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Comparison of the generic neuronal differentiation and neuron subtype specification functions of mammalian achaete-scute and atonal homologs in cultured neural progenitor cells

Liching Lo^1,2, Emma Dormand¹, Amy Greenwood^1,* and David J. Anderson^2,

¹ Division of Biology 216-76, California Institute of Technology, Pasadena, CA 91125, USA
² Howard Hughes Medical Institute, California Institute of Technology, Pasadena, CA 91125, USA
^* Present address: Department of Molecular and Cellular Biology, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138, USA

View larger version (76K): [in a new window]   Fig. 1. SOX10+ and NGN2+ cells develop in dNT cultures. (A-E), cultures grown for 48 hours in the indicated concentrations of BMP2 were stained with anti-SOX10 and counter-stained with the nuclear dye Topro-3. (E) The percentage of SOX10+ cells was scored. The data are derived from two independent experiments. Arrows indicate dense reaggregates of SOX10– neural tube cells, arrowheads indicate relatively more dispersed SOX10+ neural crest cells. (F-H) Double-labeling of a section of E10.5 rat spinal cord with anti-SOX10 and anti-NGN2. Arrows indicate NGN2+ cells at dorsal margin of the spinal cord, arrowheads indicate double-positive cells in the neural crest migration pathway. (I-K) dNT culture grown for 48 hours in 10 ng/ml BMP2 double-labeled for SOX10 and NGN2. Arrows indicate double-positive cells.

View larger version (119K): [in a new window]   Fig. 2. BRN3A+ cells in dNT cultures are peripheral sensory neurons. Cultures grown for 3 days in 10 ng/ml BMP2 were double-labeled for BRN3A (A,D,G) and the indicated markers (B,E,H). NeuN is a pan-neuronal marker; peripherin is a peripheral neuron-enriched marker. Some BRN3A+ cells also co-express RET (not shown). (G-I) The BRN3A+ cells co-expressing NeuN and peripherin were often found in close association with clusters of SOX10+ cells. However the BRN3A+ cells are SOX10– as the latter marker is rapidly downregulated during neuronal differentiation (not shown; L. L., J. Kim and D. J. A., unpublished).

View larger version (51K): [in a new window]   Fig. 3. Effect of BMP2 concentration on differentiation of BRN3A+ and PHOX2B+ cells in dNT cultures. Cultures grown for 4 days in the indicated concentrations of BMP2 were fixed and double-labeled with anti-BRN3A (A,D) and anti-PHOX2B (B,E). (G) The number of BRN3A+ or PHOX2B+ cells/cm2 was measured in a standard cloning ring. Numbers indicate the mean±s.e.m. of two independent experiments with quadruplicate dishes in each experiment. *The number of BRN3A+ cells is significantly different (P<0.01) from the number of PHOX2B+ cells at the indicated dose of BMP2.

View larger version (40K): [in a new window]   Fig. 4. Exogenous NGN1 and MASH1 promote sensory and autonomic neurogenesis, respectively, at 10 ng/ml BMP2. Cultures were infected with retroviruses encoding NGN1-IRES-GFP (A,C,E) or MASH1-IRES-GFP (B,D,F), grown for 3 days in 10 ng/ml BMP2 and triple-labeled with antibodies to BRN3A (A,B), PHOX2B (C,D) and GFP to detect infected cells (E,F; arrows). NGN1-infected cells that express BRN3A (A,E, arrows) do not co-express PHOX2B, while MASH1-infected cells that express PHOX2B (D,F, arrows) do not co-express BRN3A. (G,H) The percentage of cells infected with the indicated retroviruses that express BRN3A (G) or PHOX2B (H) is indicated. *P<0.05 indicates significantly different from control GFP-infected cells. Note that the percentage of NGN1-infected cells expressing PHOX2B in H (6%) is not significantly different from the GFP control (1%; one-way ANOVA followed by post-hoc analysis, HSD=1.33<4.34). The results were pooled from eight experiments. For additional data, see Fig. 6, Table 1 and Table 2.

View larger version (111K): [in a new window]   Fig. 5. NGN1- and MASH1-infected cells that express BRN3A or PHOX2B co-express pan-neuronal and peripheral neuron-specific markers. Cultures infected with the retroviruses indicated above the columns were grown for 3 days in 10 ng/ml BMP2, fixed and triple-labeled with antibodies to BRN3A (A,I) or PHOX2B (B,J); TuJ1 (C,D) or RET (K,L); and GFP (E,F,M,N). Arrows indicate infected cells labeled by all three antibodies. ‘Merge’ indicates superposition of the three overlying panels in the same column. I-P are shown at twice the magnification of A-H.

