BMP and FGF regulate the development of EGF-responsive neural progenitor cells

Laura Lillien ) 0 Heather Raphael 0 0 Department of Neurobiology and Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine , W1454 Biomedical Science Tower, Pittsburgh, PA 15261 , USA SUMMARY Temporal changes in progenitor cell responses to extrinsic signals play an important role in development, but little is known about the mechanisms that determine how these changes occur. In the rodent CNS, expression of epidermal growth factor receptors (EGFRs) increases during embryonic development, conferring mitotic responsiveness to EGF among multipotent stem cells. Here we show that cell-cell signaling controls this change. Whereas EGFresponsive stem cells develop on schedule in explant and aggregate cultures of embryonic cortex, co-culture with younger cortical cells delays their development. Exogenous BMP4 mimics the effect of younger cells, reversibly inhibiting changes in EGFR expression and responsiveness. Moreover, blocking endogenous BMP receptors in Proliferation and differentiation occur in temporal and spatial patterns in the CNS. These patterns are generated as a result of intrinsic differences in progenitor cells and restricted expression of extrinsic signals (reviewed in Edlund and Jessell, 1999). One of the properties of progenitor cells that changes during development is their responsiveness to extrinsic signals. This determines whether cells respond to specific signals in their environment at distinct times, and influences their choice of response to signals that are pleiotropic. We recently reported that one of the molecular mechanisms for achieving differences in responsiveness to extrinsic signals involved quantitative changes in the expression of cell-surface receptors (Lillien, 1995; Lillien and Wancio, 1998; Burrows et al., 1997). A difference in the level of receptor expression has also been implicated in threshold-dependent differences in responses to decapentaplegic in Drosophila that contribute to spatial patterning (Lecuit and Cohen, 1998). What remains to be elucidated is how differences in such intrinsic properties of progenitor cells are controlled. In the vertebrate forebrain, subsets of progenitor cells with distinct properties have been described (Levitt and Rakic, 1983; Luskin et al., 1988; Walsh and Cepko, 1988; Reynolds et al., 1992; Grove et al., 1993; Davis and Temple, 1994; Gage progenitors with a virus transducing dnBMPR1B accelerates changes in EGFR signaling. This involves a non-cell-autonomous mechanism, suggesting that BMP negatively regulates signal(s) that promote the development of EGF-responsive stem cells. FGF2 is a good candidate for such a signal, as we find that it antagonizes the inhibitory effects of younger cortical cells and exogenous BMP4. These findings suggest that a balance between antagonistic extrinsic signals regulates temporal changes in an intrinsic property of neural progenitor cells. et al., 1995; Levison and Goldman, 1997; Mayer-Proschel et al., 1997). The representation of specific subsets that differ in their proliferative and phenotypic potentials varies during development (Levitt and Rakic, 1983; Williams and Price, 1995; Kilpatrick and Bartlett, 1995). Their relative representation tends to reflect the types of cells generated at specific stages of development. For example, at earlier embryonic stages, when more neurons are generated, progenitor cells that are restricted to a neuronal fate are more abundant, while at later embryonic stages, when more glia begin to develop, progenitor cells that are restricted to a glial fate are more abundant. It has been shown that progenitor cells that are more restricted in their proliferative and phenotypic potentials are derived from multipotent stem cells (MayerProschel et al., 1997). Stem cells are normally represented in very small numbers, but it has been noted that multipotent stem cells also change during development (Burrows et al., 1997; Zhu et al., 1999). At earlier embryonic stages, stem cells have a bias for generating neuronal progeny, while later stem cells tend to generate more glia. Thus, the developmental change in cell type generation is initiated at the top of the progenitor cell hierarchy, in the stem-cell compartment. Early and late embryonic multipotent stem cells also differ in their responsiveness to mitogens. Late embryonic and adult stem cells are responsive to EGF-family ligands (Reynolds and Weiss, 1992). In contrast, at earlier stages of development, neural progenitor cells, including stem cells, are mitotically responsive to FGF2 but not to EGF (Kilpatrick and Bartlett, 1993, 1995; Ferri and Levitt, 1995; Ghosh and Greenberg, 1995; Gage et al., 1995; Ferri et al., 1996; Johe et al., 1996; Qian et al., 1997; Burrows et al., 1997). The acquisition of mitotic responsiveness to EGF is associated with the appearance of a subpopulation of progenitor cells that expresses relatively high levels of EGFRs (Burrows et al., 1997; Kornblum et al., 1997). Using a retrovirus to introduce extra copies of EGFRs into early progenitor cells, we showed that expression of a threshold number of EGFRs was required for mitotic responsiveness to EGF (Burrows et al., 1997). Early progenitor cells express lower levels of the EGFR and exhibit distinct responses to EGFR stimulation (Ferri and Levitt, 1995; Eagleson et al., 1996). Taken together, these findings suggest that differences in the level of EGFR expression determine how progenitor cells interpret an extrinsic signal at specific stages of development. Progenitor cells that express high levels of EGFR in the late embryonic cortex are lineal descendants of early progenitors that express low levels of EGFR (Burrows et al., 1997). In principle, temporal regulation of changes in EGFR expression and signaling could reflect either a cellautonomous mechanism or a response to extrinsic signals. Although cell-autonomous mechanisms have been implicated in the regulation of some differences between cortical progenitor cells (Chenn and McConnell, 1995; Zhong et al., 1996; Qian et al., 1998), extrinsic signals have been shown to modulate EGFR expression in a variety of cells. For example, retinoids reduce the transcription of EGFRs in squamous carcinoma cells (Grandis et al., 1996), EGF-family ligands increase EGFR transcription in liver epithelial cells (Earp et al., 1986), NGF reduces cell-surface expression (Brown and Carpenter, 1991; Seedorf et al., 1995) and transcription (Shibutani et al., 1998) of EGFRs in PC12 cells, and SHH produced by Drosophila photoreceptors induces EGFR expression in lamina precursor cells (Huang et al., 1998). These findings raised the possibility that the change in EGFR expression in cortical progenitor cells could be modulated by extrinsic signals. In the present study, we show that the developmental change in EGFR expression and responsiveness in cortical progenitors occurs on schedule in explant and aggregate cultures, but not in monolayer cultures, suggesting (...truncated)