Role of BRCA1 in brain development

Abstract

Breast cancer susceptibility gene 1 (BRCA1) is a breast and ovarian cancer tumor suppressor whose loss leads to DNA damage and defective centrosome functions. Despite its tumor suppression functions, BRCA1 is most highly expressed in the embryonic neuroepithelium when the neural progenitors are highly proliferative. To determine its functional significance, we deleted BRCA1 in the developing brain using a neural progenitor-specific driver. The phenotype is characterized by severe agenesis of multiple laminated cerebral structures affecting most notably the neocortex, hippocampus, cerebellum, and olfactory bulbs. Major phenotypes are caused by excess apoptosis, as these could be significantly suppressed by the concomitant deletion of p53. Certain phenotypes attributable to centrosomal and cell polarity functions could not be rescued by p53 deletion. A double KO with the DNA damage sensor kinase ATM was able to rescue BRCA1 loss to a greater extent than p53. Our results suggest distinct apoptotic and centrosomal functions of BRCA1 in neural progenitors, with important implications to understand the sensitivity of the embryonic brain to DNA damage, as well as the developmental regulation of brain size.

Fig. 1.

Embryonic BRCA1 deletion from Nestin progenitors severely disrupts brain development. (A and B) Dorsal views of BRCA1 KO (Brca11^+/−;Brca5-13cK^+/−;Nestin-Cre⁺) and control (Brca11^+/+;Brca5-13cK^+/−;Nestin-Cre⁺, CTRL) brains at E15.5 (A) and P7 (B). (C) BRCA1 KO brains show smaller sizes and failure to thrive than their CTRL littermates. (D) Body weight curve at 2 weeks old showing one third of reduction in body size of BRCA1 KO compared with their littermates in the absence of food competition. (E–G) Nissl staining of BRCA1 KO and CTRL brains at P7. (E) Gross developmental defects are observed in most of the laminated structures in the BRCA1 KO brain. (F) Upper layers (II–IV) are not developed; layer V is only partially (pV) developed, and layer VI is poorly develop in the BRCA1 KO neocortex (Ncx). (G) Pyramidal neurons in the BRCA1 KO neocortex lack of any polarity and radial processes than CTRL. (H) Nissl staining of BRCA1 KO and CTRL brains at E15.5. Arrowheads point to the ganglionic eminence. (I–K) Nissl staining of BRCA1 KO and CTRL at P7 (I and K) and P21 (J). (I) Lamination defects are observed in the olfactory bulb (Ob) of the BRCA1 KO. (J) Reduced volume, lack of foliation, and lamination are present in the cerebellum (Cb) of the BRCA1 KO. (K) CA2-CA3 lamination is disorganized with agenesis of the dentate gyrus (DG) in the hippocampus (Hc) of the BRCA1 KO. I–VI, cortical layers 1–6. (Scale bars: A, B, and E, 1 mm; F, 100 μm; G, 50 μm; H, 200 μm; I and J, 500 μm; K, 100 μm.) Also see Figs. S2–S5.

Fig. 2.

Severe lamination defects associated to BRCA1 deletion from progenitors in the neocortex and cerebellum. The lamination of the neocortex (Ncx) (A–F) and cerebellum (G–L) is severely disrupted in the BRCA1 KO vs. controls (CTRL). (A–C) Immunostaining using specific markers for layer VI (Tbr1, A), layer V (Reelin, Reln; B), and layers II–V (Bhlhb5; C and D) at P7. BRCA1 KO neocortex lack of upper layers (II–IV), only partially developed in layer V (pV) and poorly developed in layer VI. (E and F) p73-labeling Cajal-Retzius cells in the embryonic BRCA1 KO neocortex at E12.5 (E) and E15.5 (F). (G–I) Calbindin (CB) labeling Purkinje cells in the cerebellum of the BRCA1 KO and CTRL at P7. Purkinje cells are confined in a monolayer in the CTRL; however, they form clusters in the BRCA1 KO. The typical dendritic tree of Purkinje cells in CTRL is not observed in the BRCA1 KO. (J–L) GFAP labeling Bergmann glia in the cerebellum of the BRCA1 KO and CTRL at P7. Bergmann glia is distributed in clusters presenting misaligned fibers with abnormal polarity. I–VI, cortical layers 1–6; Ch, cortical hem; ChP, choroid plexus; EGL, external granular layer; Hc, hippocampus; IGL, internal granular layer; MZ, marginal zone; Ob, olfactory bulb; PCL, Purkinje cells layer; pHc, primordium of the hippocampus. (Scale bars: A and B, 125 μm; C and D, 250 μm; E and F, 125 μm; G and J, 500 μm; H and K, 200 μm; I and L, 50 μm.) Also see Figs. S3–S5.

