Lmo4 and Clim1 progressively delineate cortical projection neuron subtypes during development

Abstract

Molecular controls over the development of the exceptional neuronal subtype diversity of the cerebral cortex are now beginning to be identified. The initial subtype fate decision early in the life of a neuron, and the malleability of this fate when the balance of key postmitotic signals is modified, reveals not only that a neuron is deterministically set on a general developmental path at its birth, but also that this program must be precisely executed during postmitotic differentiation. Here, we show that callosal projection neurons (CPN) and subcerebral projection neurons (subcerebral PN) in layer V of the neocortex share aspects of molecular identity after their birth that are progressively resolved during differentiation. The LIM-homeodomain-related genes Lmo4 and Clim1 are initially expressed by both CPN and subcerebral PN in layer V, and only during mid to late differentiation does expression of Lmo4 and Clim1 become largely segregated into distinct neuronal subtypes. This progressive postmitotic resolution of molecular identity reveals similarities and possibly shared evolutionary origin between layer V CPN and subcerebral PN, and provides insight into how and when these neuronal subtypes achieve their distinct identities during cortical development.

Figure 1.

At early stages of neocortical neuronal differentiation, Lmo4 and Clim1 are coexpressed in the CP. Coronal mouse brain sections showing protein localization by immunocytochemistry. Heterozygote Clim1^+/lacZ transgenic mice, which express βgal in all Clim1-expressing neurons, were used to characterize Clim1 expression, which appears as βgal-filled cytoplasm surrounding the nucleus. (A and B) Lmo4 and Clim1 are broadly expressed in the CP at E15.5. (C–J) Boxed regions in A and B are representative locations of layer V high magnification images. (C) At E15.5, Lmo4 and Clim1 are expressed in overlapping neuronal populations in the CP (arrowheads). (D–F) Lmo4 and Clim1 are expressed at E15.5 in many neurons that express recently identified, central molecular controls over subcerebral PN development: Fezf2 (D; arrowheads), with some exceptions (D; arrows); and Ctip2 (E and F; arrowheads). (G and H) Lmo4 and Clim1 are also coexpressed at E15.5 with Satb2, a recently identified molecular control over CPN development (arrowheads), though some other neurons expressing Lmo4 or Clim1 do not express Satb2 (arrows). (I and J) Lmo4 and Clim1 are extensively coexpressed at E15.5 with Sox5, a recently identified, central molecular control over corticofugal PN development (arrowheads). CP: cortical plate; SVZ: subventricular zone; VZ: ventricular zone; LV: lateral ventricle. Scale bars: (A and B) 25 μm, (C–J) 10 μm.

Figure 2.

By intermediate stages of layer V neuronal differentiation, Lmo4 and Clim1 expression becomes partially restricted to distinct cortical PN subtypes. Coronal mouse brain sections showing protein localization by immunocytochemistry. (C-J) Boxed regions in A and B are representative locations of high magnification images. (A) At P0, Lmo4 is expressed in subsets of neurons throughout all neocortical layers, in contrast to (B) Clim1 expression, which is restricted to cortical layer V. (C) Lmo4 and Clim1 are still extensively coexpressed at P0 (arrowheads), though neurons that only express Lmo4 are apparent (arrow). (D) Lmo4 continues to be coexpressed with Fezf2 at P0 (arrowheads), though many Lmo4-expressing neurons lack Fezf2 expression (arrows). (E and F) Lmo4 is extensively coexpressed with Ctip2 at P0 (E; arrowheads), with some exceptions (E; arrows), while Clim1 is extensively coexpressed with Ctip2 (F; arrowheads). (G and H) Lmo4 is extensively coexpressed with Satb2 at P0 (G; arrowheads), with rare exceptions (G; arrow), while Clim1 is expressed by many neurons, both coexpressed with (H; arrowheads) and expressed independently from Satb2 (H; arrow). (I and J) Lmo4 and Clim1 are both extensively coexpressed with Sox5 (arrowheads), though some Lmo4-expressing neurons do not express Sox5 (I; arrows). MZ: marginal zone; CP: cortical plate; V-VI: neocortical layers V-VI; WM: white matter. Scale bars: (A and B) 50mm, (C-J) 10mm.

Figure 3.

By late stages of layer V neuronal differentiation, Lmo4 and Clim1 expression is largely segregated into CPN and subcerebral PN subtypes, respectively. Coronal mouse brain sections showing protein localization by immunocytochemistry. (A) At P6, Lmo4 is widely expressed in all cortical layers, whereas (B) Clim1 is exclusively expressed in cortical layer V (immunocytochemical background fluorescence is visible in upper layers). (C–J) Boxed regions in A and B are representative locations of high magnification images. (C) At P6, a large proportion of Lmo4-expressing neurons do not express Clim1 (arrows), although a subpopulation of Lmo4 and Clim1 coexpressing neurons exist (arrowheads). (D–F) Lmo4 expression is excluded from FG-labeled subcerebral PN (D; arrows), but it is extensively expressed in CPN (E; arrowheads), and Clim1 expression is absent from CPN (F; arrows), with rare but distinct exceptions (F; arrowhead). (G and H) Lmo4 is largely excluded from Ctip2-expressing neurons (G; arrows), with some exceptions (G; arrowheads), whereas Clim1 is extensively coexpressed with Ctip2 (H; arrowheads). (I and J) Lmo4-expressing neurons coexpress Satb2 (I; arrowheads), whereas Satb2 expression is largely excluded from Clim1-expressing neurons (J; arrows), with occasional exceptions (J; arrowhead). (D) Adapted from (Arlotta et al. 2005). I–VI: neocortical layers I–VI; WM: white matter. Scale bars: (A and B) 100 μm, (C–J) 20 μm.

Figure 4.

Model illustrating the initial coexpression and progressive segregation by late differentiation of Lmo4 and Clim1 expression into layer V CPN and subcerebral PN, respectively. The small subpopulation that continues to coexpress Lmo4, Clim1, and other molecular controls over both CPN and subcerebral PN development might represent the CStrPNi subpopulation of CPN. This model is consistent with either a scenario where fate is specified premitotically and executed and refined postmitotically, or one where fate is specified predominantly postmitotically (see Discussion).