Developmental Neuroscience · Unit 4: Making neurons

The Ventricular Zone: Radial Glia as Stem Cells

Most neurons of the cerebral cortex are made by one kind of cell, the radial glial cell, which for decades was seen mainly as a scaffold for migrating neurons. This lesson follows the nucleus of a ventricular-zone progenitor as it moves with the cell cycle, lays out the rodent evidence that radial glia are the progenitors, and separates the direct and indirect routes to a neuron. Afterwards you can use P plus Q accounting to predict how a progenitor pool grows, holds steady or runs down as it makes neurons.

Watch first The Cell Stays, the Nucleus Travels · 2:48

Look at a slice of embryonic mouse cortex and nearly every cell caught in mitosis sits against the ventricle, the fluid-filled space at the centre of the brain. The cells copying their DNA sit farther out.

Why would a cell copy its DNA in one place and divide in another? The answer leads to the cell that makes most cortical neurons, a cell long filed under a different job.

Some words first. The brain begins as the neural tube, an epithelium around a canal that becomes the ventricles (see the neurulation lesson). The surface of the wall that faces the ventricle is apical. The outer surface, covered by a basement membrane and the pia, the thin membrane over the brain, is basal. A progenitor is a cell that still divides and whose daughters will become neurons or glia. The layer beside the ventricle where progenitors live is the ventricular zone (VZ).

The idea: interkinetic nuclear migration

The early wall looks layered, with nuclei stacked at many heights, so nineteenth-century anatomists read it as several kinds of cells. In 1935 Frederick Sauer argued for a simpler picture. Each cell of the wall, a neuroepithelial cell, is long and thin and reaches from the apical surface to the basal one. Only the nucleus moves. It travels toward the ventricle to divide, and the daughters’ nuclei travel back out. A single layer of cells whose nuclei sit at many heights is called a pseudostratified epithelium.

Picture a lift: the shaft, the cell, is fixed; the car, the nucleus, rides up and down on a timetable, the cell cycle. In G1 the nucleus moves basally. S phase, when DNA is copied, happens in the basal part of the VZ. In G2 the nucleus moves back to the apical surface, and mitosis happens there. Chris calls this “Interkinetic nuclear migration”: movement between divisions.

Sauer’s cycle was an inference from fixed sections. Tritiated thymidine, a radioactive DNA building block taken up only by cells in S phase, made it a timetable. Richard Sidman, Irene Miale and Ned Feder reported in 1959 that labeled nuclei in the embryonic neural wall moved between the outer wall and the ventricle in step with the cell cycle.

The movement has motors. In embryonic rat cortex, Jin-Wu Tsai, Richard Vallee, Arnold Kriegstein and colleagues (2010) knocked down motor proteins with RNA interference and found that the kinesin Kif1a carries the nucleus basally in G1 and cytoplasmic dynein pulls it apically in G2. That division of labour was measured in rat. In zebrafish retina the apical movement has been reported to depend on actomyosin instead, so it is not a rule for every vertebrate.

As neurogenesis begins (in mouse, around embryonic days 10 to 12), neuroepithelial cells become radial glia. They keep the shape, a foot on the ventricle and a long fibre to the pia, and they keep the lift. They also switch on proteins associated with glia, such as the glutamate transporter GLAST, the brain lipid-binding protein BLBP and, in primates, GFAP. Notch signalling between neighbours helps keep them as progenitors: in mouse forebrain, an activated form of Notch1 pushed cells toward radial glial identity (Gaiano, Nye and Fishell, 2000). How that receptor works is in the Notch lesson; how Notch picks which cell leaves is in proneural genes and lateral inhibition.

One progenitor, several futures

Where cells divide does not tell you what each makes. For that you need lineage, the descendants of one cell. Chris names two ways in. In viral labeling, a virus inserts a marker gene into one progenitor’s DNA, so all its daughters inherit the mark. In clonal analysis, you study the family of a single founder.

Sally Temple did clonal analysis in the most literal way (Nature, 1989). She dissociated the forebrain of rat embryos at embryonic days 13.5 to 14.5, isolated cells able to divide, and grew them one per tiny culture, with live conditioning cells present because the founders would not divide without them. Most of the clones held only neurons or only glia, while about a fifth (22 percent) were mixed: neurons together with non-neuronal cells. One founder had made both neurons and non-neuronal cells. Chris’s terms for such cells are “Multipotent progenitors” and, where a cell can also renew itself, “Neural Stem Cells”.

