Proneural Genes and Lateral Inhibition

In the fly embryo, the cells that will make the nervous system sit in small groups. Every cell in a group can become a neural precursor, yet usually only one does.

The professor’s starting point is a single line: “All cells start off equivalent potential to take on neuroblast fate”. A neuroblast is a neural stem cell of the fly embryo; a sensory organ precursor (SOP) is the cell that will build one bristle and its neuron on the fly’s back. If the cells are equivalent at the start, something must break the tie. The answer comes in two layers. Proneural genes make a group of cells able to become neural. Then the cells signal to each other, and one wins.

The idea: a cluster becomes competent, then one cell is chosen

The proneural genes were found in the fly. achaete and scute, neighbours in a stretch of chromosome called the achaete-scute complex, are needed for bristles: remove them and bristles are missing; switch them on in extra places and extra bristles form. atonal, reported in 1993 by Andrew Jarman, Yves Grau, Lily Jan and Yuh Nung Jan, does the same job for chordotonal organs, internal stretch receptors, and in 1994 was shown to be needed for R8, the founding photoreceptor of each unit of the eye.

All of these genes encode basic helix-loop-helix (bHLH) transcription factors. The helix-loop-helix part lets two proteins pair; the basic part grips DNA at a short sequence called an E-box. A proneural protein works as a pair with a partner, Daughterless in the fly and E proteins in vertebrates. Vertebrates have relatives of both fly families: Ascl1 (once called Mash1) is related to achaete-scute, and the Neurogenins and Atoh1 to atonal.

A proneural gene first comes on in a cluster of cells. Think of it as a casting call: everyone in the cluster gets to audition. In 1993 Mar Ruiz-Gómez and Alain Ghysen followed achaete in staged fly embryos. It came on in clusters of a few cells; later one cell in each cluster held much more transcript and became the neural progenitor. They called the first state competent and the second determined. Where the clusters form was set by regulatory DNA around the gene, not by Notch: in embryos lacking Notch, the clusters still formed in their normal places, but high achaete expression was no longer restricted to one cell in each cluster. Placing the cluster and picking within it are separate events.

Lateral inhibition: “inhibiting the differentiation of neighbors”

The professor puts it this way: “Notch signaling is a pretty big deal-a model for self activation and lateral inhibition.” Here is the picking step.

You met the receptor in that lesson. Delta, a membrane protein on one cell, binds Notch on the cell it touches and pulls; two cuts free the inner part of Notch, which enters the nucleus and, with the DNA-binding protein CSL (called Suppressor of Hairless in the fly), turns on target genes. The last cut, made inside the membrane by γ-secretase, is a case of regulated proteolysis, the theme of the lesson on ubiquitin and proteolysis.

In a proneural cluster the targets that matter are the Enhancer of split, or E(spl), genes. They encode bHLH repressors that switch proneural genes off. Proneural factors, for their part, raise their own expression (the self activation) and, in the textbook model, raise Delta. Put those together and the loop closes:

more Delta on cell A → more Notch activity in cell B → more E(spl) in B → less proneural activity and less Delta in B → less Notch activity in A → more proneural activity in A

Each cell inhibits its neighbour, and the neighbour’s inhibition of it falls in return. Two inhibitions in a row make positive feedback, the switch-making arrangement described in the lesson on signaling circuits. The professor calls this “Balanced / imbalanced feedback/bistability”. A perfectly balanced pair could in principle sit in the middle forever, the way a pencil can balance on its point, but the slightest imbalance tips it. There are two stable outcomes: A neural and B not, or the reverse.

Two kinds of evidence support this picture. First, the phenotype. Fly embryos lacking Notch or Delta make far too many neuroblasts and too little skin on their underside; such genes are called neurogenic, a name used by José Campos-Ortega’s laboratory in the early 1980s for the set, after Donald Poulson’s 1930s descriptions of embryos lacking Notch. Without the receptor, no cell can be told “not you”.

Second, the comparison between neighbours. In 1991 Pascal Heitzler and Pat Simpson made fly mosaics in which patches of mutant cells, or cells with a different level of Notch activity, lay beside normal cells. Cells mutant for Notch became neural on their own, cells mutant for Delta did not, and a normal cell beside cells with less Notch activity than its own became epidermis. What decides a cell’s fate is how its signaling compares with that of the cells beside it.

