Between 2 to 5 sections per mouse were analysed. Cell death in postnatal hippocampus. Sagittal brain sections from different postnatal stages were used for TUNEL staining JNJ0966 (red). Nuclei are shown by DAPI staining (blue). Scale bar: 200 m.(TIF) pone.0024819.s004.tif (3.6M) GUID:?8B4499B6-8182-4D22-BD7B-DB8485D18CB6 Physique S5: Deletion of Rac3 leads to increased Rac1 activation in mouse brain. (A) Active GTP-bound Rac proteins were recovered from lysates of wildtype P7 brain, by pull-down on beads coupled to GST-PAK-CRIB. Lane 1, 86 g of unbound fraction (Ub) after pull down from wildtype brain lysate (WT); lane 2, 93 g of the unbound fraction after pulldown from Rac3KO P7 brain lysate (KO); lane 3, positive control: pulldown from 100 g of wildtype P7 brain lysate loaded with GTPS; lane 4 and 5, pull downs from 3 mg of brain lysates from P7 brain of wildtype and Rac3KO mice, respectively. Filters blotted for Rac1 (upper), and Rac3 (lower). (B) Quantification from two impartial experiments of Rac-GTP from wildtype brains (including both Rac1-GTP and Rac3-GTP), and from Rac3KO brains (including only Rac1-GTP).(TIF) pone.0024819.s005.tif (2.8M) GUID:?2B416871-0406-440B-ACB4-FC032788FBE9 Abstract We have previously shown that double deletion of the genes for Rac1 and Rac3 GTPases during neuronal development affects late developmental events that perturb the circuitry of the hippocampus, with ensuing epileptic phenotype. These effects include a defect in mossy cells, the major class of excitatory neurons of the hilus. Here, we have resolved the mechanisms that affect the loss of hilar mossy cells in the dorsal hippocampus of mice depleted of the two Rac GTPases. Quantification showed that the loss of mossy cells was evident already at postnatal day 8, soon after these cells become identifiable by a specific marker in the dorsal hilus. Comparative analysis of the hilar region from control and double mutant mice revealed that synaptogenesis was affected in the double mutants, with strongly reduced presynaptic input from dentate granule cells. We found that apoptosis was equally low in the hippocampus of both control and double knockout mice. Labelling with bromodeoxyuridine at embryonic day 12.5 showed no evident difference in the proliferation of neuronal precursors in the hippocampal primordium, while differences in the number of bromodeoxyuridine-labelled cells in the developing hilus revealed a defect in the migration of immature, developing mossy cells in the brain of double knockout mice. Overall, our data show that Rac1 and Rac3 GTPases participate in the normal development of hilar mossy cells, and indicate that they are involved in the CD118 regulation of the migration of the mossy cell precursor by preventing their arrival to the dorsal hilus. Introduction Rac GTPases regulate several cellular processes including actin dynamics and adhesion , and are critical for neuronal development and synaptogenesis C. Two members of the Rac family are co-expressed during development in several neuronal types: the ubiquitous Rac1 and the neural-specific Rac3/Rac1B C. Rac1 and Rac3 GTPases share about 90% protein identity, and their pattern of expression during development differs substantially, suggesting specific functions. Rac1 has been implicated in the regulation of axons, dendrites, and spines C. Recently, conditional deletion of Rac1 in ventricular zone progenitors has indicated a role of Rac1 in axon guidance while axonal outgrowth is not affected . Rac1 is JNJ0966 usually detected from very early embryonic development in the mouse (E7.5), where it is essential from early development, and its KO results in embryonic lethality, with Rac1-null mice dying during early development due to migratory defects . The transcript for Rac3 is already detectable in the mouse nervous system at E13, and is developmentally regulated in the brain, with a peak at time of intense neurite branching and synaptogenesis . While most of the work in primary neurons has been on Rac1, we have recently shown that this double deletion of the two GTPases in developing neurons leads to a strong neurological phenotype when compared to mice with single Rac1 or Rac3 deletion, showing that both GTPases are important for the development of a functional nervous system . Our previous comparative analysis of the phenotype of single and double knockout mice for the Rac1 JNJ0966 and Rac3 GTPases has shown a specific defect in the size of the dorsal hilus of the hippocampus that JNJ0966 is strongly reduced compared to single knockout or wildtype animals . This reduction correlated.