Keep the limit in view. A well shows a cell’s potential, what it can make under set conditions, not its fate, what it makes in the embryo. And her cells came from the forebrain as a whole, so they need not have been cortical progenitors.

The scaffold becomes the stem cell

Through the 1970s radial glia were known for another job. Pasko Rakic, using Golgi stains and serial electron microscopy in fetal rhesus monkey cortex, found young neurons pressed against radial fibres on their way to the cortical plate, the forming layer of neurons under the pia (Journal of Comparative Neurology, 1972). Radial glia were seen mainly as scaffolds, supports for neurons born from some other ventricular cell. Radial cells had been seen dividing, but few took them for the main source of neurons. How neurons climb the scaffold is in radial migration.

Three lines of evidence, all in rodents and all around the year 2000, changed the picture.

  1. Sorting and fate mapping, in mouse. Paolo Malatesta, Eva Hartfuss and Magdalena Götz (Development, 2000) used mice that make green fluorescent protein (GFP) from a human GFAP promoter, active in cortical radial glia, and sorted the green cells. In culture they made neurons. In 2003 the Götz group traced lineage in the living animal. Cre recombinase, an enzyme that recombines DNA at marked sites, was made from a GFAP promoter and switched on a permanent reporter in radial glia and all their descendants. Nearly all cortical projection neurons carried it, though the cortical interneurons, born in the ventral forebrain, did not.
  2. Filming lineage, in rat. Stephen Noctor, Arnold Kriegstein and colleagues (Nature, 2001) injected a GFP retrovirus into the ventricles of embryonic rats and filmed living slices of cortex. Labeled radial glia divided at the ventricle, kept their fibre, and made daughters that became neurons and moved out along the parent’s own fibre.
  3. Recording and filling, in rat. The same group (Journal of Neuroscience, 2002) recorded from precursors with an electrode, then filled each recorded cell with dye. The filled cells had the shape of radial glia, and radial glial markers labeled nearly all cells caught in S phase or mitosis.

Each method has a blind spot: a dish shows potential, a recording electrode samples only the cells it can reach, and a marker is not a family tree. The three together persuaded. In embryonic rodent cortex, the cell that guides migrating neurons is also the cell that makes most of them.

Two routes to a neuron: direct and indirect

A radial glial daughter can take one of two routes. In direct neurogenesis, a radial glial cell divides into another radial glial cell and a neuron. In indirect neurogenesis, the second daughter is an intermediate progenitor. It lets go of the ventricle, moves into the subventricular zone (SVZ), the band just outside the VZ, and divides there once more, usually into two neurons.

Noctor and colleagues (Nature Neuroscience, 2004) tracked both routes in rat cortical slices labeled with a GFP retrovirus at embryonic day 16 and followed to day 19. In this study, about 79 percent of radial glial divisions were asymmetric, giving a radial glial cell plus a neuron or an intermediate progenitor, and about 90 percent of intermediate-progenitor divisions gave two neurons. The sample was 146 cells, so read the percentages as rat, at those stages, in that study.

Transcription factors mark the steps. In mouse cortex, Chris Englund, Robert Hevner and colleagues (2005) found that Pax6 marks radial glia, Tbr2 (also called Eomes) marks intermediate progenitors and Tbr1 marks young neurons, in that order as cells move outward:

radial glia (Pax6) → intermediate progenitor (Tbr2) → neuron (Tbr1)

Are the markers only labels? In 2008 two groups removed Tbr2 from the developing mouse brain (Arnold and colleagues; Sessa and colleagues). Division at the ventricle went on, division away from it fell, and fewer cortical neurons were made. Tbr2 is needed for the indirect route, not for all neurogenesis.

What makes two daughters unequal is the subject of asymmetric division. The human cortex adds a third route, through outer radial glia far from the ventricle (Hansen and colleagues, 2010), taken up in human brain expansion.

