Changes in Epidermal Growth Factor Receptor Expression and Competence to Generate Glia Regulate Timing and Choice of Differentiation in the Retina

doi:10.1006/mcne.1997.0659

Molecular and Cellular Neuroscience

Volume 10, Issues 5–6, April 1998, Pages 296-308

https://doi.org/10.1006/mcne.1997.0659 Get rights and content

Abstract

Previous studies demonstrated that the level of epidermal growth factor receptors (EGF-Rs) expressed by progenitor cells in the newborn (P0) rat retina was limiting for the generation of Muller glial cells but not for proliferation. To determine whether EGF-R signaling biases cells to generate a specific cell type or regulates more general processes during progenitor cell development, we have introduced extra copies of the EGF-R into progenitor cells at earlier stages (E15 and E18), when different cell types are produced. We show that progenitor cells in early embryonic retina (E15) normally express lower levels of EGF-Rs than progenitor cells in later retina (E18 and P0). Whereas lower levels of stimulation of endogenous and virally transduced EGF-Rs enhanced proliferation, higher levels reduced proliferation, resulting in premature differentiation. At E15, very few EGF-R-infected progenitor cells differentiated prematurely into Muller glial cells, unlike E18 and P0 cells, even when they were exposed to an older retinal environment. Higher levels of EGF-R-mediated signaling alone therefore do not specify a glial fate, indicating that competence to generate glia is temporally regulated by additional mechanisms. The differences in EGF-R expression observed among retinal progenitor cells at distinct developmental stages may instead help to define signaling thresholds which delay or accelerate their differentiation.

References (65)

R.M. Anchan et al.
EGF and TGFα stimulate retinal neuroepithelial cell proliferation in vitro
Neuron
(1991)
C.J. Barnstable et al.
A marker of early amacrine cell development in rat retina
Dev. Brain Res.
(1985)
R.C. Burrows et al.
Response diversity and the timing of progenitor cell maturation are regulated by developmental changes in EGF-R expression in the cortex
Neuron
(1997)
R.I. Dorsky et al.
Xotch inhibits cell differentiation in theXenopus
Neuron
(1995)
M. Freeman
Reiterative use of the EGF receptor triggers differentiation of all cell types in the Drosophila eye
Cell
(1996)
W.A. Harris et al.
Two cellular inductions involved in photoreceptor determination in theXenopus
Neuron
(1992)
D. Hicks et al.
The growth and behaviour of rat retinal muller cells in vitro. 1. An improved method for isolation and culture
Exp. Eye Res.
(1990)
C.E. Holt et al.
Cellular determination in theXenopus
Neuron
(1988)
D.D. Hunter et al.
S-laminin expression in adult and developing retina: A potential cue for photoreceptor morphogenesis
Neuron
(1992)
M. Hynes et al.
Induction of midbrain dopaminergic neurons by sonic hedgehog
Neuron
(1995)

R.L. Johnson et al.

The long and short of hedgehog signaling

Cell

(1995)

W.S. Katz et al.

Different levels of the C. elegans growth factor LIN-3 promote distinct vulval precursor fates

Cell

(1995)

C.J. Marshall

Specificity of receptor tyrosine kinase signaling: Transient versus sustained extracellular signal-regulated kinase activation

Cell

(1995)

A.M. Michelsohn et al.

Changes in competence determine the timing of two sequential glucocorticoid effects on sympathoadrenal progenitor cells

Neuron

(1992)

D. Nellen et al.

Direct and long range action of a DPP morphogen gradient

Cell

(1996)

H. Roelink et al.

Floor plate and motor neuron induction by different concentrations of the amino-terminal cleavage product of sonic hedgehog autoproteolysis

Cell

(1995)

N.M. Shah et al.

Glial growth factor restricts mammalian neural crest stem cells to a glial fate

Cell

(1994)

J.S. Simske et al.

LET-23 receptor localization by the cell junction protein LIN-7 during C. elegans vulval induction

Cell

(1996)

J.R. Sparrow et al.

Cell commitment and differentiation in explants of embryonic rat neural retina. Comparison with the developmental potential of dissociated retina

Dev. Brain Res.

(1990)

K. Tomita et al.

Mammalian hairy and enhancer of split homolog 1 regulates differentiation of retinal neurons and is essential for eye morphogenesis

Neuron

(1996)

S. Traverse et al.

EGF triggers neuronal differentiation of PC12 cells that overexpress the EGF receptor

Curr. Biol.

(1994)

D.L. Turner et al.

Lineage-independent determination of cell type in the embryonic mouse retina

Neuron

(1990)

T. Watanabe et al.

Rod photoreceptor development in vitro: Intrinsic properties of proliferating neuroepithelial cells change as development proceeds in the rat retina

Neuron

(1990)

V.W. Yong et al.

