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1.
Figure 7

Figure 7. Proposed model to illustrate the role of RhoA in ephrinB3/EphA4-dependent locomotor circuit assembly.. From: The Small GTPase RhoA Is Required for Proper Locomotor Circuit Assembly.

In control mice, when EphA4-expressing CST and spinal interneuron (IN) axons encounter ephrinB3 at the spinal cord midline, they are repulsed due to the initiation of signaling pathways that involve RhoA activation and Rac1 inhibition. Loss of RhoA causes aberrant midline crossing of CST and spinal IN axons due to a failure of neurons to retract their axons and/or the absence of ephrinB3 expression at the midline.

Shalaka Mulherkar, et al. PLoS One. 2013;8(6):e67015.
2.
Figure 2

Figure 2. Expression of Rho isoforms in the developing mouse brain and spinal cord.. From: The Small GTPase RhoA Is Required for Proper Locomotor Circuit Assembly.

Brain (A) and spinal cord (C) homogenates prepared from control and RhoA cKO mice at different developmental ages were immunoblotted for the different Rho isoforms (RhoA, RhoB and RhoC) or GAPDH (loading control). Each blot is representative of 3 independent experiments. (B) Normalized levels of RhoA, RhoB and RhoC expression in the mouse brain at different developmental stages (P0, P10, P21). Protein bands from three independent experiments were quantified using NIH Image J software and normalized using GAPDH as control. Data are represented as mean ±S.E.M. (*p<0.001, Student’s t-test). (D) Quantification of different Rho isoforms (RhoA, RhoB, RhoC) from P5 mouse spinal cord was performed as stated in Fig. 2B. Data are represented as mean ±S.E.M. from three independent experiments (*p<0.001, Student’s t-test).

Shalaka Mulherkar, et al. PLoS One. 2013;8(6):e67015.
3.
Figure 3

Figure 3. RhoA cKO mice display a rabbit-like hopping gait.. From: The Small GTPase RhoA Is Required for Proper Locomotor Circuit Assembly.

(A) Control littermates display alternate limb movements, while RhoA cKO mice show a highly abnormal synchronous gait. Arrowheads indicate the position of hindlimbs with respect to each other. (B) Gait analysis of control and RhoA cKO mice. Non-toxic paint was applied to the forepaws (red) and hindpaws (black) of mice, and then they were allowed to walk on a piece of white paper to record the placement pattern of their footprints. (C) Determination of the distance between the right and left paw (a) and the distance between the same paw (b) to assess the degree of parallel movement of the limbs (comparison of a/b ratios). Data are represented as the mean ± S.E.M. (N = 6 animals per genotype), *p<0.001 (Student’s t-test).

Shalaka Mulherkar, et al. PLoS One. 2013;8(6):e67015.
4.
Figure 4

Figure 4. Aberrant midline crossing of corticospinal tract axons in RhoA cKO mice.. From: The Small GTPase RhoA Is Required for Proper Locomotor Circuit Assembly.

(A) Schematic diagram showing the strategy used to label CST axons. (B) Photomicrograph of the cervical spinal cord showing the two regions (ML and MR) that were analyzed for the presence of labeled axons. The value of axons in the region contralateral to the labeled CST was expressed as the ratio of the number of pixels in the region MR to the number of pixels in the region ML. (C) Sections from control, heterozygous, and RhoA cKO mice showed that most CST axons exited from the CST within the dorsal funiculi (marked with asterisks) and projected into the gray matter ipsilateral to the labeled CST in control, heterozygous, and RhoA cKO mice. However, in the RhoA cKO mice only, many BDA-labeled CST axons (arrows) were observed projecting into the gray matter contralateral to the labeled CST. Scale bar: 50 µm. (D) The ipsilateral/contralateral ratio of the numbers of labeled CST axons in the gray matter of the cervical spinal cords in RhoA cKO was significantly greater than that of heterozygous and control mice. N = 3–4 mice/group. Data are represented as the mean ± SD. ***p<0.001. (ANOVA followed by the Student-Newman-Keuls test).

Shalaka Mulherkar, et al. PLoS One. 2013;8(6):e67015.
5.
Figure 1

Figure 1. Generation and characterization of mice containing a conditional allele ofRhoA.. From: The Small GTPase RhoA Is Required for Proper Locomotor Circuit Assembly.

