Asymmetric Division: Numb and the Neuroblast

A fly embryo with no working copy of the gene numb still builds its small sense organs, but the sensory neurons in them are replaced by support cells. The protein Numb, it turned out, goes to only one of two sister cells.

That second fact is the subject of this lesson. A cell can make two different daughters in a single division by giving one of them something the other does not get. In the fly, the something is Numb, and what Numb does is switch off a signal you have met before.

Two flavors of stem cell division

A stem cell, or a progenitor (a dividing cell that will give rise to differentiated cells), can divide in two ways. In a symmetric division the daughters are alike: two progenitors, which enlarges the pool, or two cells that go on to differentiate, which spends it. In an asymmetric division one daughter stays a progenitor and the other moves toward a fate. A tissue that must keep its stem cells and still produce differentiated cells needs asymmetric divisions, or a balanced mix of the two symmetric kinds.

A division can become asymmetric in two ways.

1. From inside. Before dividing, the mother cell gathers a protein, called a determinant, on one side of its cortex (the layer just under the membrane) and lines its spindle up so the furrow separates that side from the other. One daughter inherits the determinant.
2. From outside. The daughters start alike, but only one stays in contact with a niche, a set of neighbouring cells that sends signals to remain a stem cell. The daughter pushed out of the niche differentiates.

Think of the first as packing a note in one lunchbox, and the second as which sister keeps her seat at the table.

The fly ovary shows the niche clearly. Each ovariole, one of the tubes in which eggs form, holds about two germ line stem cells, which sit against a small group of somatic cells. In 2000 Ting Xie and Allan Spradling showed that these somatic cells act as a niche, with the cap cells a key part: stem cells lost to differentiation were efficiently replaced, so the niche could be repopulated. The niche also answers the question “How do we maintain stem cells?” from the other side. A stem cell that loses its niche tends to differentiate, and if the niche itself is lost, the stem cells it held are not replaced. In the vertebrate neural tube, the cerebrospinal fluid on one side and the basement membrane on the other are thought to provide a niche for the progenitors of the ventricular zone.

What is Numb?

A fly bristle is a small sense organ of four cells: a neuron, a sheath cell that wraps it, and two outer support cells that build the hair and its socket. All four come from one sensory organ precursor (SOP) in two rounds of division.

In 1989 Tadashi Uemura and colleagues in the laboratory of Lily and Yuh Nung Jan reported that in numb mutant embryos, the sensory neurons are transformed into the support cells of the same lineage. In 1994 Michelle Rhyu, Lily Jan and Yuh Nung Jan stained dividing SOPs with an antibody to Numb. The protein was associated with the membrane and lay in a crescent on one half of the cell, and when the cell divided it went into one daughter only. In the scheme they used, that daughter is the inner precursor that will make the neuron and sheath; its sister makes the hair and socket. Without Numb, the lineage made four outer support cells and no neuron or sheath. With extra Numb made during the SOP division, the change went the opposite way. Later live imaging of the adult bristle revised the lineage: the inner precursor first makes a glial cell, then divides again to make the neuron and sheath. Numb’s role in choosing between the two branches stands.

So what does Numb act on? Notch, a receptor that signals only between touching cells and that, when activated, is cut so that its inner part can switch genes on. In 1996 Ming Guo, Lily Jan and Yuh Nung Jan showed that Notch works as a two-way switch at each division of the sense organ lineage and that Numb negatively regulates it, probably by binding Notch through Numb’s phosphotyrosine-binding (PTB) domain (see protein-interaction modules). In the same issue of Neuron, Eric Spana and Chris Doe studied a neural precursor in the fly CNS, MP2, that divides once into two different neurons, dMP2 and vMP2. Delta-Notch signalling was needed for vMP2, Numb went into dMP2, and flies lacking both Notch and numb made two dMP2 neurons. Numb was not needed to make dMP2. It was needed only to stop Notch.

The short version, in a phrase from the course lectures: “Notch is pro-stem cell, Numb is anti-notch.” In many lineages the daughter that keeps Notch active keeps progenitor character, and the daughter that inherits Numb cannot hear Notch. How Numb blocks Notch is still argued. In the fly sense organ lineage it is thought to act through endocytosis, pulling Notch and a partner membrane protein, Sanpodo, off the surface of the cell that inherits it.

