Bone morphogenetic proteins (BMPs), negative regulators of neural determination in the early embryo, were found to be potent inhibitors of neurogenesis in olfactory epithelium (OE) cultures. BMPs 2, 4 or 7 decreased the number of proliferating progenitor cells and blocked production of olfactory receptor neurons (ORNs). Experiments suggested that this effect was due to an action of BMPs on an early-stage progenitor in the ORN lineage. Further analysis revealed that progenitors exposed to BMPs rapidly (< 2 h) lost MASH1, a transcription factor known to be required for the production of ORNs. This disappearance was due to proteolysis of existing MASH1 protein, but new gene expression was required to trigger it. The data suggest a novel mechanism of BMP action, whereby the induced degradation of an essential transcription factor results in premature termination of a neuronal lineage.

The vertebrate olfactory epithelium neuronal progenitors-which are in close physical (OE) is a system in which behavior of neuronal proproximity to ORNs-can ''read'' the number of difgenitor cells can be observed and manipulated easily. ferentiated neurons in their environment and regulate It is morphologically and functionally similar to emproduction of new neurons accordingly. Putative neubryonic germinal neuroepithelia, but is simpler in that ronal stem cells of the OE have been identified in vitro, it produces large numbers of a single type of neuron, and studies of these cells indicate that ORNs produce the olfactory receptor neuron (ORN). The OE is amea signal that feeds back to inhibit neurogenesis. This nable to tissue culture, gene transfer, and in vivo surgiinhibitory signal may be exerted at the level of the cal approaches, and these have been exploited in exstem cell itself. Recent studies to identify this signal, periments aimed at understanding the characteristics as well as endogenous stimulatory signals that may of OE neuronal progenitor cells. This has led to the be important in regulating OE neurogenesis, are also realization that the ORN lineage contains at least discussed. ᭧ 1998 John Wiley & Sons, Inc. J Neurobiol 36: 190-three distinct stages of proliferating neuronal progeni-205, 1998 tor cells (including a stem cell), each of which repre-Keywords: neurogenesis; programmed cell death; neusents a point at which growth control can be exerted. ronal progenitor cells; stem cells; Mash1; fibroblast Neurogenesis proceeds continually in the OE, and growth factors; bone morphogenetic proteins; neurostudies in vivo have shown that this is a regulated trophins; Trk C; transgenic mouse process that serves to maintain the number of ORNs

To identify factors regulating neurogenesis and programmed cell death in mouse olfactory epithelium (OE), and to determine the mechanisms by which these factors act, we have studied mouse OE using two major experimental paradigms: tissue culture of embryonic OE and cell types isolated from it; and ablation of the olfactory bulb ('bulbectomy') of adult mice, a procedure that induces programmed cell death of olfactory receptor neurons (ORNS) and a subsequent surge of neurogenesis in the OE in vivo. Such experiments have been used to characterize the cellular stages in the ORN lineage, leading to the realization that there are at least two distinct stages of proliferating neuronal progenitor cells interposed between the ORN and the stem cell that ultimately gives rise to it. The identification of a number of different factors that act to regulate proliferation and survival of ORNs and progenitor cells suggests that these multiple cell stages may each serve as a control point at which neuron number in the OE is regulated. Our recent studies of neuronal colony-forming progenitors (putative stem cells) of the OE suggest that even these cells, at the earliest stage in the ORN lineage so far identified, are subject to such regulation: if colony-forming progenitors are cultured in the presence of a large excess of differentiated ORNs, then the production of new neurons by progenitors is dramatically inhibited. This result suggests that differentiated ORNs produce a signal that feeds back to inhibit neurogenesis by their own progenitors, and provides a possible explanation for the observation that ORN death, consequent to bulbectomy, results in increased neurogenesis in the OE in vivo: death of ORNs may release neuronal progenitor cells from this inhibitory signal, produced by the differentiated ORNs that lie near them in the OE. Our current experiments are directed toward identifying the molecular basis of this inhibitory signal, and the cellular mechanism(s) by which it acts.

In olfactory epithelium (OE) cultures, bone morphogenetic proteins (BMPs) can strongly inhibit neurogenesis. Here we provide evidence that BMPs also promote, and indeed are required, for OE neurogenesis. Addition of the BMP antagonist noggin inhibited neurogenesis in OE-stromal cell co-cultures. Bmp2, Bmp4 and Bmp7 were expressed by OE stroma, and low concentrations of BMP4 (below the threshold for inhibition of neurogenesis) stimulated neurogenesis; BMP7 did not exhibit a stimulatory effect at any concentration tested. Stromal cell conditioned medium also stimulated neurogenesis; part of this effect was due to the presence within it of a noggin-binding factor or factors. Studies of the pro-neurogenic effect of BMP4 indicated that it did not increase progenitor cell proliferation, but rather promoted survival of newly generated olfactory receptor neurons. These findings indicate that BMPs exert both positive and negative effects on neurogenesis, depending on ligand identity, ligand concentration and the particular cell in the lineage that is responding. In addition, they reveal the presence of a factor or factors, produced by OE stroma, that can synergize with BMP4 to stimulate OE neurogenesis.

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