoguchi et al., 2004) are likely to bias EZ cell progeny toward an astrocytic

fate at the expense of neurons and oligodendrocytes. Myelin-associated

inhibitors have also been demonstrated to promote differentiation of neural

progenitor cells into the glial lineage (Li et al., 2013).

Consequently, achieving extensive regeneration of the spinal cord and

production of new neurons from the EZ will need genetic (overexpression of

neurogenic transcription factors) or biochemical (infusion with neurogenic

molecules) intervention. For instance, Kojima and Tator (Kojima and Tator,

2002) have found that intrathecal administration of FGF2 and EGF boosts EZ

proliferation and leads to functional improvements, but not to production of

new neurons after spinal cord injury. In addition, Kim and others (Kim et al.,

2011) have found that treatment of exogenous progenitors in vitro or in vivo

with dibutyrylic cyclic AMP, a cell-permeable analog of cyclic AMP, enhances

their neuronal differentiation and survival after spinal cord injury. Combining

these two approaches might lead to new neurons being generated from the

EZ.

33

Migration of newly produced cells from the EZ is also an important

issue and enhancing migration could be beneficial for recovery. This could be

achieved by various strategies, such as chemokine application (Merino et al.,

2015), use of biomaterials, such as a collagen scaffold (Li et al., 2013), or

even by applying electric fields and electromagnetic waves. Indeed, following

pioneering work of Borgens in 1981, showing positive influence of applied

electric fields on spinal cord regeneration in lamprey (Borgens et al., 1981), it

has been shown that migration and differentiation of neural stem cells and

their progeny can be modulated by current (Huang et al., 2015; Yao and Li,

2016; Feng et al., 2017; Iwasa et al., 2017; Samaddar et al., 2017) or

electromagnetic field application (Cui et al., 2017).

3.6 The ependymal zone in primates

Cell types and their proliferative activity have been studied in the EZ

region of adult macaques (Alfaro‐Cervello et al., 2014). As in rodents, cellular

heterogeneity has been observed using marker analysis and electron

microscopy. One distinguishing feature of primate EZ cells compared to those

in rodents is the presence of lateral ependymocytes with multiple ciliae. These

cells do not express Nestin and do not proliferate. In comparison, dorsal and

ventral cells are uniciliated or biciliated, express Nestin and/or GFAP and

proliferate. Like in rodents, astrocytes contacting the central canal and CSF-

contacting neurons are also present in the macaque EZ.

Even though the human spinal cord EZ region has been studied since

1890 (von Lenhossék, 1891) using several histological techniques on post

mortem tissue, there is relatively little available data on it. As in rodents, this

34

region shows cellular heterogeneity during early and prenatal development.

Roof and floor plate cells differentiate first, express Nestin (Sakakibara et al.,

2007) and acquire a long radial morphology extending to the pial surface

(Sarnat, 1992). These cells constitute the midline dorsal and ventral septa

which might be involved in guiding descending and ascending axons, notably

by preventing their decussation (Sarnat, 1992). These cells could also have a

supply role for neurons before vessels appear. Indeed, ependymal cells from

the floor plate ensheath axons during spinal cord development (Gamble,

1968). Moreover, electron microscopy of the developing floor plate

neuroepithelium has also revealed that this structure has an intense secretory

activity, suggesting that, as in rodents, this region works as an organizing

centre (Tanaka et al., 1988; Wagner et al., 1990). In human infants (0.5-18

months), two subtypes of ependymocytes can be found showing either two

(exceptionally one) or more cilia (Alfaro‐Cervello et al., 2014). Astrocytes

contacting the lumen are also found. In adult humans, most ependymal cells

appear to be multi-cilialed, but a subpopulation with two cilia, resembling

macaque or rodent ependymal cells, are also present (Alfaro‐Cervello et al.,

2014). In contrast to rodents or macaques, no proliferating cells have been

found in the ependymal zone of adult humans using the Ki67 antibody

(Dromard et al., 2008; Alfaro‐Cervello et al., 2014; Garcia-Ovejero et al.,

2015). However, low levels of proliferation could have been missed. As the

spinal cord and ependyma are subjected to mechanical stress (twisting,

bending), it is likely that cells of the central canal very slowly self-renew,

similar to many epithelia of the body. Accumulation of mutations in these

proliferative cells may be at the origin of spinal cord ependymoma, an

35

infrequent tumour representing 2% of all primary brain tumours (Weller et al.,

2015).

One major structural difference between the human and rodent central

canal is that after the first decade of life, a central lumen is not patent and an

obliteration process is observed at different levels of the spinal cord in

humans. However, the central canal at the level of the medulla is maintained.

