Molecular logic of neocortical projection neuron specification, development and diversity

Abstract

The sophisticated circuitry of the neocortex is assembled from a diverse repertoire of neuronal subtypes generated during development under precise molecular regulation. In recent years, several key controls over the specification and differentiation of neocortical projection neurons have been identified. This work provides substantial insight into the 'molecular logic' underlying cortical development and increasingly supports a model in which individual progenitor-stage and postmitotic regulators are embedded within highly interconnected networks that gate sequential developmental decisions. Here, we provide an integrative account of the molecular controls that direct the progressive development and delineation of subtype and area identity of neocortical projection neurons.

Figure 1. Neocortical projection neurons are generated in an “inside-out” fashion by diverse progenitor types in the VZ and SVZ

This schematic depicts the sequential generation of neocortical projection neuron subtypes and their migration to appropriate layers over the course of mouse embryonic development. (a) Radial glia (RG) in the ventricular zone (VZ) begin to produce projection neurons around E11.5. At the same time, RG generate intermediate progenitors (IP) and outer radial glia (oRG), which establish the subventricular zone (SVZ) and act as transit-amplifying cells to increase neuronal production. After neurogenesis is complete, neural progenitors transition to a gliogenic mode, generating astrocytes and oligodendrocytes. Cajal-Retzius (CR) cells primarily migrate into neocortical layer I from non-cortical locations, while other projection neurons are born in the neocortical VZ / SVZ and migrate along radial glial processes to reach their final laminar destinations. (b) Distinct projection neuron subtypes are born in sequential waves over the course of neurogenesis. The peak birth of subplate (SP) neurons occurs around embryonic day (E) 11.5, with the peak birth of corticothalamic projection neurons (CThPN) and subcerebral projection neurons (SCPN) occuring at E12.5 and E13.5, respectively. Layer IV granular neurons (GN) are born around E14.5. Some callosal projection neurons (CPN) are born starting at E12.5, and those CPN born concurrently with CThPN and SCPN also migrate to deep layers. Most CPN are born between E14.5 and E16.5, and these late-born CPN migrate to superficial cortical layers. Peak sizes are proportional to the approximate number of neurons of each subtype born on each day. NE, neuroepithelial cell; WM, white matter

Figure 2. Models of deep-layer and superficial-layer projection neuron production by distinct progenitor lineages

(a) Fate-mapping experiments have established that most superficial-layer (commissural and associative) projection neurons derive from Cux2-positive progenitors, while deep-layer (corticofugal) neurons derive from Cux2-negative progenitors. Several models have been proposed to describe how this process occurs. (b) The “sequential competence states” model suggests that individual progenitors are able to produce a single neuronal subtype at a time as they progress through a series of competence windows, and that fate-restricted lineages do not exist. Although this model has been refuted, the precise structure of lineage trees during corticogenesis remains unknown. It is possible that progenitors commit to independent lineages before the onset of neurogenesis (c), or that some progenitors first give rise to neurons of one lineage and later commit to a different lineage (d). Similarly, progenitors might be multipotential, giving rise to more than one type of neuron (c and d), or become progressively fate restricted until they are unipotential (e).

Figure 3. Transcription factors in the VZ establish an area identity fate map

(a) Arealization of the cerebral cortex is initiated by diffusible morphogens and signaling molecules secreted from opposing sides of the neocortical periphery (left panel). These signals induce expression of complementary and orthogonal transcription factor gradients such as Pax6/Emx2 and Sp8/Couptf1, seen in a schematized flatmount view of the ventricular zone (VZ) (b) Pax6 is expressed most highly rostrolaterally, in opposition to Emx2, which is expressed most highly caudomedially. Similarly, Sp8 is expressed most highly rostromedially, in opposition to Couptf1, which is expressed most highly caudolaterally. Gradients are shown in wholemount (left) and sagittal (right) views for each. (c) Progenitors located at different medio-lateral and rostro-caudal coordinates express specific levels of these transcription factors, which combinatorially establish a fate map of cortical areas in the ventricular zone. This fate map is later translated into a definitive area map in the cortical plate (CP), shown in flatmount view (left panel). Manipulation of morphogen signaling or VZ transcription factor expression results in dramatic changes in the size and position of cortical areas (right panel). Hatching indicates mixed area identity. A1, primary auditory cortex; Ep, electroporation; M1, primary motor cortex; S1, primary somatosensory cortex; sey/sey, small eye hypomorphic mutant; V1, primary visual cortex; YAC, yeast artificial chromosome

Figure 4. Competing molecular programs direct differentiation of newly-postmitotic projection neurons into one of three broad subtype identities

(a) The subtype identities of postmitotic projection neurons are depicted within a theoretical n-dimensional “subtype space” in which individual subtype identities (as defined by gene expression, morphology, dendritic structure, projection patterns, physiology, and other characteristics) occupy distinct coordinates. Boundaries between these identities, preventing neurons of one subtype from taking on characteristics of another subtype, are established by the action of cross-repressive molecular controls. One boundary exists between neurons specified as SCPN and those specified as CThPN, and another exists between CFuPN (SCPN/CThPN) and CPN. Early in corticogenesis, undifferentiated neurons have largely overlapping subtype identities (top). As development proceeds, neurons differentiate and subtypes become more distinct from each other (bottom). (b) Known molecular controls represent key nodes of an elaborate transcriptional network, only beginning to be elucidated (top). Arrows indicate known cases of genetic or transcriptional activation or repression, and further interactions and molecular controls remain to be identified (bottom). (c) Changes in expression of these key regulators can cause boundaries between subtypes to shift, with neurons partially or completely acquiring features characteristic of other subtypes. In some mutants, neurons acquire CFuPN identity generally, rather than a well-defined CThPN or SCPN identity. The boundaries between CFuPN and deep-layer or superficial-layer CPN may shift independently of one another, represented by the dashed line between deep-layer and superficial-layer CPN. CFuPN, corticofugal projection neurons; CPN, callosal projection neurons; CThPN, corticothalamic projection neurons; SCPN, subcerebral projection neurons

Figure 5. Postmitotic regulators set up sharp gene expression boundaries between cortical areas and direct area-specific phenotypic differentiation of projection neurons

Loss of Bhlhb5 or Lmo4 function affects multiple aspects of postmitotic area identity acquisition, including gene expression, projection patterns, and cellular organization in the S1 barrel field. a) On postnatal day 7, Bhlhb5 is expressed in S1, A1, and V1, whereas Lmo4 is expressed in M1 and excluded from primary sensory areas. b) In the absence of Bhlhb5 (middle row), molecular identity of sensory areas is compromised; for example, Cdh8 expression expands into S1, from which it is normally excluded. Areally-determined projection patterns change, as CSMN in caudal motor cortex fail to reach the spinal cord. Thalamocortical axons (shown by serotonin (5-HT) immunostaining) innervate a wider area of S1 in Bhlhb5^−/−, with indistinct cortical barrels (shown by Nissl staining). Conversely, in the absence of Lmo4 (bottom row), molecular identity of motor areas is compromised, and motor expression of Cdh8 and other genes is reduced. Neurons in motor cortex are inappropriately specified, and fail to send backward collaterals. Thalamocortical axons innervate a narrower area in Lmo4 conditional null mutants, although cortical cytoarchitecture has not been investigated by Nissl staining. WT, wild-type; V1, primary visual cortex; A1, primary auditory cortex; S1, primary sensory cortex; M1, primary motor cortex