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The specification of noradrenergic locus coeruleus (LC) neurones depends on bone morphogenetic proteins (BMPs)

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

View larger version (99K): [in a new window]   Fig. 1. Expression of Phox2a (A,D,G, J, arrow), DBH (B,E,H,K, arrow) and TH (C,F,I,L, arrow) in r1 at various stages of chick development. At stage 14, transcripts of Phox2a (arrow) are observed in the dorsal aspect of r1 (A), while no expression of DBH (B) and TH (C) is detected. (D-F) At stage 20, Phox2a, DBH and TH transcripts are found in a more ventral position (arrow) and become finally localised to a region (arrow) ventrolateral to the fourth ventricle at stage 29 (G-I) and stage 34 (J-K). Arrowheads in A,D indicate the ventrally localised trochlear nuclei.

View larger version (85K): [in a new window]   Fig. 2. Expression pattern of Bmp5 (A-E) and Bmp7 (F) in r1 during early stages of chick development. (A) Whole-mount in situ hybridisation for Bmp5 at stage 10 demonstrates expression in the rostral hindbrain, including r1 (white arrow, Bmp5 expression; black arrow, midbrain-hindbrain-constriction). (B) Simultaneous detection of Bmp5 and Fgf8 at stage 11 by whole-mount in situ hybridisation demonstrates an overlap (arrowhead) of Bmp5 (white arrow) and Fgf8 (black arrow) expression in r1. (C,D) Transcripts of Bmp5 (arrows) are found at stage 11 throughout the dorsal aspect of r1, at both rostral (C) and caudal (D) regions. Note that there also seems to be expression in the epidermal ectoderm (D). At stage 14/15, transcripts of Bmp5 (arrow) are restricted to the roof plate and dorsal edges of the neural tube (E), while Bmp7 expression (arrow) is restricted to the overlying epidermal ectoderm (F). Whole-mount in situ hybridisation was used for the detection of BMP5 in C,D, resulting in the collapse of the ventricle when compared with hybridisation of sections (Fig. 4C).

View larger version (52K): [in a new window]   Fig. 3. Noggin affects development of LC neurones. Control embryos (A-C) and Noggin-treated embryos (D-F) were analysed for the expression of Phox2a (A,B,D,E, arrow) and DBH (C,F, arrow). Noggin-expressing CHO cells (D) or Noggin-soaked beads (E,F) were implanted into stage 10 chick embryos. Phox2a and DBH expression (arrow) is either lost on the Noggin-treated side (D, stage 17) or detected at the dorsal midline of r1 (E,F, stage 22).

View larger version (109K): [in a new window]   Fig. 4. Noggin application does not interfere with neural crest specification, but is required for the development of the roof plate and rhombic lip. Expression of the neural crest marker Sox10 (A,D,G, arrows) and of dorsal midline markers Wnt1 (B,E,H, arrow) and Bmp5 (arrow, C,F,I) in control embryos at stage 10 (A-C). The effect of implantation of control CHO cells (D-F) and of Noggin-expressing CHO cells (G-I) was analysed at stage 22. Note that in Noggin-treated embryos Sox10 expression is not affected (G), while expression of Wnt1 and Bmp5 is completely lost at the dorsal midline (arrows, H,I).

View larger version (94K): [in a new window]   Fig. 5. Expression of Phox2a (A,D,G, arrow), Pax6 (B,E,H, arrows) and Pax3 (C,F,I arrows) was analysed in control embryos at stage 14/15 (A-C), stage 26/27 (D,E) and stage 22 (F), and in Noggin-treated embryos at stage 26/27 (G,H) and stage 22 (I). (A-C) Wild-type embryos; (D-F) control embryos treated with CHO cells; (G-I) Noggin-expressing CHO cell treatment. Note that Phox2a expression at stage 14/15 is observed within the Pax3 and Pax6 domain (compare A-C). In controls, at stage 26/27, expression of both Phox2a and Pax6 is localised to ventrolateral regions of r1 (D,E). Pax-3 expression at stage 22 is observed in a large region that excludes dorsal midline and most parts of the ventral region (F). After application of Noggin-expressing cells, Phox2a and Pax6 expression is observed in the dorsal region of the neural tube (G,H). Pax3 expression is now expressed in the entire dorsal region (I).

View larger version (135K): [in a new window]   Fig. 6. Differential BMP dependence of dorsal Phox2a-positive LC neurones, Lhx2a-positive neurones and ventral GATA2 neurones in r1. Lhx2a-positive neurones (A, arrows) have a similar expression pattern to Phox2a-expressing LC neurones (C, arrows) during the early stages of chick r1 development (stage 22). Implantation of Noggin-producing CHO cells results in a complete loss of Lhx2a expression at stage 22 (B, arrow), while in a parallel section, Phox2a-positive cells are present in the dorsal midline (D, arrow). By contrast, at stage 22, the GATA2 expression pattern of ventral neurones (E, arrow) is unchanged after Noggin-treatment (F, arrow).

View larger version (134K): [in a new window]   Fig. 7. Phox2a is sufficient to induce noradrenergic neurones in r1. (A) At E8 DBH expression in wild-type embryos is not detected dorsal to the fourth ventricle (B, arrow). After injection of Phox2a-expressing RCAS vectors at stage 9, the dorsolateral alar plate, dorsal to the ventricle, is infected as visualised by the RT expression pattern (arrow). (B,C) In this infected E8 embryo, ectopic DBH (C) and TH-positive (D) neurones are observed dorsal to the fourth ventricle (arrows). The ventricle is indicated by a broken line.

View larger version (17K): [in a new window]   Fig. 8. A gradient of BMPs (green) produced in the dorsal half of r1 controls the specification of different cellular phenotypes. We propose that at stage 10/11, after neural crest cells have been specified and left the neuroepithelium, the specification of the precursors for noradrenergic LC neurones (broken line) and other dorsal neurones depends on BMPs. We propose that LC precursors are localised within the BMP5 and Pax3 expression domains at stage 10. Pax6 expression is not observed at this stage (Li et al., 1994). The first Phox2a-positive LC precursors (blue) are detected at stage 13/14 within the Pax3/Pax6 co-expression domain in the dorsal compartment of r1. BMPs are also required in r1 for the differentiation and/or maintenance of the dorsal midline structures, the roof plate (RP) and rhombic lip. This model differs from the classical morphogen scenario, as the BMP gradient is not generated by diffusion from a signalling centre but by a gradient of BMP synthesis. FP, floor plate.