Mapping the Cortex: Areas and Arealization

In the adult mouse, a patch of cortex near the middle of each hemisphere maps the whiskers and a patch near the back maps what the eyes see. In the embryo, the sheet that will hold both looks the same from front to back.

The cerebral cortex is the sheet of neurons on the surface of the forebrain. In 1909 Korbinian Brodmann divided the human cortex into numbered areas by how its cells were packed and layered, and many of his borders match borders of function: his area 17 is primary visual cortex, the first cortical stop for signals from the eyes. Each area has its own inputs, its own outputs and its own internal map. Yet the cortex grows from a smooth sheet of progenitors, cells that divide to make neurons, with no visible borders at all. Where do the areas come from? The process is called arealization.

The idea: a map drawn twice, by the sheet and by its inputs

Two answers were proposed a year apart. In 1988 Pasko Rakic proposed the protomap. Cortical progenitors lie in the ventricular zone, the layer of the brain wall next to the fluid-filled ventricle, and newborn neurons climb outward along radial glia, long scaffold cells that span the wall. Neurons born from one patch of the ventricular zone therefore stack up in a column above it. If the patches already differ by position, the outline of the areas is laid down where the cells are made, like a city zoned into districts before a single house is built.

In 1989 Dennis O’Leary proposed the protocortex. Here the young cortex is a sheet with broadly similar potential, and areas emerge mainly from the axons that arrive from the thalamus, the relay station that passes signals from the eyes, ears and skin to the cortex. Different thalamic nuclei send axons to different parts of the sheet, and the input tells each part what to become.

Each hypothesis predicts a result you can look for. The protomap predicts molecular differences across the progenitors, and some map that forms without thalamic input. The protocortex predicts that young cortex given new input will take on features that match the input. Both predictions came true in part, which is why the field now treats the map as drawn twice: a sketch from the sheet, then a refined copy shaped by its inputs.

”Critical for the specification of regional identities in the cortex”: Emx2 and Pax6

Cortical progenitors do differ by position. Two transcription factors, proteins that bind DNA and switch genes on or off, are made in opposing gradients across the mouse sheet. Emx2 is highest at the back and along the medial edge, near the midline. Pax6 is highest at the front and along the lateral edge. Two more factors join them. COUP-TFI (also called NR2F1) is highest at the back and lateral side; it belongs to the nuclear receptor family but has no known ligand, so it is called an orphan receptor (see that lesson). Sp8 is highest at the front and medial side. In effect, each progenitor reads its position from the mix of these proteins it holds.

A gradient is only a correlation. To show that a factor matters, you remove it and look for a change, a test of whether it is “Necessary”. In 2000 Kathie Bishop, Guy Goudreau and O’Leary reported that test for both proteins. In mice without Emx2, the front areas, motor and somatosensory, expanded toward the back, and the visual area shrank. In mice without working Pax6 (the Small eye mutant), the shift went the other way: front areas shrank and the visual area expanded forward. The same year Antonello Mallamaci, Edoardo Boncinelli and colleagues independently reported shifts of area identity in the early cortex of mice lacking Emx2.

Read these results with their limits. Both kinds of mutant mice die around birth, before the areas mature, so the “areas” were read from marker genes whose expression borders at birth line up with future areas. Mice without Pax6 have other forebrain defects as well, and later work, including a 2002 study from Luca Muzio, Mallamaci and colleagues, suggested that the late area changes in both kinds of mutant may partly reflect a poorly growing cortical sheet and not only a shifted map.

The Emx2 result passed a sharper test. In 2004 Tadashi Hamasaki and colleagues in O’Leary’s laboratory raised the level of Emx2 in progenitors while keeping its normal slope. The map moved the opposite way from the knockout: the visual area grew and the front areas shrank, with no significant change in the total area of cortex. A factor whose loss and whose excess move a border in opposite directions is allocating territory, not simply permitting cortex to exist. COUP-TFI tells a similar story. In 2007 Michèle Studer’s laboratory removed it from cortex alone, in a conditional knockout (a mouse in which a gene is deleted only in chosen cells), and frontal motor areas spread across most of the sheet while the sensory areas were squeezed toward the back.

