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doi: 10.1242/10.1242/dev.00165

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Molecular control of ciliary neuron development: BMPs and downstream transcriptional control in the parasympathetic lineage

Max-Planck-Institut für Hirnforschung, Abteilung Neurochemie, Deutschordenstrasse 46, 60528 Frankfurt/Main, Germany

View larger version (88K): [in a new window]   Fig. 1. Expression of different marker genes during ciliary ganglion development. (A) Expression of pan-neuronal (SCG10), noradrenergic (DBH) and cholinergic (VAChT, CHT1) marker genes and of autonomic transcription factors Cash1, Phox2a, Phox2b and dHand is shown at the stages indicated. Please note that only some of the SCG10/Phox2a/2b-positive cells are noradrenergic as revealed by expression of DBH at all stages. The same is true for the cholinergic markers VAChT or CHT1 at stages 19/20 and 24/25. Whereas noradrenergic genes are expressed transiently (with the exception of very few cells that still express DBH at stage 32/33), the proportion of cholinergic cells is strongly increased, so that at stage 32/33 virtually all neurons are cholinergic. Scale bars: 100 µm. (B) Expression of Cash1, Phox2b and DBH in the ciliary ganglion at stage 18. Whereas Cash1 is clearly detectable, Phox2b expression has just begun, and DBH expression cannot be detected at this stage. Arrowheads point to ciliary ganglion primordia (consecutive sections). (C) Schematic diagram summarizing the developmental expression pattern of the genes analysed in A.

View larger version (98K): [in a new window]   Fig. 2. Expression of BMP7 at the site of ciliary ganglion formation. In the environment of Phox2b-positive ciliary ganglion cells (CG, upper panel) BMP7 expression is detected in the retro-orbital mesenchyme (lower panel) by in situ hybridisation (stage 18/19). Stronger expression of BMP7 can be seen in Rathke's Pouch (RP) and in the retina. The location of the eye is indicated. Scale bar: 100 µm.

View larger version (64K): [in a new window]   Fig. 3. The BMP inhibitor noggin prevents the development of the ciliary ganglion. After unilateral implantation of noggin-expressing CHO cells in E2 chick embryos the ciliary ganglion, as shown by expression of SCG10 (A), Cash1 (B), Phox2b (C) and VAChT (D), is absent at stage 24/25 (white arrowheads, A-D). In parallel, the development of the eye is strongly reduced (A-F left side; compare with normal right side). Sox10-positive neural crest cells aggregate at the position of the ciliary ganglion (black arrowhead, F), but remain undifferentiated (SCG10-negative) on the noggin-treated side (white arrowhead, E). Unilateral ablation of the optic vesicle in E2 chick embryos also prevents eye development (G,H; white asterisks; compare normal contralateral eyes, black asterisks), but does not affect ciliary ganglion formation as demonstrated by SCG10 and Phox2b expression in the CG (G,H). The SCG10-positive structure adjacent to the CG in (G) is an enlarged oculomotor branch of the trigeminal ganglion, frequently observed in eye-ablated embryos. Scale bar: 1 mm.

View larger version (32K): [in a new window]   Fig. 4. BMP4 overexpression results in enlarged ciliary ganglia. Embryos, infected at E2 with mBMP4-RCAS display enlarged ciliary ganglia at stage 24/25 as detected by expression of Phox2b and ChAT (A). (B) Quantification of the area of Phox2b and ChAT-positive cells revealed a strong increase in comparison to the control side (Phox2b, P=0.008; ChAT, P=0.039; Student's t-test). Also the number of TH-positive cells (C) was significantly increased (P=0.002). However, the ratio between cholinergic markers and Phox2b (D) or noradrenergic markers and Phox2b (E) showed no significant difference between mBMP4 implanted and control side (P=0.051 and P=0.155, respectively), indicating that BMP4 increases ciliary neuron development but does not affect the phenotype. Scale bar: 250 µm.

View larger version (92K): [in a new window]   Fig. 5. Ciliary neurons transiently co-express cholinergic and noradrenergic marker genes. Double in situ hybridization for VAChT (red) and TH (black) at stage 24 shows the presence of cells that co-express VAChT and TH (white arrowheads) or only express VAChT (red arrowhead) or TH (black arrowhead). The VAChT signal in double-stained cells (white arrowheads) is low but significantly above background, as illustrated by comparison with cells devoid of VAChT expression (black arrowhead). Scale bar: 25 µm.

View larger version (53K): [in a new window]   Fig. 6. The number of noradrenergic cells in ciliary ganglia is strongly increased by ectopic expression of dHand. (A) Embryos infected at E2 with dHand-RCAS display an increased number of TH-positive cells at E8. Phox2b and TH stainings were done on alternate sections. (B) The quantification of TH-positive cells per ciliary ganglion area (Phox2b-positive) revealed a strong increase compared to controls (n=9; P=0.009). Scale bar: 100 µm.

View larger version (20K): [in a new window]   Fig. 7. Schematic diagram summarizing the role of BMPs in the development of noradrenergic sympathetic and parasympathetic ciliary neurons. BMPs are sufficient and essential for the development of both noradrenergic sympathetic (Varley et al., 1995; Reissmann et al., 1996; Shah et al., 1996; Schneider et al., 1999) and parasympathetic ciliary neurons (the present study). The differential expression of the BMP-downstream transcription factor dHand suggests that sympathetic and ciliary neuron precursors display differences that determine BMP downstream signaling and in turn neuron identity (illustrated by the different shading of sympathetic and parasympathetic precursor cells). dHand is implicated in the maintenance of noradrenergic differentiation (TH, DBH) in sympathetic neurons.

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