”CSF and basement membrane provide niche”: counting what leaves

A niche is the local setting that keeps a stem cell a stem cell. A radial glial cell touches two. Its apical foot faces the cerebrospinal fluid (CSF) in the ventricle; in mouse that fluid carries growth factors, among them IGF2, that support progenitor proliferation (Lehtinen and colleagues, 2011). Its basal foot rests on the basement membrane under the pia. In Chris’s words, “CSF and basement membrane provide niche.” In an asymmetric division, the daughter that leaves loses its contact with the apical surface.

Chris writes the cell population as P plus Q: P, progenitors; Q, cells that quit the cycle. Treat them as fractions of the daughters made each round, so P + Q = 1. A pool of N progenitors divides into 2N daughters:

next pool = 2N × P; neurons made this round = 2N × Q

Below Q = 0.5 the pool grows; at 0.5 it holds steady, as when every division is asymmetric; above it, it shrinks. In mouse cortex, Takahashi, Nowakowski and Caviness estimated that over the eleven cycles of neurogenesis Q passes 0.5 in the eighth cycle and reaches about 0.8 in the tenth.

The same group timed the clock (1995). In mouse dorsal cortex the cycle lengthens from about 8 hours at embryonic day 11 to about 18 hours near the end of neurogenesis, and nearly all the added time is in G1. One hypothesis links the two trends: a longer G1 gives fate determinants more time to act. In cultured mouse embryos, a drug that lengthened G1 without stopping the cycle led to early neurogenesis in the forebrain neuroepithelium (Calegari and Huttner, 2003). Layer order is in birth order and layers; the later switch to glia, in origins of glia.

Design problem

One radial glial cell divides six times, each time keeping one radial glial daughter. How many neurons does it make if every division is direct (radial glia plus neuron)? How many if every division is indirect (radial glia plus an intermediate progenitor that divides once into two neurons)? Which change in division mode would most enlarge the cortex?

One way to do it

Direct: six divisions make 6 neurons, and one radial glial cell remains.

Indirect: each division adds one intermediate progenitor, which makes two neurons, so six divisions make 12 neurons, and one radial glial cell still remains. Routing output through intermediate progenitors doubles the neurons per radial glial division without spending the radial glial cell.

The bigger lever is earlier. A symmetric proliferative division, radial glia into two radial glia, makes no neurons now but doubles every later output. One such division before the six indirect ones gives two radial glial cells and 24 neurons; two such divisions give four and 48. In P plus Q terms, keeping Q low for a few extra early cycles multiplies the pool, and the pool multiplies everything after it.

How we measure it

Pulse labeling of DNA synthesis

A labeled DNA building block, tritiated thymidine or the analogue BrdU, is given briefly and is taken up only by cells copying DNA. Fixing tissue at later times shows where the labeled nuclei go and when they divide, which gives the position of S phase, the movement of the nucleus and the length of the cell cycle.

Retroviral lineage labeling with time-lapse imaging

A retrovirus carrying a marker gene such as GFP is injected into the ventricle, where it enters dividing progenitors and inserts into their DNA, so all their descendants are marked. Filming living slices then shows which cell divided and what each daughter became.

Cell sorting and Cre-based fate mapping

Cells that make a fluorescent protein from a cell-type promoter can be sorted and grown alone to test what they can make. Cre recombinase made from the same promoter switches on a reporter permanently, so every descendant stays marked and lineage can be read in the animal.

Whole-cell recording with dye filling

An electrode records a single cell's electrical properties and then fills it with a dye that does not pass easily to neighbours. The cell's full shape can then be matched to its physiology, which tied precursor-like recordings to radial glial shape.

Finish line

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Proneural Genes and Lateral Inhibition

Every cell in the cluster could become the neuron. Why does only one?

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Pax6 paired domain on DNA, the radial glial transcription factor
Pax6 paired domain on DNA, the radial glial transcription factor ● LIVE · RCSB PDB 6PAX
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Loading live structure 6PAX from RCSB PDB…

Two small helical subdomains sit on either side of the DNA, one in the major groove at each end of the binding site. Pax6 is the factor that marks cortical radial glia in the Pax6 to Tbr2 to Tbr1 sequence.

