Abstract
ABSTRACTThe concept of a morphogen – a molecule that specifies two or more cell fates in a concentration-dependent manner – is paradigmatic in developmental biology. Much remains unknown, however, about the existence of morphogens in the developing vertebrate central nervous system (CNS), including the mouse dorsal telencephalic midline (DTM). Bone morphogenetic proteins (BMPs) are candidate DTM morphogens, and our previous work demonstrated BMP4 sufficiency to induce one DTM cell fate – that of choroid plexus epithelial cells (CPECs) – in a mouse embryonic stem cell (mESC) culture system. Here we used BMP4 in a modified mESC culture system to derive a second DTM fate, the cortical hem (CH). CH and CPEC markers were induced by BMP4 in a concentration-dependent manner consistent with in vivo development. BMP4 concentrations that led to CH fate also promoted markers for Cajal–Retzius neurons, which are known CH derivatives. Interestingly, single BMP4 administrations also sufficed for appropriate temporal regulation of CH, CPEC, and cortical genes, with initially broad and overlapping dose-response profiles that sharpened over time. BMP4 concentrations that yielded CH- or CPEC-enriched populations also had different steady-state levels of phospho-SMAD1/5/8, suggesting that differences in BMP signaling intensity underlie DTM fate choice. Surprisingly, inactivation of the cortical selector gene Lhx2 did not affect DTM expression levels, dose-response profiles, or timing in response to BMP4, although neural progenitor genes were downregulated. These data indicate that BMP4 can act as a classic morphogen to orchestrate both spatial and temporal aspects of DTM fate acquisition, and can do so in the absence of Lhx2.
Highlights
By definition, a morphogen is an instructive molecule that can specify two or more cell fates in a concentration-dependent manner (Ashe and Briscoe, 2006)
In contrast to choroid plexus epithelial cells (CPECs), cortical hem (CH) cells should be negative for Ttr while being positive for Msx1, Lmx1a, and Wnt3a (Fig. 1A); we did not find a BMP4 concentration that induced a CH-like profile in the aggregate system, as all three genes displayed similar dose-response profiles (Fig. S1B)
Using two different mouse embryonic stem cell (mESC) lines, we found that, unlike the aggregate-only system, moderate BMP4 concentrations (0.5-5 ng/ml for M2 and 1.5-5 ng/ml for M1 cells) induced a CH-like profile, with relatively high Wnt3a, Lmx1a, and Msx1 compared to Ttr; this was clear for the M2 line (Fig. 1B)
Summary
A morphogen is an instructive molecule that can specify two or more cell fates in a concentration-dependent manner (Ashe and Briscoe, 2006). In vertebrate CNS model systems, classic morphogens are thought to exist, including Sonic hedgehog (SHH) in the spinal cord, retinoic acid (RA) in the hindbrain, and fibroblast growth factors (FGFs) in the rostral-most telencephalon (Dessaud et al, 2007; Ericson et al, 1997; Schilling et al, 2012; Stamataki et al, 2005; Toyoda et al, 2010). These examples have relied largely on in vivo models, in which potentially overlapping, redundant, or interacting positional systems remain intact and are challenging to eliminate experimentally. In vitro systems, which allow for homogenization of positional information and enable formal testing for morphogen activity, have been more difficult to come by
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