Design problem

Two neighbouring cells in a fly proneural cluster start with equal Delta, and both express the proneural genes. Predict the outcome when (a) feedback works normally, (b) one cell has half the normal amount of Notch, (c) neither cell has Notch, and (d) both cells have Notch switched on constantly, whatever Delta they receive.

In the fly epidermis this produces a neat, fine-grained pattern: one precursor surrounded by cells that become skin.

”Early on, Notch signaling is anti-differentiation”: Hes in vertebrate progenitors

The same parts are at work in the vertebrate neural tube and cortex, but the result looks different. The vertebrate relatives of E(spl) are the Hes genes, and the professor’s short version is “Hes inhibits proneural genes”. Progenitors express Notch, the ligand Dll1, Hes1 and Hes5, and proneural factors such as Ascl1 and Neurog2. A cell that starts to differentiate raises Dll1 and activates Notch in the progenitors around it, which keeps them as progenitors. The neural tube does not show the fly’s tidy pattern, and neurons leave the progenitor pool over days. Lateral inhibition here works as a brake on how fast progenitors are used up.

The mouse genetics fits. In 1995 Ryoichiro Kageyama’s laboratory, with François Guillemot and others, reported mice lacking Hes1: their neural tubes expressed more Ascl1, made neurons too early, and had severe neural tube defects. Later mutants lacking Hes5 as well lost progenitors still faster.

Then came a surprise about timing. In 2002 Hiromi Hirata and colleagues found that in several kinds of cultured cells Hes1 rises and falls with a period of about two hours, because Hes1 protein represses its own gene and both its messenger RNA and protein are short-lived. In 2008 Hiromi Shimojo, Toshiyuki Ohtsuka and Kageyama imaged reporters in mouse neural progenitors and found that Hes1 oscillates there too, with a period of about two to three hours, and that Neurog2 and Dll1 oscillate in the opposite phase. In cells becoming neurons, Hes1 falls and Neurog2 stays high. One reading is that oscillating Dll1 lets progenitors keep signaling to each other without any of them committing; a steady high level of a proneural factor is what pushes a cell to differentiate.

This answers a question a student once asked in his class: “we are confused about whether the Notch is in stem cell or non-stem cell?” Both cells have the receptor. The cell whose Notch is more strongly activated stays a progenitor; the cell sending more Delta goes on to differentiate. How a dividing progenitor gives its two daughters different amounts of Notch activity, using Numb, is the subject of asymmetric division; the cells themselves are described in radial glia as stem cells, and the later role of Notch in making glia in where glia come from.

Do proneural genes carry identity? The Ascl1 and Neurog2 swap

Proneural factors start neurogenesis. Do they also say what kind of neuron to make? The mouse forebrain offered a test. Neurog2 is high in dorsal progenitors, which make the excitatory neurons of the cortex. Ascl1 is high in ventral progenitors of the ganglionic eminences, which make inhibitory neurons, although some Ascl1 is present dorsally too.

In 2000 Carol Fode, Guillemot and colleagues found that removing Neurog2 let Ascl1 rise in the dorsal forebrain, and some dorsal cells then switched on a subset of ventral markers such as Dlx1 and GAD67, a change that required Ascl1. Other ventral markers did not appear. Placing the Ascl1 coding region at the Neurog2 locus induced some ventral markers in dorsal cells as well.

In 2002 Carlos Parras, Carol Schuurmans and colleagues also did the reverse, putting Neurog2 in place of Ascl1. In the ventral forebrain, Neurog2 restored neuron production and the ventral markers, yet did not switch on dorsal markers. The two swaps were not mirror images. Ascl1 could push dorsal cells partly ventral; Neurog2 did not push ventral cells dorsal.

So proneural factors carry partly different identity information, and how much depends on the factor and the tissue. That fits the fly: atonal starts both a stretch receptor and a photoreceptor, and the surrounding tissue decides which. Competence comes from the proneural factor; much of the identity comes from where the cell is and what it has already been told. When in a lineage those instructions arrive is the subject of birth order and fate.