A new double labelling immunofluorescence technique for the determination of proliferation of human astrocytes in culture

J. Neurosci. Methods

(1987)

N.B. Zak et al.

Localization of DER and the pattern of cell divisions in wild-type and Ellipse eye imaginal discs

Dev. Biol.

(1992)

R. Adler et al.

Plasticity and differentiation of embryonic retinal cells after terminal mitosis

Science

(1989)

K. Akgawa et al.

Identification and characterization of cell types in monolayer cultures of rat retina using monoclonal antibodies

Brain Res.

(1986)

M.R. Alexiades et al.

Quantitative analysis of proliferation and cell cycle length during development of the rat retina

Dev. Dynam.

(1996)

M.R. Alexiades et al.

Subsets of retinal progenitors display temporally regulated and distinct biases in the fates of their progeny

Development

(1997)

D.M. Altshuler et al.

A temporally regulated, diffusible activity is required for rod photoreceptor development in vitro

Development

(1992)

D.M. Altshuler et al.

Taurine promotes the differentiation of a vertebrate retinal cell type in vitro

Development

(1993)

R.M. Anchan et al.

EGF and TGFα control cell fate determination in developing rat retina

Soc. Neurosci. Abstr.

(1993)

Cited by (61)

Interleukin-4 activates divergent cell-intrinsic signals to regulate retinal cell proliferation induced by classical growth factors
2022, Molecular and Cellular Neuroscience
Citation Excerpt :
Most progenitor cells in the rat developing retina express EGFR, and early progenitor cells express lower levels of EGFR than late progenitors (Lillien, 1995; Lillien and Wancio, 1998). Boosting EGFR signaling promotes neuronal fate among early retinal progenitor cells (Anchan et al., 1991; Lillien and Wancio, 1998), whereas EGFR stimulation in late progenitors can induce a Müller glia fate in vitro and in vivo (Anchan et al., 1991; Lillien, 1995; Lillien and Wancio, 1998), suggesting that EGFR signaling contributes to the timing of differentiation rather than just specifying a particular cell fate. EGFR is also highly expressed in Müller glia, and EGF acts as mitogen for Müller glia in vitro and in vivo in the rodent retina (Close et al., 2006; Hicks and Courtois, 1992; Karl et al., 2008; Roque et al., 1992; Ueki and Reh, 2013).
In the developing retina, precise coordination of cell proliferation, differentiation, and survival is essential for proper retinal maturation and function. We have previously reported evidence that interleukin-4 (IL-4) plays critical roles in neuronal differentiation and survival during retinal development. However, little is known about the role of IL-4 on retinal cell proliferation. In the current study, we investigated if IL-4 regulates cell proliferation induced by epidermal growth factor (EGF) and by fibroblast growth factor 2 (FGF2) in primary retinal cell cultures obtained from newborn rats. First, we show that EGF and FGF2 act as mitogens for glial cells, increasing proliferation of these cells in the retina. EGF- and FGF2-induced mitogenesis requires activation of distinct cell-intrinsic signals. In retinal cells exposed to FGF2, IL-4 downregulates p53 levels (a protein whose activation induces cell-cycle arrest) and increases mitogenic responsiveness to FGF2 through activation of protein kinase A (PKA) pathway. Conversely, in retinal cells exposed to EGF, IL-4 downregulates cyclin D1 levels (a protein required for cell-cycle progression), upregulates p53 levels, and decreases mitogenic responsiveness to EGF. The inhibitory effect induced by IL-4 on retinal cells exposed to EGF requires activation of Janus kinase 3 (JAK3), but not activation of PKA. Based on previous and current findings, we propose that IL-4 serves as a node of signal divergence, modulating multiple cell-intrinsic signals (e.g., cyclin D1, p53, JAK3, and PKA) and mitogenic responsiveness to cell-extrinsic signals (e.g., FGF2 and EGF) to control cell proliferation, differentiation, and survival during retinal development.
A Chimeric Egfr Protein Reporter Mouse Reveals Egfr Localization and Trafficking In Vivo
2017, Cell Reports
Citation Excerpt :
Coronal cryostat sections revealed Egfr-Em present in the periocular mesenchyme surrounding the optic cup, in the extraocular muscles, in mesenchymal cells giving rise to the hyaloid vasculature in the vitreous, in the corneal mesenchyme and epithelium, in the eyelid epithelium (palpebral epidermis), as well as the palpebral and bulbar conjunctiva (Figures 2F and 2F′). This labeling pattern is consistent with previous studies reporting endogenous Egfr mRNA expression and corresponds to roles of EGFR in migration and proliferation of periocular epithelia and mesenchyme (Lillien and Wancio, 1998; Reneker et al., 1995, 2000; Xia and Kao, 2004). The paired-like homeodomain transcription factor Pitx2 is expressed in the periocular mesenchyme and extraocular muscles at this age in the mouse embryo (Gage et al., 2005), and Egfr-Em co-stained with Pitx2 in the periocular mesenchyme, corneal mesenchyme, presumptive hyaloid vasculature, and extraocular muscles (Figures 2F and 2F′).
EGF receptor (EGFR) is a critical signaling node throughout life. However, it has not been possible to directly visualize endogenous Egfr in mice. Using CRISPR/Cas9 genome editing, we appended a fluorescent reporter to the C terminus of the Egfr. Homozygous reporter mice appear normal and EGFR signaling is intact in vitro and in vivo. We detect distinct patterns of Egfr expression in progenitor and differentiated compartments in embryonic and adult mice. Systemic delivery of EGF or amphiregulin results in markedly different patterns of Egfr internalization and trafficking in hepatocytes. In the normal intestine, Egfr localizes to the crypt rather than villus compartment, expression is higher in adjacent epithelium than in intestinal tumors, and following colonic injury expression appears in distinct cell populations in the stroma. This reporter, under control of its endogenous regulatory elements, enables in vivo monitoring of the dynamics of Egfr localization and trafficking in normal and disease states.
Involvement of connexin43 in the EGF/EGFR signalling during self-renewal and differentiation of neural progenitor cells
2013, Cellular Signalling