This observation was initially made by histology (Motavkin and Bakhtinov,

1973; Milhorat et al., 1994; Yasui et al., 1999) but was then confirmed using

MRI on 59 healthy volunteers (Garcia-Ovejero et al., 2015). The lumen is

replaced by a cellular mass, which is highly vascularized. This mass is

surrounded by radially arranged cells, giving a pseudo-rosette appearance.

More rarely, pseudo-canals, surrounded with epithelial cells with beta-catenin+

junctions, are found in these masses (Garcia-Ovejero et al., 2015). Multi-

ciliated VIM+ ependymocytes remain in this structure but astrocytes are also

found, suggesting a process of gliosis (Alfaro‐Cervello et al., 2014). Some

authors have proposed that this obliteration should not be regarded as an

entirely passive process but rather as formation of a secretory organ with

physiological functions (Motavkin and Bakhtinov, 1973). However, this

remains to be fully established.

In contrast to macaques, rodents, and fish, the presence of CSF-N has

not yet been reported for the human spinal cord (Dromard et al., 2008; Alfaro‐

Cervello et al., 2014; Djenoune et al., 2014). In zebrafish, these cells are

implicated in locomotor behaviour by responding to both passive and active

bending of the spinal cord and regulating the tail beat frequency (Böhm et al.,

2016). It is tempting to speculate that loss of the tail, which occurred during

36

human and chimpanzee evolution around 4-7 millions ago (Kumar et al.,

2005), or acquisition of bipedalism, was accompanied by a reduction or loss

of CSF-N. Despite the presumptive lack of CSF-N, the EZ receives

innervation which appears to increase with age (Motavkin and Bakhtinov,

1973). The role of this innervation, which is also present in rodents, is

unknown, but may be involved in the proliferation of EZ cells as in the SVZ,

where various nervous fibre subtypes (notably 5-HT and dopamine) control

proliferation and fate of neural stem and progenitor cells (Bond et al., 2015).

This innervation may also control some secretory functions of EZ cells, which

has been suggested in salamanders (Zamora, 1978). In zebrafish, this

innervation controls regenerative neurogenesis from the EZ (Reimer et al.,

2013; Barreiro-Iglesias et al., 2015).

With regard to expression of markers by adult human EZ cells, only a

few studies have been carried out and results are not always consistent. This

might be due to the quality of human samples (post-mortem delay), the

precise anatomical location of the studied tissues and the implemented

techniques (paraffin-embedding or cryosection). In addition, for some markers

with many isoforms, like GFAP with 10 isoforms (Hol and Pekny, 2015),

different antibodies may only detect specific forms of the protein. This could

lead to discrepancy between findings of different laboratories. However, the

available data indicate that in young adults (second decade), subsets of cells

express several markers typically found in immature neural cells such as

CD15, FOXA2, NESTIN, PAX6, SOX2, SOX11, and VIMENTIN (Hugnot,

unpublished results) (Dromard et al., 2008; Cawsey et al., 2015). There is

obvious cellular heterogeneity, as only a subset of EZ cells express GFAP or

37

NESTIN. In addition, some markers are mostly expressed in the dorsal part

(CD15, PAX6), while radial NESTIN+ cells are observed in the ventral and

dorsal parts of the EZ (Dromard et al., 2008; Cawsey et al., 2015) (Hugnot,

unpublished results). This indicates a dorsal-ventral regionalization of the

adult human spinal cord EZ, similar to that observed in the mouse (Sabourin

et al., 2009; Yu et al., 2013; Khazanov et al., 2016) and zebrafish (Figs 1, 2).

In older individuals, even after EZ obstruction, cells of the remaining mass

may retain some immature features, as indicated by the presence of CD15,

SOX2, PAX6, PAX7, and several HOX proteins. This has been detected by

tissue labeling and/or PCR (Garcia-Ovejero et al., 2015).

Evidence for the persistence of neural progenitor cells in the adult

human spinal cord also comes from in vitro studies. Dromard et al, were able

to generate SOX2+/NESTIN+ neurospheres from the gray matter central

region using defined media containing EGF and FGF2 (Dromard et al., 2008).

These neurospheres were multipotent and generated neurons and glial cells

after differentiation. However, the neurospheres could not be passaged,

suggesting that they were derived from proliferation-limited neural progenitors

rather than bona fide stem cells. Using different culture conditions (adherent

substrate and Matrigel) Mothe et al. were able to isolate SOX2+/NESTIN+

multipotent cells that could be passaged for at least 9 months (Mothe et al.,

2011). These cells generate glial and neuronal cells after transplantation into

spinal-injured rats and are thus of great interest with respect to understanding

the molecular mechanisms controlling their proliferation and fate.

There are several critical points when considering human EZ cells as a

suitable cellular source for regeneration. Human EZ cells appear to react to

38

spinal cord injury with an increase in NESTIN expression (Cawsey et al.,

2015), but whether these cells proliferate and migrate has not been reported.