Pax6 has an older fame. It is needed to build the eye. Mice with one broken copy have small eyes, which named the Small eye mutant; mice with two broken copies have no eyes. People with one broken copy of PAX6 have aniridia, an eye lacking most of its iris. When the fly version, eyeless, was switched on in places where it is normally off, flies grew eyes on their legs and wings. That is the other kind of test, “Sufficient”: adding a gene where it is normally absent induces or enlarges a structure. In the cortex the same protein does a quieter job, biasing position within a tissue that already exists.

Moving a border with FGF8

A gradient of transcription factors needs a source of position. In the mouse, part of it comes from signaling centres at the edges of the cortical sheet: the cortical hem at the medial edge, which releases Wnt and BMP proteins, and a source of the growth factor FGF8 at the front. FGF8 acts through FGF receptors, which are receptor tyrosine kinases (see that lesson).

In 2001 Tomomi Fukuchi-Shimogori and Elizabeth Grove tested FGF8 in mouse embryos by electroporation: they injected DNA encoding FGF8 into the brain and used brief electric pulses to drive it into progenitors at a chosen spot. Their readout was the barrel field. In the somatosensory cortex of rodents, layer 4 holds clusters of cells called barrels, one for each large whisker, laid out in rows that copy the whisker rows on the snout. The field can be seen in the first postnatal week and makes a sharp landmark.

Extra FGF8 added at the front shifted the barrel field toward the back. Think of FGF8 as a lamp at the front of a room: cells read how bright it is to judge how far forward they sit, so a brighter lamp makes every cell think it sits farther forward than it does.

Design problem

You can place an extra source of FGF8 anywhere in the mouse cortical primordium. Put one at the back, far from the normal source, and predict what happens to the barrel field. Then predict the map if you reduce FGF8 at the front instead. Say which result tests sufficiency and which tests necessity.

The signal and the gradients are one system. In 2003 Fukuchi-Shimogori and Grove reported that Emx2 shapes the map by regulating FGF signaling, and, in a separate study, reducing Fgf8 shifted early gradients of gene expression toward the front (Garel, Huffman and Rubenstein, 2003). That cross-regulation helps explain how a single source at one edge can move every border.

Input matters too: transplanted and rewired cortex

Genes sketch the map, but input shapes much of it. In 1991 Bradley Schlaggar and O’Leary grafted visual cortex from fetal rats into the somatosensory cortex of newborn rats. Thalamic axons that carry whisker signals grew into the graft, and the graft formed barrels. Tissue that would have become visual cortex could still build a feature of a different area when it received that area’s input.

The ferret experiments from Mriganka Sur’s laboratory went further. Ferrets are born with immature visual pathways, so in newborns the retina’s normal targets and the normal inputs to the auditory thalamus could be removed, and retinal axons then grew into the auditory thalamus. Auditory cortex received visual activity. In 2000 Jitendra Sharma, Alessandra Angelucci and Sur reported that in adult ferrets this rewired auditory cortex held neurons tuned to edge orientation, arranged in a map like that of visual cortex, though less regular. A companion paper reported that the animals responded to light seen through the rewired pathway as a visual stimulus.

The protomap is not undone by these results. In mice lacking the transcription factor Gbx2, thalamic axons never reach the cortex, yet in 1999 Betty Miyashita-Lin, John Rubenstein and colleagues found that several area marker genes still had their normal borders at birth. The sketch forms without input. Later experiments in mice suggest that thalamic input then sharpens it: when thalamic axons from the visual thalamus were removed by gene deletion, the genetic distinction between primary visual cortex and the higher-order visual areas around it failed to develop. Spontaneous and sensory activity then refine the maps inside each area, the subject of later lessons in this course.