In explants co cultured with BMP7 expressing COS 1 cells, ectopic Lhx2 9 expression was observed. The dorsal to ventral extent of Lhx2 9 expression was expanded, reflecting a 3. 1 fold improve in dI1 neurons,3C. DM treatment substantially decreased ectopic Lhx2 9 expression induced by BMP7 in explants by 39%,3C corroborating the lead to explants and giving further evidence that BMP mediated specification of dI1 neurons need form I BMP receptor kinase activity. The impact of DM on the orienta tion of TAG 1 dI axons was examined within the same explants as those utilised to measure Lhx2 9 induction. In explants cultured adjacent to manage COS 1 cells expressing empty vector, dI axons extended having a D V trajectory of 5. 1 1. three, Figure 3D,E.
In explants exposed to BMP7 trans fected COS 1 cells, TAG 1 axons have been repelled, extend ing away in the BMP7 source, with an angle of reorientation of 30 three. 1. Despite the fact that ectopic Lhx2 9 expression was lowered when DM was applied towards the explants, inhibitor MEK162 no transform was observed within the response of dI axons to BMP7 inside the pre sence of DM. The lack of impact of DM on dI axon orientation parallels the resistance of BMP7 evoked growth cone col lapse to blockade of form I BMP receptor kinase activity. From these outcomes we infer that form I BMP receptor activity, potentially acting via the Smad cascade, initiates the BMP7 and BMP6 evoked pathway of inductive specification, but although type I receptor sub units may perhaps be needed as part of the functional receptor complicated, type I BMP receptor kinase activity isn’t required for BMP7 evoked axon orientation.
Taken together, our these details benefits argue that divergence of dI neuron inductive and orienting responses stems from distinct BMP,receptor interactions in which BMP7, at low con centrations, and BMP7 and BMP6, at higher concentra tions, engage different receptors within the receptor complicated. These considerations led us to examine which type II BMP receptors and connected downstream signaling elements could help axon orientation selectively. dI neurons express kind II BMP receptors Of the three form II BMP receptors, only ActRIIA and BMPRII are needed for Smad independent BMP7 evoked chemotaxis of monocytic cells. Nothing is recognized in detail of the in vivo distribution of sort II BMP receptors in embryonic spinal cord.
To start to discover the possibility that BMP7 evoked development cone collapse and orientation includes specific sort II BMP receptors, we determined the distribution of all 3 variety II BMP receptors in dissociated dI neurons. Wes tern blots of dI neuronal lysates showed expression of all 3 form II BMP receptors. Immuno fluorescence analysis of dI cultures, in conjunction with phase contrast imaging and DAPI nuclear staining to supply total neuronal counts, revealed that ActRIIA and BMPRII are the predominant sort II BMP receptors expressed in dI neurons, in 86% and 79% of neurons, respectively. In con trast, ActRIIB was expressed in only 33% of neurons.