In macaques, production of new glial cells and neurons has been observed

after injury (Yang et al., 2006; Vessal et al., 2007). However, whether these

cells originate from the EZ remains to be established.

An essential difference between rodents and primates is the presence of a

high level of GFAP intermediate filaments in a hypocellular layer, which

surrounds the EZ region in primates (Dromard et al., 2008; Alfaro‐Cervello et

al., 2014) (Fig. 2). A similar hypocellular GFAP+ layer is also found adjacent to

the SVZ neural stem cell niche in marmosets, macaques, and humans (Sanai

et al., 2004; Quiñones‐Hinojosa et al., 2006; Gil‐Perotin et al., 2009;

Sawamoto et al., 2011) and thus appears to be a distinctive feature of

primates. The role of this layer is currently unknown but its presence may

represent an obstacle for regeneration, as EZ cells would have to migrate

through this dense layer to reach lesioned areas. The large size of the spinal

cord might also be an obstacle to regeneration for three reasons. Firstly,

cellular regeneration in anamniotes is achieved notably by ependymo-radial

glia spanning from the EZ to the pial surface. While radial glial cells are clearly

present in rodents and macaque around the canal, it is not known whether

these cells reach the meninges, which are an important source of signals for

neural stem cell maintenance and fate determination during CNS

development (Siegenthaler and Pleasure, 2011). It can be speculated that

with the increase in size, radial cells around the EZ can no longer extend to

the pial surface in mammals and have lost some of their capacity for

39

regeneration. Secondly, longer spinal cords with a larger central canal cavity

could have favoured selection of multi-ciliated ependymocytes during

evolution (Nakayama and Kohno, 1974), so as to move the CSF fluid over a

greater distance. To support this hypothesis, it would be informative to study

the cellular composition of the EZ region in mammals with a long vertebral

column, such as giraffes and whales, to see if multi-ciliated ependymocytes

found in primates are also present. The presence of multi-ciliated

ependymocytes could have been selected for at the expense of proliferative

capacity and stemness, as multi-ciliated cells are post-mitotic (Brooks and

Wallingford, 2014). Thirdly, differences in myelin between anamniotes and

mammals may also account for the contrasting ability to regenerate neurons

from EZ cells. Whereas the basic structure of myelin is conserved between

fishes and mammals, some differences exist. Myelin Protein zero, a protein

possibly involved in zebrafish CNS regeneration (Schweitzer et al., 2003), is

present in both CNS and PNS myelin of fish, in contrast to the mammalian

homolog, which is expressed only in PNS myelin (Bai et al., 2011). Recent

results indicated that proteins of myelin, such as myelin basic protein, can

inhibit spinal cord neural stem cell proliferation in vitro and such mechanisms

could also operate in vivo to limit regeneration in mammals (Xu et al., 2017).

4. Comparing regenerating and non-regenerating species

From the above comparisons, far reaching similarities between EZ cells

in anamniotes and mammals become apparent (summarized in Table 1 and

Fig. 2). Morphologically, cells in the EZ are ciliated and have radial processes.

In anamniotes, these processes form endfeet at the pial surface. In mammals,

40

only EZ cells at the dorsal and ventral midline appear to have endfeet at the

pial surface, whereas the radial processes of most EZ cells are shorter and

end on parenchymal blood vessels. EZ cells in anamniotes and amniotes also

share expression of structural and functional genes, such as GFAP and

Vimentin.

Spinal EZ cells are not a homogeneous population, with dorso-ventral

and rostro-caudal differences in expression of developmental transcription

factors that confer regional identity. This may be related to a degree of

lineage restriction of progenitor cells. Remarkably, both anamniote and

mammalian EZ cells express transcription factors related to « stemness »,

such as Sox2. However, in anmniotes, the neuronal lineages of progenitor

cells are fully expressed, whereas in mammals environmental factors appear

to restrict this potential.

After a lesion, EZ cells in both anamniotes and mammals react to

lesion signals with strongly increased proliferation. Hence, any lesion stimulus

is adequate in anamniotes and mammals to bring EZ cells out of relative

quiescence to start proliferating. This suggests that lesion-induced

intracellular mechanisms may be similar. Indeed, as we discuss above, the

Notch pathway, which attenuates neurogenesis, is upregulated in EZ cells of

regenerating and non-regenerating species. Moreover, activity of the

hedgehog pathway is observed across vertebrates. It is therefore likely that

differences in intracellular signalling are not fundamental, but that different

levels of activity of these pathways lead to dramatically different net outcomes

in terms of gliogenesis versus neurogenesis.

