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Mash1 and Ngn1 control distinct steps of determination and differentiation in the olfactory sensory neuron lineage

Elise Cau^*, Simona Casarosa and François Guillemot

IGBMC, 1 rue Laurent Fries, 67404 Illkirch cedex, CU de Strasbourg, France
^* Present address: Department of Anatomy and Developmental Biology, University College London, Gower Street, London WC1E 6BT, UK
Present address: Department of Physiology and Biochemistry, University of Pisa, 56010 Ghezzano-Pisa, Italy

View larger version (139K): [in a new window]   Fig. 1. Sequential expression of Mash1 and Ngn1 in progenitors of the olfactory epithelium. (A) In situ hybridisation with RNA probes for Mash1 (in blue) and Ngn1 (in brown) on frontal sections of OE at E12.5 shows that the two genes are expressed in distinct and partially overlapping progenitor populations. Mash1 is expressed in cells located in apical, intermediate and basal positions while Ngn1 expression is restricted to basal progenitors. The two genes are co-expressed in a subset of basal progenitors (arrowheads in the inset). The top right panel shows the apical (a) and basal (b) sides on a schematic representation of a OE section. (B,B') Hybridisation with a Ngn1 probe on E12.5 wild-type (B) and Mash1 mutant (B') OE showing that Ngn1 expression in basal progenitors requires Mash1 function. (C,C') Hybridisation with a Mash1 probe on E12.5 wild-type (C) and Ngn1 mutant (C') OE showing that in contrast, Mash1 expression in OE progenitors is independent of Ngn1 function. Scale bars, 25 µm.

View larger version (132K): [in a new window]   Fig. 2. Mash1 and Ngn1 are both required for the production of olfactory sensory neurons but only Mash1 is required for the generation of basal OE progenitors. (A-C,A'-C',A'',C'') In situ hybridisation with a probe for the pan-neuronal marker SCG10 on sections of wild-type (A,C), Mash1 mutant (A',B,C') and Ngn1 mutant (A'',B',C'') OE at E12.5 (A-A'',B,B') and on whole-mount olfactory placodes at E10.0 (C-C''). There is a drastic reduction in the number of neurons differentiating in the OE of both Mash1 and Ngn1 mutant embryos at E12.5, apparent in the medial part of the OE (A',A'') and more pronounced in the rostral part of the Mash1 mutant OE (B) and in the caudal part of the Ngn1 mutant OE (B'). Mash1 is not required for the generation of neurons at placodal stage (C'), while these early born neurons are missing in a Ngn1 mutant placode (C''). (D-D'') Immunocytochemistry for BrdU after a short period of incorporation followed by Haematoxylin staining reveals progenitors cells in S phase of the cell cycle (brown nuclei) and in mitosis (mitotic figures indicated by arrowheads) in wild-type (D), Mash1 mutant (D') and Ngn1 mutant (D'') OE at E12.5. Dividing progenitors are present in apical (top) and basal (bottom) positions in wild-type OE (D). Basal progenitors are missing in the Mash1 mutant OE (D'). Because post-mitotic neurons are also absent, apical progenitors are now present in the whole thickness of the OE. In contrast, basal progenitors are present in the Ngn1 mutant OE (D''). Scale bars, 25 µm (A-A'',B',B''); 50 µm (C-C''); 10 µm (D-D'').

View larger version (153K): [in a new window]   Fig. 3. Mash1 and not Ngn1 is required for the expression of Notch signalling genes in the OE. Probes for Notch ligands delta-like 3 (A-A''), Serrate 1 (B-B'') and Serrate 2 (C-C'') and for the Notch bHLH effector genes Hes1 (D-D'') and Hes5 (E-E'') hybridised to wild-type (A-E), Mash1 mutant (A'-E') and Ngn1 mutant (A''-E'') OE at E12.5. Expression of Ser1 and Hes1 in apical cells and of Dll3, Ser2 and Hes5 in basal cells is abolished in Mash1 mutant OE. In contrast, expression of these genes is unaffected in Ngn1 mutant OE. Note that Hes1 and Ser1 are also expressed in the mesenchyme surrounding the OE. Scale bar, 25 µm.

