Epidermal growth factor receptors control competence to interpret leukemia inhibitory factor as an astrocyte inducer in developing cortex

Abstract

Cortical progenitors begin to interpret leukemia inhibitory factor (LIF) and bone morphogenetic protein (BMP) as astrocyte-inducing signals during late embryonic cortical development, coincident with an increase in their expression of epidermal growth factor receptors (EGFRs). To determine whether the developmental change in EGFRs regulates the change in responsiveness to LIF and BMP, we analyzed cortical progenitors induced to express EGFRs prematurely and progenitors from late embryonic EGFR-null cortex. Premature elevation of EGFRs conferred premature competence to interpret LIF, but not BMP, as an astrocyte-inducing signal. EGFR-null progenitors from late embryonic cortex did not interpret LIF as an astrocyte-inducing signal but responded to BMP4. LIF responsiveness in EGFR-null cells was rescued by the addition of EGFRs but not by the stimulation of fibroblast growth factor receptors. Astrocyte differentiation induced by LIF depends on signal transducer and activator of transcription 3 (STAT3). We show that the level of STAT3 increases during late embryonic development in a subset of progenitors. EGFRs regulate this change in STAT3 and increase STAT3 phosphorylation in response to LIF. Increasing STAT3 prematurely with a retrovirus also increased the phosphorylation of STAT3 by LIF. In contrast to the finding with EGFRs, however, increasing STAT3 did not cause LIF to induce astrocytes, although it reduced expression of the neurogenic factor PAX6 (paired box gene 6 ). Our findings show that developmental changes in EGFRs regulate the competence of progenitors to interpret LIF as an astrocyte-inducing signal. EGFRs elevate STAT3 expression and increase its phosphorylation by LIF, but this is not sufficient to change LIF responsiveness to astrocyte induction, suggesting that EGFRs also regulate LIF responsiveness downstream of STAT3.

Fig. 1.

Premature expression of EGFRs induces a premature change in responsiveness to LIF. Progenitors in cortical explants were infected with either control virus or virus expressing EGFRs at E11, E13, or E16, and the proportion of infected cells (β-gal⁺) (D, F) that express the astrocyte marker GFAP (A, B, G) or the neuron marker TuJ1 (C, E) was determined 4 d later. Cultures were grown in the absence (−) or presence (+) of LIF (L) (A, C) or BMP4 (B) (B, C). EGFRs induce premature competence to interpret LIF as an astrocyte-inducing signal (A, F, G). Some EGFR-infected cells differentiate into neurons (C–E), but this is reduced compared with control-infected cells. Arrows point to double-labeled cells.D–G, E13 progenitors were infected with EGFR virus, grown without (D, E) or with (F, G) LIF, and then stained for β-gal (D) and TuJ1 (E) or β-gal (F) and GFAP (G). C, Comparisons are between EGFR virus and control virus. *p = 0.01; **p ≤ 0.006; ***p < 0.0001.

Fig. 2.

The developmental change in responsiveness to LIF depends on EGFRs. Explants of E16–E17 cortex from EGFR-null mice (−/−) and wild-type littermates (+/+) were infected with control virus (A) or EGFR virus (B), and the proportion of infected cells (β-gal⁺) (C) that express the early astrocyte marker S-100β (A, D) or the late astrocyte marker GFAP (B) was determined after 3 d in the absence (−) or presence (+) of LIF. Competence to generate astrocytes in response to LIF is lost by EGFR-null cells (A) and restored by infecting progenitors with EGFR virus (B). Arrows point to a double-labeled cell expressing β-gal (C) and S-100β (D). *p = 0.03; **p = 0.001; ***p < 0.0001, comparing LIF with untreated cells.

Fig. 3.

The developmental change in responsiveness to BMP4 does not depend on EGFRs. Explants of E16–E17 cortex from EGFR-null mice (−/−) and wild-type littermates (+/+) were infected with a control virus, and the proportion of cells that expressed the late astrocyte marker GFAP was determined after 3 d in the absence (−) or presence (+) of BMP4. *p = 0.009, comparing BMP4 with untreated −/− cells.

Fig. 4.

FGF2-dependent change in responsiveness to LIF requires EGFRs. Explants of E16–E17 cortex from EGFR-null mice (−/−) and wild-type littermates (+/+) were infected with a control virus, and the proportion of infected cells that express the astrocyte marker GFAP was determined after 3 d in the absence (−) or presence (+) of FGF2 or the combination of FGF2 and LIF. FGF2 alone increases astrocyte development in wild-type and EGFR-null cells but does not enhance astrocyte development in response to LIF in EGFR-null cells. *p ≤ 0.0004.

Fig. 5.

STAT3 expression changes during development and is regulated by EGFRs. A, E17 dorsolateral cortex was stained for STAT3. Note that four cells (arrows) express a high level of STAT3 immunoreactivity, whereas the majority express intermediate or low levels (see Results for quantification of the populations). The population of cells that expresses a high level of STAT3 appears at E15 and increases in size over 2 d, coexpressing the progenitor markers PCNA (B) or RC2 (C). D, Premature elevation of EGFRs induces a high level of STAT3 prematurely. Cells infected with the EGFR virus at E13 express the viral marker β-gal (E; arrows) and a high level of STAT3 (F; arrows). G, The size of the population of cells that expresses a high level of STAT3 is reduced in EGFR-null cortical explants (−/−), compared with wild type. *p ≤ 0.02; **p < 0.0001.

Fig. 6.

Premature expression of a high level of STAT3 reduces PAX6 but does not change responsiveness to LIF. A retrovirus that coexpresses eGFP (A) and wild-type STAT3 (B) was used to infect progenitors in E11.5 cortical explants. C, Four days after infection, STAT3 virus increases the proportion of cells that express the early astrocyte marker S-100β in the absence (−) of LIF but does not cause more cells to respond to LIF as an astrocyte-inducing signal. +, Addition of LIF. The proportion of infected E11.5 cells (D; arrow) that express the neurogenic factor PAX6 (E; arrow) is reduced by STAT3 virus compared with a control virus (F). *p = 0.02; **p = 0.003. GFP, Green fluorescent protein.

Fig. 7.

LIF induces STAT3 phosphorylation. E13 explants were infected with EGFR virus (A, B) or STAT3 virus (C, D), dissociated 4 d later, stimulated with LIF for 20 min, and then stained with antibodies to phospho-STAT3 (A, C) and viral markers (B, D).E, The proportion of cells that respond to LIF by phosphorylating STAT3 (pSTAT3) is greater among EGFR-infected and STAT3-infected cells compared with control-infected cells at E11 and E13. −, No LIF; +, addition of LIF. *p ≤ 0.01; **p < 0.0001, comparing LIF with no LIF. More EGFR-infected and STAT3-infected cells responded to LIF at E13 than at E11 (p < 0.0025).

Fig. 8.

Relationship between EGFR expression and responsiveness to LIF. Progenitors that express low (A) or no (D) EGFRs generate neurons when stimulated with LIF, whereas progenitors that express a high level of EGFRs (B, C) generate astrocytes in response to LIF.