1. Response Diversity and the Timing of Progenitor Cell Maturation Are Regulated by Developmental Changes in EGFR Expression in the Cortex 
Robert C Burrows, Deborah Wancio, Pat Levitt, Laura Lillien 
Neuron 
Volume 19, Issue 2, Pages (August 1997)
DOI: /S (00)80937-X

2. Figure 1 Virus Infects Progenitor Cells in the VZ of Cortical Explants
Explants of E15 cortex were infected and stained 1 day later with X-gal (A), diamidophenylindole (B), or BrdU (C). The infected cells are located in the lower third of the explant, below the developing CP, and within the region labeled by BrdU (C) that defines the VZ. The tissue in (C) was exposed to BrdU for 2 hr prior to fixation. Over the 4 days in culture, progenitor cells in explants continue to divide, differentiate, and migrate, illustrated by a thickening of the MAP-2+ CP. Scale bar = 250 μm.
Neuron  , DOI: ( /S (00)80937-X)

3. Figure 2 Expression of Endogenous and Virally Transduced EGFR
(A) Micrograph of E21 cortex (just below the CP) infected with EGFR virus at E15. The section was stained with an antiserum that recognizes both the virally transduced and endogenous forms of the EGFR (left section), and double labeled with rabbit anti-β-gal to identify infected cells (right section). Note the staining of both EGFR-infected cells and uninfected neighboring cells at this age.
(B) Section of an E15 explant stained with the same EGFR antiserum 1 day after infection with EGFR virus. The section was double labeled with anti-β-gal to identify infected cells. The only cells that appear to be labeled with the EGFR antibody are the virally infected cells.
(C) Histogram comparing the mean fluorescence intensity of virally transduced and endogenous EGFRs at E21 in vivo. EGFR-infected cells (42) and 25 uninfected cells (10 in cortex, 15 in SVZ) were analyzed in sections from three animals. Note that the level of virally transduced EGFR expression is at most only severalfold higher than the level expressed by normal progenitor cells in the SVZ at this late embryonic age. Scale bar = 50 μm.
Neuron  , DOI: ( /S (00)80937-X)

4. Figure 3
Quantitative Analysis of the Laminar Distribution of EGFR and Control Infected Cells In Vivo
(a) Brains infected on E15, analyzed 2 days later. Note the modest shift of the EGFR- infected cells away from the deepest zone, representing the VZ. Chi-square analysis for trends showed the groups to be statistically different at the P < level.
(b) Animals infected on E15, analyzed 5 days later exhibit a pronounced skewing of the distribution of cells away from the VZ and toward the marginal zone (MZ). Chi-square analysis showed the groups to be different at the P < level.
(c) Analysis of the distribution of E infected cells within the subplate (CP1), deep (CP2), and superficial (CP3–CP4) CP shows a similar pattern.
(d) Analysis of complete laminar distribution of cells from control and EGFR-infected animals (E15 + 5) shows the striking bilaminar pattern of the EGFR-infected cells in CP1 (subplate) and the MZ and a more classic distribution of the control infected cells.
(e) E
(f–h) E Analysis was done as described for the E15-injected group. Chi-square analysis showed the groups to be different at the P < level.
Neuron  , DOI: ( /S (00)80937-X)

5. Figure 4
Micrographs Show the Anatomical Distribution of EGFR-Infected Cells in the Developing Cortex of an E (Top) and an E Fetus
Infected cells were visualized immunocytochemically using anti-β-gal. At both ages, there is a striking bilaminar distribution of cells in the subplate and deep CP (CP1–CP2) and superficial MZ. Scale bar = 50 μm.
Neuron  , DOI: ( /S (00)80937-X)

6. Figure 5
Quantitative Analysis of Cell Phenotype Following Infections In Vivo at E15 and in Explants Infected at E12, E15, and E18
(a) In vivo, phenotype was analyzed 5 days postinfection with control or EGFR virus. The percentage of cells labeled with both β-gal and either GFAP or S100β reveals an increase in glial phenotype among EGFR-infected cells.
(b) Laminar distribution of infected cells expressing β-gal and S100β in animals infected on E15 and sacrificed 5 days postinfection on E20. Closed bars represent control infected cells, and open bars represent the EGFR-infected cells.
(c–f) Explants were infected with control virus or EGFR virus at either E15, E12, or E18 and cultured for 3–4 days with or without TGFα (1–10 ng/ml, added daily). As in vivo, adding extra EGFRs to <E18 progenitor cells enhanced astrocyte development.
Neuron  , DOI: ( /S (00)80937-X)

7. Figure 6
Clonal Analysis of E12, E15, and E18 Cortical Progenitor Cells in Monolayer Cultures
Progenitor cells were infected with control virus or EGFR virus and cultured as monolayers 1 day later. After 3 days in monolayer culture in the absence or presence of ligand (TGFα, 1–10 ng/ml, added daily), cultures were double labeled with anti-β-gal to identify infected clones, and either anti-MAP-2 to identify neurons, and anti-GFAP or anti-S100β to identify astrocytes. Each bar represents the mean ± SEM from three to five experiments in which 42–206 clones were counted per condition.
Neuron  , DOI: ( /S (00)80937-X)

8. Figure 7
Generation of Spheres in the Absence or Presence of Virally Transduced EGFRs
(a) Explants were infected with control virus at either E12, E15, E16, or E18, dissociated 1 day later, and cultured in EGF or TGFα (1–10 ng/ml, added every fourth day) under conditions that support the generation of spheres of multipotential progenitor cells. The number of spheres per culture was counted 10–12 days later. The data represent the mean ± SEM from 3–11 experiments.
(b) Explants of E12 cerebral hemispheres (hippocampus + dorsolateral cortex) and E15 dorsolateral cortex were infected with either control virus or EGFR virus, and cultured under the same conditions. Each bar represents the mean ± SEM from 5–12 experiments.
Neuron  , DOI: ( /S (00)80937-X)

9. Figure 8
Cells in Spheres Generated Prematurely from E12 Progenitor Cells Following Infection with EGFR Virus Express the Virally Transduced Human EGFR and Can Differentiate into Neurons, Oligodendrocytes, or Astrocytes
The spheres shown in (A) and (B) were generated after 14 days of exposure to EGF (0.1 ng/ml).
(A) A phase image of the spheres.
(B) Spheres were stained with an antibody specific for the virally transduced huEGFR. Spheres were then grown without ligand for 5 days and stained with anti-huEGFR ([C], [E], and [G]) and either anti-MAP-2 (D) to identify cells that differentiated into neurons, anti-Rip (F) to identify oligodendrocytes, or anti-GFAP (H) to identify astrocytes. Scale bar = 30 μm.
Neuron  , DOI: ( /S (00)80937-X)

10. Figure 9
Model Illustrating the Different Combinations of Receptor and Ligand Levels That May Be Used to Generate Different Thresholds of EGFR Stimulation, Resulting in Distinct Cellular Responses in Cortical Progenitor Cells at Different Stages of Maturation
Previous studies (Ferri and Levitt 1995) demonstrated that early VZ cortical progenitor cells were responsive to EGF and TGFα, but unlike later SVZ cells, they responded by expressing a phenotype characteristic of neurons in limbic cortex rather than dividing or differentiating into astrocytes.
Neuron  , DOI: ( /S (00)80937-X)