View larger version (158K): [in a new window]   Fig. 4. Ngn1 function is required for the expression of a subset of transcriptional regulators expressed in basal progenitors. Expression of NeuroD (A-A''), Phd1 (B-B''), Ebf1 (C-C'') and Lhx2 (D-D'') analysed by in situ hybridisation on sections of wild-type (A-D), Mash1 mutant (A'-D') and Ngn1 mutant (A''-D'') OE at E12.5. In wild-type OE, NeuroD is expressed in basal progenitors and only transiently in differentiating neurons (A) (Cau et al., 1997), while Phd1, Ebf1 and Lhx2 are expressed both in dividing basal progenitors and in post-mitotic OSNs (B-D; data not shown). Expression of the four genes is abolished in Mash1 mutants OE (because of the loss of both basal progenitors and post-mitotic neurons in these mutants) except in restricted areas that maintain neurogenesis (A'-D'). In contrast, expression of Ebf1 and Lhx2 is maintained in basal progenitors in Ngn1 mutant OE (C'',D''), while expression of NeuroD and Phd1 is missing (A'',B''). Ebf1 is also expressed in the mesenchyme surrounding the OE. Scale bar, 25 µm.

View larger version (7K): [in a new window]   Fig. 5. A model of the regulatory interactions involved in the determination and differentiation of the olfactory sensory neuron lineage. Mash1 is expressed in both apical and basal progenitors in the OE. Apical progenitors are primary neuroepithelial cells that give rise to secondary progenitors that settle basally. Mash1, which is required for the expression of the Notch signalling genes Serrate 1 and Hes1 in apical progenitors, and for the generation of basal progenitors, has the characteristics of a determination gene in the OE. A number of genes encoding transcriptional regulators are activated downstream of Mash1 in basal progenitors, including Ngn1. Ngn1 function is required for expression of the neuronal differentiation gene NeuroD and the neuronal subtype determinant Phd1 and for differentiation of olfactory sensory neurons. In the absence of Ngn1, however, basal progenitors express the Notch signalling genes Ser2, Dll3 and Hes5 and the transcriptional regulators Ebf1 and Lhx2. Ngn1 is therefore required to initiate one of several parallel programs of OSN differentiation activated downstream of Mash1 in basal progenitors. Note that the pathway depicted in this figure only operates in a subset of OE progenitors (e.g. most progenitors at E12.5; see Discussion).

View larger version (144K): [in a new window]   Fig. 6. Ngn1 is redundant with Mash1 for the determination of a subset of olfactory sensory neuron progenitors. Expression of the pan-neuronal marker SCG10 (A-A''',C-C''') and of the progenitor and neuronal marker Ebf1 (B-B''') in the OE (A-A''') and the vomeronasal organ (C-C''') of wild-type (A-C), Mash1 mutant (A'-C'), Ngn1 mutant (A''-C''), and Mash1, Ngn1 double mutant (A'''-C''') embryos at E12.5. Subsets of OSNs differentiate in the absence of Mash1 or Ngn1 function (A',A'',B',B''), while in the absence of both genes, Ebf1-positive progenitors and SCG10-positive OSNs are completely missing (A''',B'''). In contrast, some OSNs persist in the VNO of Mash1, Ngn1 double mutants (C'''). Expression of the Notch ligand Dll1 (D-D''') and the Notch effector Hes5 (E-E''') in wild-type (D,E), Mash1 mutant (D',E'), Ngn1 mutant (D''-E'') and Mash1, Ngn1 double mutants (D'''-E''') in E10.0 olfactory placodes. Dll1 is expressed in isolated cells and Hes5 in cell clusters in wild-type and single mutant placodes, while expression of the two genes is missing in Mash1, Ngn1 double mutant placodes (D''',E'''), indicating that Mash1 and Ngn1 have redundant functions in the determination of placodal progenitors. Scale bars, 25 µm (A-A''',B-B''',C-C'''); 50 µm (D-D''',E-E''').