Fig. 3.

p53-dependent apoptosis activation on BRCA1 deletion from neuronal progenitors. (A–H) Short pulses (2 hours) of BrdU in BRCA1 KO and control (CTRL) at E12.5 (A–F) and E15.5 (G–I). Immunostaining with BrdU (red), Caspase3 (green), and DAPI (blue) as counterstaining are shown. High power views of neocortex (Ncx) and ganglionic eminences (GE) are shown in E and G, respectively. Arrowheads in B and F indicate Caspase3⁺ GE. Arrow in E points to a BrdU⁺/Caspase3⁺ cell. Arrows in H indicate the same Caspase3⁺ cells in the same series. Arrowheads in G indicate Caspase3⁺ cells in the same series. (I) BrdU-positive cell quantification at E15.5 showing a pronounced significant reduction in proliferation in the KO brains vs. controls (n = 3, P = 0.0012). (J–L) Immunohistochemistry showing high levels of p53 in the ventricular zone (VZ) and preplate (PrP). p53 is normally expressed in Cajal-Retzius cells in control (arrowheads in J) and KO (arrowheads in K and L). CP, cortical plate; LV, lateral ventricle; SVZ, subventricular zone. (Scale bars: A and B, 250 μm; C and D, 125 μm; E and F, 50 μm; G and H, 100 μm; J and K, 100 μm; L, 50 μm.) Also see Fig. S1.

Fig. 4.

Survival of embryonic neuronal progenitors is dependent of BRCA1. (A–H) BRCA1 KO brains immunostained with the early neuronal differentiated marker Tuj1 (red), Caspase3 (green), and DAPI (blue) as counterstaining at E12.5 (A–D) and E15.5 (E–H). C and D and G and H are high-power views. Arrowheads in F and H indicate the same cell. (I–L) BRCA1 KO brains immunostained with the neuronal progenitor markers Sox2 (I–K) or Pax6 (L), Caspase 3 (green), and DAPI (blue) as counterstaining at E12.5. Arrows point to Caspase3⁺ cells colabeled with Pax6 (L) and Sox2 (K). K is a high-power view. IZ, intermediate zone; MZ, marginal zone; PrP, preplate; LV, lateral ventricle; SVZ, subventricular zone; VZ, ventricular zone. (Scale bars: A, 250 μm; B, 100 μm; C and D, 75 μm; E, 250 μm; F–H, 75 μm; I, 125 μm; J, 75 μm; K and L, 50 μm.) Also see Fig. S1.

Fig. 5.

Proliferation and apoptosis defects induced by BRCA1 loss are cell autonomous. In vitro assay of embryonic neural progenitor cells (ENPCs) isolated from P7 BRCA1 floxed forebrains. ENPCs were cultured and infected with retrovirus or lentivirus delivering the Cre-GFP fusion protein. (A) Concomitant labeling with BrdU indicates that ENPCs have a reduced proliferation on removal of BRCA1 (BRCA1 floxed over KO) as shown by the decreased amount of BrdU⁺/GFP⁺ cells in BRCA1 KO vs. control (BRCA1 floxed over control; CTRL). (B) Statistical analysis showing the BrdU incorporation efficiency in CTRL ENPCs infected with virus expressing GFP and Cre-GFP. Delivery of GFP alone did not have a significant effect in proliferation, whereas Cre-GFP showed about 20% decrease. (C) Statistically significant analysis (Student t test, P < 0.005) showing that BRCA1 KO ENPCs infected with Cre-GFP have a reduction of 80% in BrdU incorporation compared with GFP alone. (D) TUNEL assays showing an increase in twice as much apoptosis in BRCA1-deficient ENPCs than in CTRL (Student t test, P < 0.005) at 2 dpi. (E) Statistic showing a significant fivefold increase in the percentage of TUNEL⁺ cells in Cre-GFP infected ENPCs in comparison with GFP alone (Student t test, P < 0.005). (F) FACS analysis of apoptotic cells stained with Annexin V and DAPI at 3 dpi. (G and H) In vitro assay shows neuronal differentiation deficiency measured by the ratio of Map2(a+b) positivity over total GFP⁺ cells quantitated (H; Student t test, P < 0.005) in BRCA1 KO ENPCs infected with Cre-GFP or GFP alone. Astrocytic differentiation measured by GFAP was not affected. (Scale bars: A, D, and G, 45 μm.) Also see Fig. S6.