View larger version (23K): [in a new window]   Fig. 6. Neuronal subtype-inducing properties of NGN1 and MASH1 at different concentrations of BMP2. At each concentration of BMP2, cultures infected with NGN1 (A), MASH1 (B) or control GFP (A,B) retroviruses were fixed and double-labeled with antibodies to BRN3A or PHOX2B, and GFP. The percentage of infected (GFP+) cells co-expressing BRN3A or PHOX2B is plotted. The data are pooled from 11 experiments, although not all BMP2 concentrations were tested in every experiment. For additional quantification see Table 2. Note that in B, although the percentage of MASH1-infected cells expressing PHOX2B in 10 ng/ml BMP2 (25%; red triangles) is similar to the percentage of MASH1- or GFP-infected cells expressing BRN3A (red and blue dots), this similarity is coincidental; PHOX2B and BRN3A are never co-expressed (not shown). (C) The percentage of BRN3A+ and neuronal (TuJ1+) cells among NGN1-infected cells is directly compared. Note that the enhancement of the sensory marker is always greater than the enhancement of neuronal differentiation. The fold-induction of BRN3A expression declines with increasing BMP concentration because the level of baseline BRN3A expression increases (see A); see also Table 2. *P<.05; other values are not significantly different from each other.

View larger version (77K): [in a new window]   Fig. 7. NGN1 induces BRN3A and PHOX2B in non-overlapping cell populations at 25 ng/ml BMP2. NGN1 retrovirus-infected cultures were grown for 3 days in 25 ng/ml BMP2, fixed and triple-labeled with antibodies to BRN3A (A, red), PHOX2B (B, blue) and GFP (C, green). (D,E) The BRN3A+, GFP+ (arrows) and PHOX2B+, GFP+ cells (arrowheads) are non-overlapping.

View larger version (101K): [in a new window]   Fig. 8. NGN1 and MASH1 both induce autonomic neurogenesis in NCSC clonal cultures. Colonies were grown for 4 days after infection with the retroviruses indicated above the columns, before fixation and labeling. (A-H) Representative colonies from NGN1- and MASH1-infected cultures labeled with antibodies to NF160 (C,D), GFP (A,B) and with DAPI to visualize cell nuclei (E,F); (G,H) Phase-contrast views of the fields shown in A,C,E and B,D,F, respectively. Arrows in B,D,F,H indicate a neuron; arrowheads in B,F,H indicate a non-neuronal cell. All cells in the NGN1 colony illustrated are neurons. No neurons have differentiated at this time point in control GFP-infected cultures (not shown). (I-P) Colonies from a similar experiment triple-labeled with antibodies to GFP (I,J), BRN3A (K,L) and PHOX2B (M,N); ‘Merge’ indicates superposition of the three labels.

View larger version (113K): [in a new window]   Fig. 9. Differences in cell cycle withdrawal-promoting activity and Notch sensitivity between NGN1 and MASH1 in NCSCs. (A-H) BrdU labeling in NGN1- or MASH1-infected colonies pulsed for 24 hours 3 days after infection. Colonies were labeled antibody to NF-M and the nuclear dye Topro-3 (A,B), anti-BrdU (C,D) and anti-GFP (E,F). Arrows in B,D,F,H indicate a neuronal cell, arrowheads indicate a non-neuronal cell (see Table 4 for quantification). (I-P) MASH1 is more sensitive than NGN1 to inhibition by Delta-Fc. Colonies were double-labeled with anti-GFP (green) and anti-NF-M (red). In K,L,O,P only the NF staining is shown for clarity. Arrows indicate process-bearing, NF-M+ neurons, arrowheads indicate NF-M– non-neuronal cells. Note that there are still some neurons in NGN1-infected colonies treated with Delta-Fc (P) (see Table 4 for quantification).

View larger version (23K): [in a new window]   Fig. 10. Role of NGNs in the specification of neuronal identity in the PNS. (A) Summary of the results obtained with NGN1 retrovirus in dNT cells (left) and NCSCs (right). (B) Model to explain results obtained in dNT cultures. IDS1, n indicates postulated sensory neuron identity co-determinants; IDAn indicates postulated autonomic identity co-determinants; the former are thought to be induced by low, and the latter by high concentrations of BMP2 (Fig. 3). The existence of such identity co-determinants is inferred from the fact that BMP2 dominantly controls the neuronal subtype promoted by NGNs, depending on its concentration (Fig. 6 and Table 2). The fact that NGNs can significantly enhance either sensory or autonomic differentiation above that in control GFP-infected cells (Fig. 6) implies that NGNs promote either the expression and/or function of the collaborating identity co-determinants. Blunt symmetrical arrows: the reciprocal inhibition between sensory and autonomic identity co-determinants is inferred from the fact that NGN1 promotes sensory and autonomic fates in a mutually exclusive manner at 25 ng/ml (Fig. 7). The induction of NGNs by BMP2 (broken arrow) is inferred from the observation that increasing BMP2 concentrations enhance the expression of endogenous NGN2. MASH1 promotes autonomic but not sensory neurogenesis at all concentrations of BMP2, because it cannot upregulate, or collaborate with, sensory identity co-determinants.