Birth Order and Fate: Temporal Identity and the Inside-Out Cortex

In the mouse cortex, a neuron born early in cortical neurogenesis ends up deepest, beside the white matter. A neuron born days later ends up near the surface, after climbing past every neuron made before it.

You have seen how Notch and Numb make two sister cells different (see asymmetric division). That gives a yes or no. The professor’s question is the one it leaves open: “If Numb only makes daughter cells different, how does a daughter cell decide which fate to choose?” A fly neuroblast and a mammalian cortex give the same answer from opposite ends. Part of a neuron’s identity is set by when it is born.

The idea: a neuron’s birthday predicts its layer

The neocortex is the six-layered outer sheet of the mammalian forebrain. Its excitatory neurons are made by progenitors in the ventricular zone, the layer of dividing cells that lines the fluid-filled ventricle (see the ventricular zone). The new neurons then travel outward. “Cerebral Cortex Histogenesis: 3 main steps” runs like this:

1. Preplate. The first neurons to leave the ventricular zone form a thin sheet under the outer surface, the preplate.
2. Split. The next wave of neurons settles inside the preplate and splits it in two. The outer part becomes the marginal zone (future layer 1), home of the Cajal-Retzius cells. The inner part becomes the subplate. The sheet of neurons between them is the cortical plate.
3. Inside-out. Each later wave passes the neurons already in the cortical plate and settles above them. Layer 6 is made first, layer 2 last.

How do you know when a neuron was born? You date its last S phase, the part of the cell cycle when DNA is copied. Give a pregnant mouse one dose of thymidine carrying tritium, a radioactive form of hydrogen. Cells take up thymidine by the salvage pathway, in which thymidine kinase adds the first phosphate (see nucleotide metabolism), and only DNA uses it, so only cells copying DNA in the next hour or so are marked. A cell that divides again halves its label. A neuron that stays heavily labeled weeks later made its last DNA copy soon after the pulse. The label marks that S phase. It does not tell the cell anything.

In 1961 Jay Angevine and Richard Sidman injected pregnant mice with tritiated thymidine on different embryonic days and looked at where the labeled cells lay in the cortex of the offspring. Cells labeled earliest lay deepest, and cells labeled on later days lay progressively nearer the surface. The rule is a strong tendency, with overlap between days. The order also ruled out a simple alternative. If new cells just shoved older ones outward, the earliest cells would end up on top. They ended up on the bottom: later cells passed them.

The same order was found, in 1974, by Pasko Rakic in the visual cortex of rhesus monkeys. In the mouse, the neurons of layers 2 to 6 are made over about six embryonic days, E11 to E17, with subplate and layer 6 neurons first and the upper-layer neurons last. These are mouse numbers, and the classes overlap.

Inside-out is a rule of the mammalian neocortex, not of the whole nervous system. In the retina, birth order decides which cell type a progenitor makes, and the layers are not built by later cells passing earlier ones. The professor’s line marks the contrast: “Different strategy In the CNS…precursors tend to migrate to more superficial layers.” How neurons make the climb belongs to radial migration, and what goes wrong in the reeler mouse to Reelin and the reeler mouse.

How do 30 neuroblasts make hundreds of cell types?

The fly asks the question in its cleanest form. In each half-segment of the Drosophila embryo’s nerve cord there are about 30 neuroblasts, the fly’s neural stem cells. Each one buds off a series of smaller cells called ganglion mother cells, and each of those divides to make neurons or glia. “Numb versus notch is a yes or no? This still doesn’t address diversification.” Something else must tell the third daughter of a lineage that it is not the first.

The answer is a clock of transcription factors. “Importance of birth order in diversification: start the transcriptional clock.” In 2001 Takako Isshiki, Bret Pearson, Scott Holbrook and Chris Doe reported that most neuroblasts express four factors in turn: Hunchback, then Krüppel, then Pdm, then Castor. A ganglion mother cell keeps the factor that was on when it budded off, and so do its neurons. The windows overlap, so the sequence is an outline rather than a set of sharp steps.

Think of a bakery whose recipe card changes through the day. Which oven the loaf comes from (the neuroblast’s position in the embryo) sets the kind of bread; which hour it comes out (the factor present) sets which loaf in the day’s series it is.