Fig. 6.

Concomitant deletion of p53 in BRCA1 KO mice rescues apoptosis defects and restore lamination but not neuronal polarity. BRCA1 KO mice were crossed onto a p53 KO background. (A) Nissl staining at P7 showing cortical cytoarchitecture in control (CTRL), BRCA1 KO (B1KO) crossed with heterozygous p53 (B1KOp53^+/−), and B1KO crossed with homozygous p53 (B1KOp53KO). B1KOp53^+/− behaves as B1KO, whereas B1KOp53KO behaves more like a CTRL in terms of lamination in the cortex. (B) Series of high-power views of A. (C) Series of high-power views of B. Lamination is rescued in the B1KOp53KO but not polarity of the pyramidal neurons in the cortex that persist disoriented as in B1KOp53^+/− or B1KO. Arrowheads in C indicate examples of pyramidal neurons not radially oriented. (D) Immunofluorescence in the cerebellum using proliferating cell nuclear antigen (green) to label dividing granular cells, Caspase3 (red, arrowheads), and DAPI as counterstaining. In the B1KOp53KO, the apoptosis defect is rescue and only present basal levels of apoptosis as in CTRL. However, the B1KOp53^+/− presents high levels of apoptosis and behaves as in B1KO. I–VI, cortical layers 1–6. (Scale bars: A, 125 μm; B, 75 μm; C, 50 μm; D, 125 μm.) Also see Fig. S7.

Fig. 7.

BRCA1 KO defects are fully rescued by codeletion with the tumor suppressor gene ATM. BRCA1 conditional KO (B1KO) mice crossed onto a p53 (B and D–F; B1KOp53KO) or ATM (C and G–K; B1KOATMKO) backgrounds. (A–C) Nissl staining showing P7 brain sections in control (CTRL; A), B1KOp53KO (B), and B1KOATMKO (C). Nissl sections at the level of the neocortex (Ncx) and cerebelllum (Cb) are also shown. Brain structures in B1KOATMKO resemble CTRL ones, whereas in the B1KOp53KO they are substantially recovered but still present some brain structural defects. (D) Nissl staining series showing a partial recovery of the hippocampal lamination in the B1KOp53KO compared with the defects still present in B1KOp53^+/− (similar to B1KO) and CTRL. (E–H) Immunofluorescence in the hippocampus (Hc) showing Bhlhb5 labeling of granular cells in the dentate gyrus (DG) in the B1KOp53KO compared with B1KOATMKO and CTRL. DAPI is shown as counterstaining. Bhlhb5 expression is similar between B1KOATMKO and CTRL at difference than in B1KOp53KO. (I) Ku55933-induced ATM inhibition promotes the proliferation of BRCA1-deficient ENSCs. (J) Western blotting and detection of phosphorylated ATM (ATMp) or total ATM (ATM) protein showing that ATM is activated in BRCA1-deficient ENSCs. (K) Western blotting and detection of ATMp or total ATM protein after treatment with the ATM inhibitor Ku55933. ATM is inactivated after treatment with Ku55933. (Scale bars: A–C, panoramic at 1 mm, Ncx at 100 μm, and Cb at 500 μm; D, 100 μm; E–H, 100 μm.) Also see Fig. S7.