Direct visualization of microtubules using a genetic tool to analyse radial progenitor-astrocyte continuum in brain

Abstract

Microtubule cytoskeletal dynamics of cortical progenitors and astroglial cells have critical roles in the emergence of normal functional organization of cerebral cortex and in disease processes such as tumorigenesis. However, tools to efficiently visualize these events are lacking. Here we describe a mouse genetic model to efficiently visualize and analyse radial progenitors, their astroglial progeny, and the microtubule cytoskeleton of these cells in the developing and adult brain. Using this tool, we demonstrate altered microtubule organization and capture dynamics in adenomatous polyposis coli-deficient radial progenitors. Further, using multiphoton microscopy, we show the utility of this tool in real-time imaging of astrocytes in living mouse brain and the short-term stable nature of astrocytes in cerebral cortex. Thus, this model will help explore the dynamics of radial progenitor/astrocyte development or dysfunction and the influence of microtubule functions during these events.

Figure 1. Generation of Glast1 promoter-EMTB-GFP mice

(A) Glast1 BAC clone (RP23-63O21), indicating the location of Glast1 gene. (B) Insertion of transgene (EMTB-3GFP-pA) into Glast1 BAC clone. Integration of the transgene by homologous recombination, followed by selection of the targeted BAC clone, and removal of the Neo cassette. Glast1-EMTB-3GFP-pA transgene thus generated was used to drive astroglial specific GFP labeling of stable microtubules in the developing and mature brain. (C) Founder lines were identified by Southern blot analysis of tail DNA and independent breeding lines (named Glast-EMTB-GFP^, ) were established from two of the founders. (D) PCR products of 808 and 844-bp size were detected with two sets of primers (#1, 2) complementary to 5' and 3′ arms of Glast-EMTB-GFP DNA. The transgene plasmid was used as a positive control (+ lane) and DNA isolated from wild type (wt) mice was used as a negative control. Size markers (bp) are indicated in panels C and D.

Figure 2. Selective labeling of radial progenitors in Glast-EMTB-GFP mice

(A) Prominent labeling of radial progenitors is evident in coronal section from E16 cerebral cortex. (B, C) Microtubule cytoskeleton is clearly labeled in isolated radial progenitors from E16 cerebral cortex. (D) Higher magnification image from E16 dorsal cortex illustrates labeled radial glial scaffold. (E, F) Postnatally, as radial glia transform into astrocytes, both radial processes (arrowheads) and newly formed astrocytes (asterisks) are labeled in P10 cerebral cortex. P, pial surface; V, ventricular surface. Scale bar: A, 300 μm; B, 20 μm; C, 7μm; D, 75μm; E–F, 175 μm.

Figure 3. Labeling of astrocytes in distinct compartments of the adult brain

(A) A sagittal section from adult Glast-EMTB-GFP brain shows widespread astrocyte labeling in cortical parenchyma (asterisk), hippocampus (HC), rostral migratory stream (RMS), and cerebellum (CBM). (B) Higher magnification view of labeled astrocytes in cortical parenchyma. (C) Imaging of individual cortical astrocytes illustrates the labeling of the astrocyte process network. (D) Labeled astroglial progenitors in the anterior subventricular zone and (E) astrocytes in the rostral migratory stream. (F) In hippocampus, prominent labeling of radial progenitors in dentate gyrus (DG) region, where neurogenesis occurs, is evident. (G) Higher magnification view of outlined area in panel F. Arrowhead indicates radial processes of progenitors. (H) Labeled Bergmann glial cells in cerebellum. (I) Higher magnification view of outlined area in panel H. Arrowhead indicates radial processes of cerebellar Bergmann glial cells. P, pial surface; V, ventricle. Dotted lines in panel E outlines the top and bottom edges of the rostral migratory stream. Scale bar: A, 800μm; B, 480μm; C, 52μm; D–E, 120μm; F, 330μm; G, 60μm; H, 600μm; I, 50μm.

Figure 4. In vivo multiphoton microscopy of labeled astrocytes in living adult Glast-EMTB-GFP mice

Labeled astrocytes in somatosensory region of cerebral cortex were imaged through a cranial window at a depth of 250μm (A) or 350μm (B) from the pial surface. The complex morphology of astrocytes (arrow, A), their corresponding processes (arrowhead, B), and the astrocytic end-feet contacts with the blood vessels (BV, panel A) can be distinguished. (C, D) Time lapse imaging of astroglial processes in vivo indicates that these processes are stable over the short term (compare processes adjacent to asterisks). Time elapsed between frames are indicated in minutes. Scale bar: 27μm.

Figure 5. Dynamics of microtubule filaments

Growth, shortening or branching of individual microtubule filaments can be seen in this time-lapse panel (see filaments adjacent to arrowhead [growth and shortening] or asterisk [branching]). Merged panel shows the extent of rearrangements over time. Panels were taken from a section of Supplementary Movie 4 (from 1.00 to 1.20 time frames). Time elapsed between frames are indicated in minutes. Scale bar: 5μm.

Figure 6. Disrupted microtubule organization in APC deficient radial progenitors

Labeled microtubule filaments in cortical regions of control (hGFAP-Cre; APC^Lox/+; Glast-EMTB-GFP) and APC deficient (hGFAP-Cre; APC^Lox/Lox; Glast-EMTB-GFP [APC cKO]) radial progenitors were repeatedly imaged. Growth, shortening, and branching of individual microtubule filaments (arrowheads, A) can be seen in control cells. In contrast, significant numbers of APC deficient microtubule filaments are kinked and appear to have hairpin loop-like shape (arrowheads, B). (C) Quantification of kinked, hairpin loop-like microtubule filaments in control (n=66) and APC cKO (n=37) radial progenitors indicates a significant increase in APC cKO cells. % of hairpin loop-like microtubule filaments in 150μm² cortical area were measured and used as microtubule (MT) disruption index. Time elapsed between frames are indicated in seconds. Data shown are mean ±SEM; asterisk, significant when compared with controls at p<0.01 (Student's t test). Scale bar: 2.5μm. Also see supplemental movie #9.