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KIF1A motor domain, the kinesin that carries the nucleus basally
KIF1A motor domain, the kinesin that carries the nucleus basally ● LIVE · RCSB PDB 1I5S
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A compact motor domain with a nucleotide in its pocket. Kinesins of this kind walk toward the plus ends of microtubules, which in rat ventricular-zone cells lies the direction of the basal G1 movement.

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Cytoplasmic dynein motor domain, the motor that pulls the nucleus apically
Cytoplasmic dynein motor domain, the motor that pulls the nucleus apically ● LIVE · RCSB PDB 3VKG
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Loading live structure 3VKG from RCSB PDB…

Six AAA+ modules form a ring, with a long stalk that reaches the microtubule. Dynein walks toward microtubule minus ends, the opposite direction to KIF1A, which fits its role in the apical G2 movement.

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BrdU, the DNA label that marks cells in S phase
BrdU, the DNA label that marks cells in S phase ● LIVE · PubChem CID 6035
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Loading live structure 6035 from PubChem…

A thymidine look-alike with a bromine atom in place of the methyl group. Cells copying DNA take it in, and an antibody then finds it.

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Frederick C. Sauer

Frederick C. Sauer

Argued in 1935 that the wall of the embryonic neural tube is one population of elongated cells whose nuclei move to the lumen to divide and back out afterwards, not a mixture of separate dividing and supporting cells.

In the nineteenth century the wall of the embryonic neural tube looked like a crowd of different cells. Nuclei sat at every height, and the round cells caught dividing beside the central canal looked unlike the long cells around them. Wilhelm His took those round cells for a separate class of germinal cells. In 1935, in the Journal of Comparative Neurology, Frederick Sauer argued for a simpler reading. The wall, he proposed, is made of one kind of cell, each long enough to reach from the inner surface to the outer one, and what differs from cell to cell is only where its nucleus happens to be. Nuclei move toward the lumen and round up to divide, and the daughter nuclei move back out. Sauer worked from fixed sections, so his cycle was an inference from positions. In 1959 Richard Sidman, Irene Miale and Ned Feder followed nuclei labeled with tritiated thymidine and found the sequence he had predicted. His reading made it possible to treat the whole wall as one pool of progenitors.

✓ verbatim Sauer, J. Comp. Neurol. 62:377 (1935), Mitosis in the neural tube; Sidman, Miale & Feder, Exp. Neurol. 1:322 (1959).
Pasko Rakic

Pasko Rakic

Showed with Golgi staining and serial electron microscopy of fetal rhesus monkey cortex that migrating neurons lie against radial glial fibres, and proposed that the fibres guide them, the view of radial glia as a scaffold.

Rakic worked on fetal rhesus monkeys, whose cerebral wall in mid-gestation is much thicker than a rodent's, so a neuron born at the ventricle has a long way to go before it reaches the cortex. In 1972, in the Journal of Comparative Neurology, he combined two methods. Golgi staining fills a few cells completely, so their whole shape can be seen. Serial electron micrographs show which membranes touch which. Young neurons on their way to the upper layers of the cortex lay pressed against long radial fibres that crossed the wall from the ventricle toward the pial surface, and they followed those fibres through the intermediate zone. He proposed, building on a 1971 report in Brain Research, that the fibres, which belong to radial glial cells, guide migration. He later built this into the radial unit hypothesis (Science, 1988): neurons born in one patch of the ventricular zone settle in one column of cortex. The work made radial glia famous as a scaffold, the frame against which their role as progenitors would be tested a generation later.

✓ verbatim Rakic, J. Comp. Neurol. 145:61 (1972), Mode of cell migration to the superficial layers of fetal monkey neocortex; Rakic, Science 241:170 (1988).
Magdalena Götz

Magdalena Götz

With Paolo Malatesta and Eva Hartfuss, sorted radial glia from embryonic mouse cortex and found that they make neurons in culture, then traced their lineage in the mouse and found that nearly all cortical projection neurons descend from them.

