In perfused brains from animals subjected to in utero electroporation, we did not observe variable dendritic protrusion morphologies at the resolution imaged (Figure 4C). The predominant protrusion type observed in vivo was mushroom spine. Therefore, we measured spine head diameters and spine density as the functional SP600125 mw parameters of these postsynaptic structures. We found that NDR1-KD and NDR1siRNA + NDR2siRNA decreased dendritic spine head diameter (Figure 4G), suggesting that NDR1/2 is required for dendritic spine development in vivo. These results are in agreement with our hippocampal culture results, in which NDR1/2 promoted mushroom

spines and active synapses and limited immature protrusions. It is possible that certain factors that contribute to spine formation and stabilization, which are present in vivo, are largely absent in cultures. Such differences between cultures and in vivo studies, caused by similar manipulations of NDR1/2 activity, might result in the different spine phenotypes we observe (Figures 3A–3D and 4G). We also found that dendritic spine density was reduced in NDR1-CA-expressing neurons and was increased by NDR1siRNA + NDR2siRNA in vivo, while we did not observe a significant change with NDR1-KD ( Figure 4H). It

is possible that the NDR1-KD expression level was not sufficient to cause increased spine density in vivo. NDR1/2 participates in limiting dendritic spine density as is demonstrated in cultured NDR1-CA-expressing neurons ( Figures 3A–3D). Our data supports that NDR1 activity is necessary in limiting dendritic spine numbers in vivo as Selleck Cabozantinib well. Overall, our data shows that NDR1/2 regulates spine morphology by enlarging spine heads and limiting spine numbers in vivo. These data, together with data from neuronal cultures (Figure 3), support a role for NDR1/2 function in dendritic spine morphogenesis. Having found NDR1/2 function important

for dendrite arborization and synaptic development, we next looked into the underlying mechanisms. Since there were no known substrates of NDR1/2, we utilized the chemical genetic substrate labeling below method followed by phospho-specific covalent capture (Blethrow et al., 2008) to identify NDR1 substrates. This method utilizes analog-sensitive kinases, in which the hydrophobic gatekeeper residue is replaced by a smaller amino acid, to allow binding and utilization of ATP analogs modified with bulky substitutions. The crystal structure of the Src ATP binding pocket in analog-sensitive mutants depicts how larger ATP analogs (Figure 5C) can fit the binding pocket of Src-as (Figure 5A). We generated two analog-sensitive NDR1-CAs (M166A and M166G). In order to identify which bulky ATP-γ-S analog is most compatible with the mutant kinase, we performed an in vitro kinase reaction with these mutants using NDR1′s target peptide as described previously (Stegert et al., 2005).