PROX1: a lineage tracer for cortical interneurons originating in the lateral/caudal ganglionic eminence and preoptic area

Abstract

The homeobox-encoding gene Prox1 and its Drosophila homologue prospero are key regulators of cell fate-specification. In the developing rodent cortex a sparse population of cells thought to correspond to late-generated cortical pyramidal neuron precursors expresses PROX1. Using a series of transgenic mice that mark cell lineages in the subcortical telencephalon and, more specifically, different populations of cortical interneurons, we demonstrate that neurons expressing PROX1 do not represent pyramidal neurons or their precursors but are instead subsets of cortical interneurons. These correspond to interneurons originating in the lateral/caudal ganglionic eminence (LGE/CGE) and a small number of preoptic area (POA)-derived neurons. Expression within the cortex can be detected from late embryonic stages onwards when cortical interneurons are still migrating. There is persistent expression in postmitotic cells in the mature brain mainly in the outer cortical layers. PROX1(+ve) interneurons express neurochemical markers such as calretinin, neuropeptide Y, reelin and vasoactive intestinal peptide, all of which are enriched in LGE/CGE- and some POA-derived cells. Unlike in the cortex, in the striatum PROX1 marks nearly all interneurons regardless of their origin. Weak expression of PROX1 can also be detected in oligodendrocyte lineage cells throughout the forebrain. Our data show that PROX1 can be used as a genetic lineage tracer of nearly all LGE/CGE- and subsets POA-derived cortical interneurons at all developmental and postnatal stages in vivo.

Figure 1. PROX1 expression in the embryonic forebrain.

(A–D) Immunolabeling for PROX1 in coronal sections of the forebrain at E12.5 and E14.5. Expression can be seen in the SVZ of the MGE, dorsal LGE (dLGE), CGE and POA as well as in the dentate neuroepithelium (DNE), thalamus (Thal) and hypothalamus (Hyp). (E) Co-localization between PROX1 and Ki67 in the SVZ of the MGE at E14.5. (F–G) At E16.5, sparse PROX1^+ve cells are visible in the cortex (shown at higher magnification in G). (H–M) Immunolabeling for PROX1 and Venus/GFP in transgenic mouse lines that label either all cortical interneurons (H,I), MGE/POA-derived ones (J,K) or LGE/CGE–derived ones (L,M). PROX1^+ve cells in the cortex at E16.5 correspond to migrating immature interneurons expressing Venus (H, I). MGE/POA-derived migrating interneurons are largely immunonegative for PROX1 (J, K). Most PROX1^+ve cells in the cortex represent LGE/CGE-derived migrating interneurons (L, M). Arrows indicate PROX1^+ve Venus/GFP^+ve interneurons, arrowheads indicate PROX1^+ve Venus/GFP-negative cells. Open arrowheads point to autofluorescence from red blood cells. Scale bars: A–D, F, 500 µm; E, 25 µm; G, 100 µm; H, J, L, 100 µm; I, K, M, 20 µm.

Figure 2. PROX1 is expressed in cortical interneurons and oligodendrocyte lineage cells at P30.

(A) PROX1, Venus and SOX10 expression in the primary somatosensory cortex of the Dlx1-Venus^fl mouse. The cortex is divided into 10 equal bins for quantification. (B) Higher magnification of the boxed area in A. Nuclear PROX1 expression is evident in interneurons (arrows) and oligodendrocyte lineage cells (arrowheads) throughout all the layers of the cortex. Interneurons generally showed more intense labelling for PROX1. (C) Distribution of PROX1^+ve Venus^+ve interneurons quantified across cortical bins. (D) Quantification of the percentage of PROX1^+ve cells expressing Venus (interneurons or INs) or SOX10 (oligos) in each bin. (E) Contribution of PROX1^+ve cells to the total oligodendrocyte lineage population. WM, white matter. Scale bars: A, 100 µm; B, 50 µm.

Figure 3. PROX1 labels cortical interneurons derived from the LGE/CGE and POA.

(A) Co-expression of PROX1 and Venus/YFP in transgenic mouse lines that label interneurons derived from different progenitor regions. Arrows indicate co-expression, arrowheads indicate Venus/YFP-negative PROX1^+ve cells. Open arrowheads indicate YFP-labeled oligodendrocytes. (B) Contribution of the different progenitor regions to the PROX1^+ve interneuron population. (C) Co-localization of PROX1 and Venus/YFP expressed as a percentage of the total interneurons derived from each progenitor region. (D) The contribution of LGE/CGE/POA (light grey) or POA only (dark grey) to the PROX1^+ve population in each bin. The POA contributes mainly upper layer PROX1^+ve interneurons. Scale bar: A, 50 µm.

Figure 4. Co-expression of PROX1 with cortical interneuron markers.

(A) PROX1 is expressed in CR, NPY, RLN and VIP cortical interneurons. Arrows indicate PROX1^+ve marker^+ve cells, arrowheads indicate PROX1^−ve cells. (B) Quantification of the contribution of PROX1^+ve cells to each of the marker^+ve populations (white), and specifically to the LGE/CGE/POA-derived populations labelled with Venus (grey) in the Lhx6-Cre;Dlx1-Venus^fl mouse. (C) The percentage of PROX1^+ve interneurons co-expressing each of the interneuron markers. (D) Laminar distribution of the interneurons co-expressing PROX1 and CR, NPY or RLN (light grey) as well as the total LGE/CGE/POA-derived marker^+ve populations (dark grey). Scale bar: A, 50 µm.

Figure 5. Expression of PROX1 in the hippocampus at P30.

(A, C, E, G) PROX1 co-expression with Venus/YFP in the hippocampus of the different transgenic mouse lines. (B, D, F, G) Higher magnification images of the boxed areas. Arrows indicate PROX1^+ve Venus/YFP^+ve interneurons, arrowheads indicate PROX1^+veSOX10^+ve oligodendrocytes, open arrowheads indicate YFP^+ve oligodendrocytes in Nkx5.1-Cre;R26R-YFP transgenic mice. Scale bars: A, C, E, G, 500 µm; B, D, F, H, 50 µm.

Figure 6. Expression of PROX1 in the striatum at P30.

(A) PROX1 labels most Venus^+ve striatal interneurons (arrows) in the Dlx1-Venus^fl mouse, and some oligodendrocyte lineage cells (arrowheads). PROX1^+ve interneurons include the MGE-derived subtypes expressing PV (B) and NPY (C). (D) PROX1 co-localizes with MGE-derived striatal interneurons expressing YFP in the Lhx6-Cre;R26R-YFP mouse. Scale bar: A–D, 50 µm.