Götz's group asked the question by isolating the cells. Paolo Malatesta, Eva Hartfuss and Götz used mice that make green fluorescent protein (GFP) from a human GFAP promoter, which is active in radial glia of the embryonic cortex, and separated the green cells from the rest with a fluorescence-activated cell sorter. In culture the sorted radial glia made neurons (Development, 2000). To trace lineage in the living mouse (Neuron, 2003), Cre recombinase made from the same kind of promoter switched on a permanent reporter in radial glia and in every cell descended from them. In the cortex, nearly all projection neurons carried the reporter, but the interneurons, which come from the ventral forebrain, did not. In the basal ganglia few neurons carried it, so the result was different there. A 2004 fate-mapping study (Anthony and colleagues) found that radial glia made the neurons of every region, so that regional difference is contested. The work let a marker of glia be used to follow neurons.

✓ verbatim Malatesta, Hartfuss & Götz, Development 127:5253 (2000); Malatesta et al., Neuron 37:751 (2003).
Arnold R. Kriegstein

Arnold R. Kriegstein

His laboratory, with Stephen Noctor as first author, filmed retrovirally labeled radial glia in rat cortical slices dividing to make neurons, then found that many of their daughters are intermediate progenitors that divide again in the subventricular zone.

In Kriegstein's laboratory, Stephen Noctor and colleagues injected a retrovirus carrying the gene for GFP into the brain ventricles of embryonic rats. A retrovirus inserts its genes into the DNA of the dividing cell it infects, so every descendant of that cell stays green. They then cut living slices of the cortex and filmed the labeled cells for hours to days. Radial glial cells divided at the ventricle, kept their long fibre, and made daughters that became neurons and moved outward along the parent's own fibre (Nature, 2001). A 2002 paper in the Journal of Neuroscience recorded from precursors with an electrode, filled them with dye, and found radial glial shape and radial glial markers on nearly all dividing cells. In 2004, in Nature Neuroscience, the group followed daughters further and found a second progenitor that leaves the ventricle and divides once more, usually into two neurons. The papers made radial glia the main progenitors of rat cortex and opened the study of intermediate progenitors.

✓ verbatim Noctor et al., Nature 409:714 (2001); Noctor et al., Nat. Neurosci. 7:136 (2004).
Test yourself
Question 1 of 8 Score 0 / 0

In the ventricular zone of the embryonic cortex, where does a progenitor's nucleus sit when the cell divides?
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Every structure, slide, simulation, and clip on this page is listed below with the source it came from. None of the 3D structures is drawn by hand or by AI: each one loads by accession ID from RCSB PDB or PubChem. This panel is generated from the lesson's own data file, so what it lists is what the page loads.

Molecular structures live data

  • Pax6 paired domain on DNA, the radial glial transcription factor — RCSB PDB 6PAX · coordinates CC0 · Human PAX6 paired domain bound to DNA, X-ray crystal structure (Xu et al., Genes Dev., 1999)
  • KIF1A motor domain, the kinesin that carries the nucleus basally — RCSB PDB 1I5S · coordinates CC0 · Mouse KIF1A motor domain with ADP, X-ray crystal structure (Kikkawa et al., Nature, 2001)
  • Cytoplasmic dynein motor domain, the motor that pulls the nucleus apically — RCSB PDB 3VKG · coordinates CC0 · Dictyostelium cytoplasmic dynein motor domain, X-ray crystal structure (Kon et al., Nature, 2012)
  • BrdU, the DNA label that marks cells in S phase — PubChem CID 6035 · 5-Bromo-2'-deoxyuridine, PubChem compound record

Scientist profiles & quotes

  • Frederick C. Sauer — Sauer, J. Comp. Neurol. 62:377 (1935), Mitosis in the neural tube; Sidman, Miale & Feder, Exp. Neurol. 1:322 (1959).
  • Pasko Rakic — Rakic, J. Comp. Neurol. 145:61 (1972), Mode of cell migration to the superficial layers of fetal monkey neocortex; Rakic, Science 241:170 (1988).
  • Magdalena Götz — Malatesta, Hartfuss & Götz, Development 127:5253 (2000); Malatesta et al., Neuron 37:751 (2003).
  • Arnold R. Kriegstein — Noctor et al., Nature 409:714 (2001); Noctor et al., Nat. Neurosci. 7:136 (2004).