Expression alone could be a clock face with no hands. The test was genetic. Hunchback and Krüppel also pattern the early embryo, which complicates tests in the nervous system. Without Hunchback in the nervous system, first-born identities were lost in the lineages they examined. With Hunchback kept on, lineages made extra cells with first-born features: in the NB7-1 lineage, which normally makes the motor neurons U1 to U5 in that order, extra cells resembling U1 or U2. Prolonged Krüppel made extra second-born cells. Hunchback did not make every first-born cell the same; each lineage made its own first-born type. Time and place work together. In 2006 the same laboratory showed that Pdm and Castor specify the later U4 and U5 identities in NB7-1.

Moving a birthday: competence narrows

A mammalian birthday predicts a layer, but the cortex offers two explanations. The progenitor might carry its own schedule. Or the surroundings at the time of birth might tell its daughter where to go. Susan McConnell set the two calendars against each other in ferrets, whose visual cortex keeps making neurons after birth, so a newborn kit is a host cortex at the upper-layer stage.

In 1991 McConnell and Christine Kaznowski labeled ferret progenitors with tritiated thymidine, took them from embryos at a stage that makes deep-layer neurons, and put them into older hosts making layers 2 and 3. What mattered was where each cell was in its cycle when it was moved. Cells moved while in S phase mostly settled in layers 2 and 3, like the host’s own neurons. Cells moved later in the cycle mostly settled in the deep layers. Their fate had been fixed before the move.

Could an old progenitor go back? In 1996 Gregory Frantz and McConnell put late progenitors into a young host making deep layers. They still made upper-layer neurons. In 2000 A. R. Desai and McConnell found that progenitors at an intermediate stage, making layer 4, could be pushed forward to upper layers but not back to layer 6. Competence narrows one way.

Three limits apply. These experiments read only the cells that reached the cortex, the cue in the host is still unknown, and a layer position is not a whole identity.

Design problem

Progenitors that normally make deep-layer neurons are moved into an older ferret cortex whose own progenitors make upper-layer neurons. Predict where their daughters settle if they are moved (a) in S phase, and (b) late in the cell cycle. Then predict (c) the reverse experiment: late progenitors moved into a young host.

Does a mammalian progenitor run a clock like the fly’s? That is not settled. When the laboratory of Sally Temple grew single mouse cortical progenitors alone in culture (Shen and colleagues, 2006), clones made neurons with early, deep-layer markers before neurons with later markers. That points to a schedule within the lineage. Homologs of the fly factors do have temporal jobs in mouse: Ikaros, a relative of Hunchback, in the retina, and the COUP-TF genes, relatives of the fly’s Seven-up, in the switch from making neurons to making glia. A full cascade like the fly’s has not been shown in cortex.

The production schedule and the identity it leaves

How many neurons come out at each stage? Every division gives two daughters. Each daughter either divides again (the proliferative fraction, P) or leaves the cycle to become a neuron (the leaving fraction, Q).

P + Q = 1. Progenitors after one cycle = 2 × P × progenitors before. Neurons made in that cycle = 2 × Q × progenitors before.

When Q is below 0.5 the pool grows; above 0.5 it shrinks. In 1996 Takao Takahashi, Richard Nowakowski and Verne Caviness measured Q in mouse dorsomedial cortex in the ventricular epithelium. Q was low early, crossed 0.5 on E14, in the eighth cycle, and rose to about 0.8 by E16. Their model spanned eleven cell cycles over E11 to E17. The cycle also lengthens over the same days. So the cortex first builds its progenitor pool and then spends it, and the order in which it spends it is the order of the layers.

Each layer’s neurons carry identity genes that serve as markers. In mouse, Tbr1 is high in subplate and layer 6 neurons; Ctip2 and Fezf2 are high in layer 5 neurons that project below the cortex; Satb2 marks callosal neurons, which are mostly in the upper layers. Expression overlaps, and position alone does not define a class.

One more caution. The inside-out schedule describes the excitatory, glutamate-releasing neurons made by dorsal progenitors. Most cortical interneurons, which release GABA, are born in rodents in the ganglionic eminences of the ventral telencephalon and travel into the cortex (see tangential migration). Two birthplaces, two transmitters, two schedules sharing one sheet.