41

However, fundamental differences are likely to exist between

progenitor cells in anamniotes and mammals. For example, ERGs in

anmniotes have to fulfil the functions of ependymal cells and astrocytes, as

there is little evidence for the presence of “free” astrocytes in anamniotes. In

mammals, there is a clear distinction between ependymal cells and

parenchymal astrocytes. Nevertheless, a closer comparison between cells in

the EZ between regenerating and non-regenerating species may be

informative in order to determine where lesion reactions diverge. Analyses

should also be performed in less-studies species such as turtles (Marichal et

al., 2009), birds, large or aquatic mammals and non-human primates such as

marmoset to bring additional molecular and cellular insights into the functions

of the EZ regions across species.

5. Outlook:

Based on transgenic marker expression, specific EZ cell populations

can be highly purified and subjected to gene expression profiling techniques.

This can be done in a systematic comparison between regenerating and non-

regenerating species and on an EZ domain-specific basis, in order to follow

up on potential lineage restrictions of progenitor domains. Moreover, single

cell sequencing has the capacity to identify unanticipated subpopulations of

progenitor cells (Cuevas-Diaz Duran et al., 2017). This has the potential to

unravel the gene expression programmes that are being switched on after a

lesion.

Three-dimensional ultrastructural analyses by serial block-face

scanning electron microscopy (Denk and Horstmann, 2004; Titze and

42

Genoud, 2016) can serve to describe the morphological diversity of cell types

in the EZ and be correlated with expression profiles. This can address

whether similar niches, with the presence of stem cells and transit amplifying

cells, exist as in neurogenic areas in the brain (Chang et al., 2016).

It will also be important to elucidate how EZ cells receive signals that

are crucial for regeneration. For example, the ventral source of Shh is much

more remote from target cells during regeneration of the adult spinal cord than

during neural tube development. A common denominator between cells in the

EZ is their close and often direct contact with the central canal, which may

transport signals (Zappaterra and Lehtinen, 2012). Hence, analysing the

composition of the CSF and receptors on EZ cells after injury should be

informative.

Finally, zebrafish are the foremost vertebrate in vivo model for

automated drug screening (White et al., 2016). Screening whole zebrafish

larvae for drugs that enhance lesion-induced neurogenesis (Reimer et al.,

2013) combined with those on spinal neurospheres from mammals (Cheng et

al., 2017) may yield important tools to tweak the lesion-induced gene

expression programmes in EZ cells towards a more pro-regenerative

phenotype. We conclude that a comparative approach to the analysis of

spinal EZ cells holds considerable promise to identify critical steps that decide

between inhibitory gliogenesis and pro-regenerative neurogenesis after spinal

cord injury.

43

ACKNOWLEDGEMENTS:

We thank the 2017 cohort of the Wellcome Trust 4 year PhD

programme in Tissue Repair at the University of Edinburgh for critically

reading the manuscript. This work was supported by BBSRC to CGB and TB;

INSERM, AFM, IRME and IRP to JPH’s team; ERA-NET NEURON CoFund

Consortium NEURONICHE (MRC, Spinal Research, ANR) to all authors.

44

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TABLES:

Table 1: Evolutionary comparison of cells in the ependymal zone of the spinal cord

Cilia CSF-N Dorsol-ventral

regionalisation

Rostro-caudal

regionalisa-tion

Retention of developmental

gene expression

Shared ependyma

specific gene

expression

Expression of other genes

Cell types generated

after lesion

Zebrafish ✓ ✓ ✓ Not known FoxA2 ?, Nkx6.1,

Pax6, Shh, Sox2, Sox11

FoxJ1 GFAP, Vim, nestin, CD15 ?

Motor neurons,

interneurons, astrocyte-like bridging glia

Rodents ✓ ✓ ✓ ✓ FoxA2, Nkx6.1,

Pax6, Shh, Sox2, Sox9

FoxJ1 GFAP, Vim, Nestin, CD15

Astrocytes, Oligodendro-

cytes

Primates ✓(presence of multi-ciliated cells)

✓(macaque)

No evidence in

human

✓

Not known FoxA2, Nkx6.1,

Pax6, Sox2

FoxJ1 GFAP, Vim, Nestin, CD15

Not known

60

FIGURE LEGENDS:

Fig. 1: Distinct progenitor domains give rise to different types of neurons after

spinal cord transection of adult zebrafish. A schematic cross-section through

the adult spinal cord is shown, indicating domains of ependymo-radial glial

cells (ERGs) that are defined by expression of the indicated transcription

factors. These ERG domains give rise to the indicated neuronal cell types in

response to recapitulated developmental signals and injury-related signals.

See sections 2.3 and 2.4 for relevant references.

Fig. 2: The organisation of the ependymal zone is complex across

vertebrates. A schematic representation of the EZ is shown for different

species. Note that for humans, the EZ is represented for a young adult before

obstruction of the central canal (see text). The presence of radial cells

contacting vessels in humans is extrapolated from the macaque EZ, but has

not been directly shown.

Figure References:

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