(A) Schematic diagram of the strategy used to generate RhoA conditional knockout mice. A conditional RhoA allele was created by inserting two LoxP sites in a region of the RhoA gene flanking exon 3 and part of intron 3 (RhoAfl/fl). An internal Frt-flanked neomycin (Neo) casette was also introduced as a selection marker, which was subsequently removed by crossing mutant mice with mice expressing Flippase (Flp recombination). The region of the RhoA gene between the two loxP sites was then excised in neuroprogenitor cells by crossing the RhoAfl/fl mice with Nestin-Cre mice (Cre recombination). (B) PCR genotyping of RhoAfl/fl (control), RhoAfl/+;Nestin-Cre (RhoA het), and RhoAfl/fl;Nestin-Cre (RhoA cKO) mice. Top gel, RhoA allele; bottom gel, Cre. (C) Protein isolated from the brains of adult control, RhoA het, and RhoA cKO mice were immunoblotted with antibodies against RhoA or GAPDH (loading control) to assess loss of RhoA expression.

Shalaka Mulherkar, et al. PLoS One. 2013;8(6):e67015.
6.
Figure 6

Figure 6. Loss of RhoA inhibits ephrinB3-induced growth cone collapse and disrupts ephrinB3 midline expression.. From: The Small GTPase RhoA Is Required for Proper Locomotor Circuit Assembly.

(A) Representative images of growth cones from control and RhoA cKO cortical neurons after a 30 min incubation with preclustered ephrinB3-Fc or Fc alone. Scale bar:10 µm. (B) Quantification of the growth cone collapse response of control and RhoA cKO neurons treated with ephrinB3-Fc or Fc (*p<0.001, ANOVA followed by the Tukey test). (C) Cortical neurons from RhoA cKO mice express EphA4 at levels similar to control neurons. (D) Percentage of ephrinB3-induced growth cone collapse in rat cortical neurons pretreated or not with the ROCK inhibitor Y-27632 (*p<0.001, ANOVA followed by the Tukey test). (E) Expression of ephrinB3 is lost from the midline at L2 in RhoA cKO mice. Arrowheads show ephrinB3 positive staining in the spinal cord of P5 mice. Scale bar: 10 µm. Three sections per animal and N = 3 animals were analyzed per genotype.

Shalaka Mulherkar, et al. PLoS One. 2013;8(6):e67015.
7.
Figure 5

Figure 5. Abnormal morphology and incorrect innervation in the spinal cord of RhoA cKO mice.. From: The Small GTPase RhoA Is Required for Proper Locomotor Circuit Assembly.

(A) Dark field photos demonstrated that the dorsal funiculi of RhoA cKO mutant mice at the cervical spinal cord were more shallow and widened compared to the control and heterozygous mice. RhoA cKO mice exhibited a remarkable increase in the amount of gray matter at the midline. The width (horizontal line in A, left panel) and the height (vertical line in A, left panel) of funiculi were measured. Scale bar: 500 µm. (B) The ratio of width to height measurements for the dorsal funiculus of RhoA cKO mice was significantly greater than that of control and heterozygous mice (p<0.001). No significant differences were observed between control and heterozygous mice (p>0.05) (C). At the midline, the space between the dorsal column and ventral column of gray matter of RhoA cKO mice was significantly greater compared to control and heterozygous mice (p<0.001). n = 3–4 mice per group. Data are represented as the mean ± SD. *** = p<0.001 (ANOVA followed by the Student-Newman-Keuls test). (D) Schematic drawing illustrating the strategy used for labeling spinal interneurons. Crystals of rhodamine dextran were applied unilaterally to control and RhoA cKO isolated mouse spinal cords at L4 (red rectangle). Contralateral projections were then visualized by imaging the labeled spinal cords at L2 (black dashed box) with an epifluorescence microscope. (E) Many spinal interneuron axons aberrantly cross the midline at L2 in the spinal cords of RhoA cKO mice, but not control littermates. Lower panels show an enlarged image of the area boxed in the upper panels. Scale bar represents 200 µm. Three sections per animal and N = 4 animals were analyzed per genotype.

Shalaka Mulherkar, et al. PLoS One. 2013;8(6):e67015.

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