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Like I'm five

Deep inside the growing brain is a layer of tall, thin cells that stretch all the way across it like poles in a tent. Each pole can split into two. One half stays a pole and the other half becomes a nerve cell, which climbs up the pole to its place. Sometimes the new half splits once more first, so the pole makes two nerve cells instead of one.
High school

The brain starts as a tube, and its wall is made of long cells that reach from the inside of the tube to the outside. Each cell's nucleus moves up and down inside it as the cell gets ready to divide, always dividing at the inner surface. These cells, called radial glia, were long known as ladders that young neurons climb. Experiments in mice and rats around the year 2000 showed that they are also the stem cells that make most neurons of the cortex. Each division keeps one radial glial cell and makes either a neuron or a helper cell that divides once more into two neurons. Early on, more daughters stay as stem cells; later, more leave to become neurons, until the stem cells are used up.
College

The ventricular zone is a pseudostratified epithelium of neuroepithelial cells, which become radial glia as neurogenesis begins. Each cell spans the wall from its apical foot at the ventricle to its basal foot on the pial basement membrane, and its nucleus moves with the cell cycle: basally in G1, S phase in the basal ventricular zone, apically in G2, mitosis at the apical surface. In rat cortex the basal movement uses the kinesin Kif1a and the apical movement uses cytoplasmic dynein. Sorting and Cre fate mapping in mouse, time-lapse films of retrovirally labeled cells in rat slices, and dye-filled recordings showed that radial glia are the main cortical progenitors. They make neurons directly or through Tbr2 intermediate progenitors that divide again in the subventricular zone, and Pax6, Tbr2 and Tbr1 mark the three steps. With P the fraction of daughters that stay progenitors and Q the fraction that quit, the pool grows when Q is below 0.5 and runs down when Q is above it.
Graduate

Cumulative labeling in mouse dorsal cortex gives a cycle of about 8 hours at E11 rising to about 18 hours near the end of neurogenesis, with almost all of the increase in G1, while Q rises from near zero to about 0.8 by the tenth of eleven cycles. A pool of N progenitors yields 2NQ neurons and 2NP progenitors per cycle, so output peaks when the pool is still large and Q has passed 0.5. Routing output through Tbr2 intermediate progenitors doubles neurons per radial glial division without spending a radial glial cell. In rat slices labeled at E16 (Noctor et al., 2004), about 79 percent of radial glial divisions were asymmetric and about 90 percent of intermediate-progenitor divisions gave two neurons, in a sample of 146 cells. Lengthening G1 pharmacologically in cultured mouse embryos promoted neurogenesis, and shortening it by Cdk4 and cyclin D1 overexpression delayed neurogenesis and expanded the basal progenitor pool.

An open question is causation between the clock and the fate: whether a longer G1 lets fate determinants act, whether it follows from a fate already chosen, or both, and how signals from the CSF at the apical foot and the basement membrane at the basal foot are read by a nucleus that moves between them.
One cell, one family

To follow one cell's family, Sally Temple isolated dividing cells from embryonic rat forebrain and grew each one alone in a tiny culture. The cells would not divide unless live conditioning cells were present, which told her that signals from the environment help drive proliferation. Every family could then be traced to a single founder. About a fifth of the families mixed neurons with non-neuronal cells.

— Temple, Nature 340:471 (1989).
A clock read from film

Atlas and Bond followed tritiated thymidine through eleven-day mouse embryos, sampling at intervals from 25 minutes to 18 hours after the injection. No mitotic figure was labeled until an hour had passed, and after that labeled mitoses were seen for about five and a half hours in the primitive ependymal cells, the dividing cells of the embryonic neural tube. The share of labeled cells rose and fell in waves, which they read as cells moving into and out of the mitotic zone.

— Atlas & Bond, J. Cell Biol. 26:19 (1965).
Germinal cells that were not a separate class

Nineteenth-century anatomists saw large round cells dividing beside the canal of the neural tube and long cells everywhere else, and Wilhelm His treated the round ones as their own class of germinal cells. Frederick Sauer's 1935 paper argued that the round cells were the long cells caught at mitosis, having pulled their nuclei to the lumen. The argument rested on fixed sections and positions alone. Pulse labeling of DNA synthesis confirmed it about a quarter of a century later.

— Sauer, J. Comp. Neurol. 62:377 (1935).

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