Temporal identity transition from Purkinje cell progenitors to GABAergic interneuron progenitors in the cerebellum

Abstract

In the cerebellum, all GABAergic neurons are generated from the Ptf1a-expressing ventricular zone (Ptf1a domain). However, the machinery to produce different types of GABAergic neurons remains elusive. Here we show temporal regulation of distinct GABAergic neuron progenitors in the cerebellum. Within the Ptf1a domain at early stages, we find two subpopulations; dorsally and ventrally located progenitors that express Olig2 and Gsx1, respectively. Lineage tracing reveals the former are exclusively Purkinje cell progenitors (PCPs) and the latter Pax2-positive interneuron progenitors (PIPs). As development proceeds, PCPs gradually become PIPs starting from ventral to dorsal. In gain- and loss-of-function mutants for Gsx1 and Olig1/2, we observe abnormal transitioning from PCPs to PIPs at inappropriate developmental stages. Our findings suggest that the temporal identity transition of cerebellar GABAergic neuron progenitors from PCPs to PIPs is negatively regulated by Olig2 and positively by Gsx1, and contributes to understanding temporal control of neuronal progenitor identities.

Figure 1. Mutually exclusive expression of Olig2 and Gsx1 in the cerebellar VZ at E12.5

(a–d) Wild-type cerebellar primordium at E12.5 stained with antibodies for Gsx1, Olig2 and Ptf1a. Coloured lines indicate ventral (green), intermediate (yellow) and dorsal (red) regions of cerebellar VZ, respectively. Arrowheads indicate the boundary between Gsx1 and Olig2-expressing progenitors. (e–g) Higher-magnification images of the rectangular regions in (a), (b) and (d), respectively. Scale bars represent (a–d) 100 μm, (e–g) 50 μm.

Figure 2. Cerebellar progenitors for Purkinje cells and Pax2 + INs

(a–p) Lineage tracing analyses using Olig2^GFP/+ (a–d,i–l) and Gsx1^GFP/+ (e–h,m–p) embryos. (a–h) In the cerebellum, Purkinje cells are specifically labelled with an antibody for Corl2. (i–p) GABAergic interneurons are labelled with an antibody for Pax2. Arrowheads highlight double-stained cells. Stages, genotypes and antibodies are indicated. Scale bars represent (a,e,i and m) 100 μm, (b–d,f–h,j–l and n–p) 25 μm.

Figure 3. Temporal identity transition of cerebellar GABAergic neuron progenitors

(a–d) Developmental changes of localization of PCPs and PIPs in the embryonic cerebellar VZ. (a–c) Immunostaining with Gsx1, Olig2 and Ptf1a in (a) E12.5, (b) E13.5 and (c) E14.5 cerebella. (d) Schematic diagram of developmental changes of Gsx1- and Olig2-expressing regions in the cerebellar VZ. As development proceeds, Gsx1-expressing region expands to the dorsal region, while Olig2-expressing region shrinks. (e–g) Intermediate-term lineage trace using Olig2^GFP/+ embryos to investigate the lineage relationship between PCPs and PIPs. Cerebellar primordium of Olig2^GFP/+ at E13.5 (e,f) or E14.5 (g) was immunostained with indicated antibodies. Arrowheads highlight double-positive cells for GFP and the marker. (f) High magnification of the rectangle in (e). (h) Schematic diagram of temporal identity transition of cerebellar GABAergic neuron progenitors from PCPs to PIPs. PCPs at early stages change their character to become PIPs at late embryonic stages and start to generate Pax2 + INs. Scale bars represent: (a–c,e) 100 μm, (f) 25 μm, (g) 10 μm.