Neural progenitor cells (NPCs) are sensitive to epidermal growth factor (EGF), which is essential for their self-renewal. Recently we showed that high level of connexin43 (Cx43) expression and gap junctional intercellular communication (GJIC) are also required to maintain NPCs in a proliferative state. In this study the connection between EGF/EGFR signalling and Cx43 expression was investigated during proliferation and differentiation of cultured ReNcell VM197 human NPCs. We found that EGF, but not basic fibroblast growth factor (bFGF), strongly stimulated both Cx43 expression and GJIC in proliferating cells. This stimulatory effect was blocked by AG1478, a specific inhibitor for EGFR kinase. Notably, knockdown of Cx43 strongly inhibited the cell proliferation promoted by EGF/EGFR signalling. High sensitivity to EGF was still maintained in differentiated NPCs. Administration of EGF to differentiating cells led to a pronounced increase (9-fold) of Cx43 expression and a re-induction of proliferation. This strong impact of EGF was found to correlate with a surprisingly massive 60-fold up-regulation of EGFR expression in differentiated cells. Our data argue for a mutual regulation between Cx43 expression and EGF/EGFR signalling during self-renewal and differentiation of NPCs.
The group E Sox genes Sox8 and Sox9 are regulated by Notch signaling and are required for Müller glial cell development in mouse retina
2009, Experimental Eye Research
Citation Excerpt :
Multiple lines of evidence indicate the involvement of several molecules in Müller glial cell development. For example, bHLH transcription factors, NeuroD, Math3, and Math5, are believed to act as negative regulators (Morrow et al., 1999; Inoue et al., 2002; Le et al., 2006), while TGFα signaling via epidermal growth factor receptor (EGF-R) and ciliary neurotrophic factor (CNTF) are thought to function as positive regulators (Lillien, 1995; Lillien and Wancio, 1998; Goureau et al., 2004). However, it is unknown how these molecules regulate Müller glial cell development and whether they interact with the Notch pathway.
Although Müller glial cells play pivotal roles in the vertebrate retina, the regulation of their development is poorly understood. While Notch-Hes5 signaling has been shown to be involved in this developmental process, the presence of Müller glial cells in Hes5-deficient mice suggests the involvement of other molecules. We found that two group E Sox genes, Sox8 and Sox9, are expressed in proliferating progenitors and then exclusively in Müller glial cells in mouse retina. Knocking-down Sox8 and Sox9 by shRNA significantly reduced the population of Müller glial cells and relatively increased the proportion of rod photoreceptors, suggesting that the Sox genes play roles in the specification of Müller glial cells. Using an activated form of Notch and the γ-secretase inhibitor DAPT, we also found that Notch signaling regulates the transcription of Sox8 and Sox9. This is the first evidence that group E Sox genes play important roles in the developing vertebrate retina.
Development and neurogenic potential of Müller glial cells in the vertebrate retina
2009, Progress in Retinal and Eye Research
Citation Excerpt :
Expression analysis of the EGF receptor demonstrated a low level of receptor in early progenitor cells and higher levels in later progenitor cells. Further increasing receptor levels by viral misexpression increased glial cell production in late progenitor cells and not early progenitor cells (Lillien, 1995; Lillien and Wancio, 1998). Müller glial production has also recently been shown to be dependent upon Sox 9 (Poche et al., 2008).
Considerable research on normal and diseased states within the retina has focused on neurons. Recent research on glia throughout the central nervous system, including within the retina where Müller glia are the main type of glia, has provided a more in depth view of glial functions in health and disease. Glial cells have been recognized as being vital for the maintenance of a healthy tissue environment, where they actively participate in neuronal activity. More recently, Müller glia have been recognized as being very similar to retinal progenitor cells, particularly when compared at the molecular level using comprehensive expression profiling techniques. The molecular similarities, as well as the developmental events that occur at the end of the genesis period of retinal cells, have led us to propose that Müller glia are a form of late stage retinal progenitor cells. These late stage progenitor cells acquire some specialized glial functions, but do not irreversibly leave the progenitor state. Indeed, Müller glia appear to be able to behave as a progenitor in that they have been shown to proliferate and produce neurons in several instances when an acute injury has been applied to the retina. Enhancement of this response is thus an exciting strategy for retinal repair.
Nf1 Mutation Expands an EGFR-Dependent Peripheral Nerve Progenitor that Confers Neurofibroma Tumorigenic Potential
2008, Cell Stem Cell
Citation Excerpt :
Ligand (EGF) exposure confers multipotency to CNS subventricular zone progenitors at the transit-amplifying stage (Doetsch et al., 2002; Fernandes et al., 2004; Reynolds and Weiss, 1992). EGFR signaling influences fate choices and chemotaxis of neural progenitors (Aguirre et al., 2005; Burrows et al., 1997; Lillien and Wancio, 1998). EGF has been included in culture medium for neural progenitors not only in the CNS but also in PNS boundary cap cells and dermal neural crest-derived progenitors (Fernandes et al., 2004; Hjerling-Leffler et al., 2005; Maro et al., 2004; Sieber-Blum et al., 2004), yet the effects of EGFR and its ligands have not been analyzed in DRG or peripheral nerve progenitors.
Defining growth factor requirements for progenitors facilitates their characterization and amplification. We characterize a peripheral nervous system embryonic dorsal root ganglion progenitor population using in vitro clonal sphere-formation assays. Cells differentiate into glial cells, smooth muscle/fibroblast (SM/Fb)-like cells, and neurons. Genetic and pharmacologic tools revealed that sphere formation requires signaling from the EGFR tyrosine kinase. Nf1 loss of function amplifies this progenitor pool, which becomes hypersensitive to growth factors and confers tumorigenesis. DhhCre;Nf1^fl/fl mouse neurofibromas contain a progenitor population with similar growth requirements, potential, and marker expression. In humans, NF1 mutation predisposes to benign neurofibromas, incurable peripheral nerve tumors. Prospective identification of human EGFR⁺;P75⁺ neurofibroma cells enriched EGF-dependent sphere-forming cells. Neurofibroma spheres contain glial-like progenitors that differentiate into neurons and SM/Fb-like cells in vitro and form benign neurofibroma-like lesions in nude mice. We suggest that expansion of an EGFR-expressing early glial progenitor contributes to neurofibroma formation.