For later differentiation events, this is how we flip a coin

Here is the logic. Both daughters sit next to each other and could signal through Notch. The coin is Numb: the daughter that gets it is the Notch-off side. Numb carries no instruction of its own about what to become. It biases how one cell answers a signal from its sister, so the cell inherits a private veto on an outside message.

The Notch half of the coin was shown with timing. In 1990 Volker Hartenstein and James Posakony used flies with a temperature-sensitive Notch allele and switched Notch off at chosen times in the pupa. Early loss gave extra bristle precursors. Loss just after the precursors divided gave sense organs made only of neurons, with no shaft or socket. Notch is used once to pick the precursor, and again to split its progeny.

Design problem

In the fly bristle lineage, predict the cells and the bristle you would see if (a) there is no Notch signalling in the SOP and its descendants (“What happens if we’re all heads?”), (b) there is no Numb (“What happens if we’re all Tails?”), or (c) Numb is inherited by both daughters of the first division.

Great for a sensory organ, but what about the CNS?

The fly’s central nervous system is built by neuroblasts, stem cells that leave the neural epithelium and divide again and again. Each division is unequal in size and fate: a large apical daughter stays a neuroblast, and a small basal daughter, the ganglion mother cell (GMC), divides once more to make two neurons or glia.

The machinery has two halves.

The top sets the axis. At the apical cortex, Bazooka (the fly Par3), Par6 and the kinase aPKC form a complex with Inscuteable. Inscuteable recruits Pins, which with the G protein subunit Gαi and the protein Mud tethers microtubules from one spindle pole, so the spindle lines up along the apical-basal axis.

The bottom gets the cargo. aPKC phosphorylates proteins that would otherwise sit all around the cortex, including Lgl and Numb, which releases them from the apical side (see phosphorylation switches). The determinants end up basal. The adaptor Miranda carries the transcription factor Prospero and the translational repressor Brat; Numb uses its own adaptor. In the GMC, Prospero enters the nucleus and shuts down genes for self-renewal and division.

“A difference in polarity between NB and SOP: both rely on asymmetric division.” The SOP divides within the plane of the epithelium, along the head-to-tail axis, with Pins and Numb on the anterior side and the Par complex on the posterior side. The neuroblast divides across the epithelium, with Pins and the Par complex together at the apical side and Numb opposite. And “still using notch signaling in the CNS”: when a GMC divides, Numb again goes to one sibling and Notch decides between the two, as in MP2.

When the coin is rigged

If the cargo is not sorted, the small daughter may not stop being a stem cell. In 2005 Emmanuel Caussinus and Cayetano Gonzalez transplanted larval brain tissue from flies mutant for pins, miranda, numb or prospero into adult flies. The tissue grew to more than a hundred times its starting size, invaded other tissues, killed its hosts within about two weeks, and could be moved to new hosts for years. In 2006 Jens Betschinger, Karl Mechtler and Jürgen Knoblich showed that Brat and Prospero together keep the GMC from self-renewing in larval neuroblasts; in brat or prospero mutants both daughters grew like neuroblasts and formed larval brain tumours. Two other laboratories reported brat overgrowth that year.

Vertebrates are less clear. In 1996 Weimin Zhong and colleagues found a mouse Numb at the apical membrane of dividing cortical progenitors; depending on the cleavage plane, it could go to one daughter or both. Mice have a second relative, Numblike. Removing both early in mouse neurogenesis used up progenitors, while a more restricted removal in the dorsal forebrain, studied across the later peak of neurogenesis, caused progenitors to overproliferate. In mammals the plane of division predicts fate less reliably than in the fly, and Numb also works in the junctions between progenitors and in endocytosis, so not every phenotype need come from blocking Notch.

A closing question from the course lectures points onward: “Numb versus notch is a yes or no? This still doesn’t address diversification.” Numb makes two sisters different. It does not say which of many neuron types a GMC’s progeny become; that depends on when it was born, the subject of birth order and fate.