Figure 4. Phenotypes of pN3-Gsx1 Tg and Gsx1 KO mice

(a–d) Cerebellar primordium of E12.5 pN3-Gsx1 Tg mouse stained with antibodies for Gsx1, Ptf1a and Olig2. Coloured lines indicate ventral (green), intermediate (yellow) and dorsal (red) regions of cerebellar VZ, respectively. (e–h) Cerebellar primordium of E12.5 (e,f) wild-type or (g,h) pN3-Gsx1 Tg mouse stained with antibodies for (e,g) Pax2 or (f,h) Corl2 and Lhx1/5. Postmitotic GABAergic neurons are labelled with an antibody for Lhx1/5 at this stage. (i) The average number of Pax2 + INs per slice of control (wild type and Gsx1^GFP/+), Gsx1^GFP/GFP and pN3-Gsx1 Tg embryos (n = 3, *P<0.05, **P<0.01 by t-test, mean±s.e.m.). (j–q) Immunostaining of E12.5 Gsx1^GFP/+ (j–m) and Gsx1^GFP/GFP (n–q) cerebella with indicated antibodies. Scale bars represent (a–h,l,p) 100 μm, (j,m,n,q) 50 μm, (k,o) 10 μm.

Figure 5. Phenotypes of Olig1/2 dKO mice

(a,e) Cerebellar primordium of E13.5 (a) wild-type and (e) Olig1/2 dKO mouse stained with antibodies for Corl2 and Lhx1/5. Coloured lines indicate ventral (green), intermediate (yellow) and dorsal (red) regions of cerebellar primordia, respectively. (b–d,f–h) Higher-magnification images of the rectangular regions in (a) and (e), respectively. (i,m) Cerebellar primordium of E13.5 (i) wild-type and (m) Olig1/2 dKO mouse stained with antibodies for Pax2 and Lhx1/5. (j–l,n–p) Higher-magnification images of the rectangular regions in (i) and (m), respectively. Scale bars represent (a,e,i and m) 100 μm, (b–d,f–h,j–l and n–p) 50 μm.

Figure 6. Altered fates of GABAergic neurons generated around E12.5 in Olig1/2 dKO mice

(a) Schematic diagram of BrdU incorporation experiment. At E12.5, intraperitoneal injection of BrdU was performed and cells in S-phase (progenitors) incorporated BrdU. At E13.5 postmitotic neurons generated from BrdU-labelled progenitors can also be labelled with BrdU. The number and the position of BrdU-labelled GABAergic interneurons and Purkinje cells predicts a general distribution of PIPs and PCPs at E12.5, respectively. (b,c) Examples of immunostaining of E13.5 wild-type cerebellum using antibodies for BrdU, Lhx1/5 and (b) Pax2 or (c) Corl2. Immunostaining of Lhx1/5 enables the distinction of BrdU-labelled postmitotic neurons from BrdU-labelled mitotic progenitors. (d,e) Representative images of plots of indicated cells in wild-type and Olig1/2 dKO cerebella. (f) The ratio of BrdU-labelled Pax2 + INs or Purkinje cells to BrdU-labelled GABAergic neurons in wild-type and Olig1/2 dKO cerebella (n =3, ***P<0.001 by t-test, mean±s.e.m.). (g,h) Short-term lineage tracing analyses using (g) heterozygotes and (h) homozygotes of Olig1/2 dKO line. Arrowheads indicate GFP-positive Pax2 INs. Scale bars represent (b,c) 100 μm, (g,h) 25 μm.

Figure 7. Ectopic expression of Olig2 in Gsx1-expressing progenitors

(a) Schematic diagram of in utero electroporation. Plasmid DNA was injected into the fourth ventricle and electroporated to the Gsx1-expression region at E12.5. The electroporated brains were collected at E13.5 or E14.5. (b) Immunostaining of E13.5 wild-type cerebellum electroporated with Olig2 at E12.5. Antibodies are indicated. (c–f) Sections of E14.5 wild-type mice electroporated with (c,d) control and (e,f) Olig2. Regions just above the Gsx1-expressing cells are shown. Arrowheads indicate co-immunostaining of GFP and the marker. (g,h) Quantification of the percentages of (g) Pax2-positive cells and (h) Lhx1/5-positive cells in electroporated cells (n =3, NS, not significant, **P<0.01 by t-test, mean±s.e.m.). (i) Summary of this study. Scale bars represent (b) 25 μm, (inset of b,c–f) 10 μm.