View all citing articles on Scopus

View full text

Regular ArticleChanges in Epidermal Growth Factor Receptor Expression and Competence to Generate Glia Regulate Timing and Choice of Differentiation in the Retina

Abstract

Neuron

Dev. Brain Res.

Neuron

Neuron

Cell

Neuron

Exp. Eye Res.

Neuron

Neuron

Neuron

Cell

Cell

Cell

Neuron

Cell

Cell

Cell

Cell

Dev. Brain Res.

Neuron

Curr. Biol.

Neuron

Neuron

J. Neurosci. Methods

Dev. Biol.

Plasticity and differentiation of embryonic retinal cells after terminal mitosis

Science

Identification and characterization of cell types in monolayer cultures of rat retina using monoclonal antibodies

Brain Res.

Quantitative analysis of proliferation and cell cycle length during development of the rat retina

Dev. Dynam.

Subsets of retinal progenitors display temporally regulated and distinct biases in the fates of their progeny

Development

A temporally regulated, diffusible activity is required for rod photoreceptor development in vitro

Development

Taurine promotes the differentiation of a vertebrate retinal cell type in vitro

Development

EGF and TGFα control cell fate determination in developing rat retina

Soc. Neurosci. Abstr.

Regular Article
Changes in Epidermal Growth Factor Receptor Expression and Competence to Generate Glia Regulate Timing and Choice of Differentiation in the Retina