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Fig. 4 | Neural Development

Fig. 4

From: Rac1 plays an essential role in axon growth and guidance and in neuronal survival in the central and peripheral nervous systems

Fig. 4

Reduced diameters of limb nerves and reduction in the number of LMC motor neurons in Olig2-Cre;Rac1 CKO/− embryos. a Diagrams traced from Fig. 2e’, g’ showing where the nerve diameters were measured. D Proximal and D Distal, measurements of dorsal nerve diameter immediately proximal and distal, respectively, to the point where Fz3 −/− dorsal axons stall. V, measurement of ventral nerve diameter at a similar distance from the plexus. b At the locations shown in (a), the nerve diameters were measured from neurofilament-immunostained whole-mount forelimbs (top) and hindlimbs (bottom) from one litter at E12.5 and a second litter at E13.5 (left and right, respectively, separated by gray dashed lines). Numbers above each set of data points are as follows: (top) P values for the WT littermate vs. Olig2-Cre;Rac1 CKO/− comparison (Student’s two-tailed two-sample unequal variance t-tests) and (bottom) the average thickness of Olig2-Cre;Rac1 CKO/− nerves as a percentage of the WT controls. c Motor neuron apoptosis visualized with cleaved caspase3 and Islet1/2 immunostaining in cross sections of E11.5 lumbar spinal cords. The ventrolateral region of the spinal cord is shown with the left border of the spinal cord demarcated by a white dashed line. Very few apoptotic cells are present in Rac1 CKO/+ controls (left), whereas cleaved caspase3 is seen in multiple Islet1/2+ cells in the Olig2-Cre;Rac1 CKO/− ventral spinal cord (right). N = 3 for each genotype. Scale bar, 500 μm. d Diagram showing the expression pattern of transcription factors in LMCL and LMCM motor neurons. SC spinal cord, D dorsal, V ventral. Adopted from Figure 5A of Hua et al. [17]. e LMCM and LMCL motor neurons in the ventrolateral spinal cord were identified as Islet1+/Foxp1+ and Islet1−/Foxp1+ populations, respectively, in cross sections of E12.5 Rac1 CKO/− (left panel; lateral is to the left) and Olig2-Cre;Rac1 CKO/− (right panel; lateral is to the right) lumbar spinal cords. White dashed lines encircle the LMC. The solid white line demarcates the border of the spinal cord. L lateral, M medial. Scale bar, 100 μm. f Quantification of the number of LMCM (top) and LMCL (bottom) motor neurons per 14-μm-thick cross section of E12.5 lumbar spinal cords. Motor neurons were counted from 10 serial sections and averaged from three pairs of WT (blue) and Olig2-Cre;Rac1 CKO/− (red) littermates, with adjacent counted sections separated by four uncounted sections. g Top, GFP immunostaining shows defects in axon growth among primary spinal cord motor neurons in dissociated cell cultures from E11.5 Hb9-GFP;Olig2-Cre;Rac1 CKO/− (right) and littermate control Hb9-GFP;Olig2-Cre;Rac1 CKO/+ (left) embryos. Red arrows, axons and axon branches. Bottom, quantification of axon lengths. Scale bar, 100 μm

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