Research reportRemarkable complexity and variability of corticospinal tract defects in adult Semaphorin 6A knockout mice
Introduction
Originating in the cerebral cortex and descending to the spinal cord, the corticospinal tract (CST) plays a role in controlling voluntary movement. Because of the length and complexity of its trajectory, a number of axon guidance molecules and their receptors have been implicated in steering the growing axons towards their proper targets during development (Canty and Murphy, 2008, Leyva-Diaz and Lopez-Bendito, 2013). CST axons extend out of the cortical plate and direct towards the internal capsule at embryonic days 13–14 (E13-14). In these initial steps, Semaphorin 3A (Sema3A), Sema3C, and Netrin-1 guide the CST axons (Bagnard et al., 1998, Metin et al., 1997). At E14-15, the CST axons pass through the internal capsule and cerebral peduncle. Slit/Robo signaling is required for preventing the CST axons from entering the ventral forebrain and growing towards the midline (Bagri et al., 2002, Lopez-Bendito et al., 2007). After descending through the pons and medulla, the CST axons cross the midline at the pyramidal decussation and enter the dorsal funiculus at postnatal day 0. Netrin-1, L1, and NCAM are required to regulate the formation of the pyramidal decussation (Cohen et al., 1998, Finger et al., 2002, Rolf et al., 2002). In the spinal cord, Wnt signaling controls caudal growth of axons (Liu et al., 2005), and Ephrin-B3/EphA4 signaling prevents the CST axons from recrossing the midline (Coonan et al., 2001, Dottori et al., 1998, Kullander et al., 2001, Paixao et al., 2013).
The semaphorins are a large family of secreted and membrane-associated proteins that regulate nervous system development (Jongbloets and Pasterkamp, 2014, Kruger et al., 2005). They are classified into several groups on the basis of their structural characteristics: among vertebrate semaphorins, class 3 semaphorins are secreted proteins, whereas class 4–6 semaphorins are transmembrane proteins and class 7 semaphorins are glycophosphatidylinositol-anchored proteins. Semaphorin signals are mediated mainly by receptors of the plexin family. In addition to acting as ligands, transmembrane semaphorins including Sema6A also serve as receptors, mediating reverse signaling (Jongbloets and Pasterkamp, 2014, Kruger et al., 2005, Perez-Branguli et al., 2016).
During development, Sema6A plays a crucial role in guiding CST axons. In Sema6A knockout (KO) mice, several aberrant projections were observed in the pontine and medullary areas, whereas no obvious abnormalities were present rostral to the midbrain (Rünker et al., 2008). At the midbrain–hindbrain boundary, dorsal turning at the caudal end of the cerebral peduncle, abnormal midline crossing, and splitting into several bundles were observed (Rünker et al., 2008). At the level of the inferior olive, most fibers showed misrouting on the ventrolateral surface of the medulla (Faulkner et al., 2008, Rünker et al., 2008). Sema6A acts as a chemorepellent for axons that express its receptors Plexin-A2 (PlxnA2) and PlxnA4. Because Sema6A is highly expressed in the inferior olive, it was postulated that lateral expansion of the CST axons near the inferior olive in Sema6A KO mice was caused by the loss of Sema6A that constrains CST axons close to the midline through repulsion (Rünker et al., 2008). In addition, PlxnA2 and PlxnA4 are expressed in layer 5 cortical neurons, and the CST defects similar to those in Sema6A KO mice were observed in PlxnA4 KO mice, but not in PlxnA2 KO mice. It thus appears that Sema6A repels CST axons through PlxnA4 in this region (Faulkner et al., 2008, Rünker et al., 2008). This prominent defect in the caudal medulla was shown to be present in adulthood (Rünker et al., 2008), although analysis of other aberrant CST fibers in the adult brain has not been performed. In particular, because these previous studies were done using brain sections, the routes that the aberrant fibers take throughout the brain remain unknown. In addition, because neurodevelopmental abnormalities cause neurologic and psychiatric diseases (Rünker et al., 2011, Welniarz et al., 2016), phenotypic analysis of the adult brain is as important as that of the brain in its developmental stages.
In this study, we performed 3D reconstruction of the abnormal CST fibers in adult Sema6A mutant brains. This method enabled us to track the long and complex trajectory of the CST and to detect phenotypic variability among individual mutant mice. Our results clearly showed that the defects described in early postnatal mutants persist to adulthood. Moreover, we identified several patterns of CST defects that have not been previously described. Immunostaining combined with 3D reconstruction facilitates analysis of nerve tracts, especially long and complex projections such as the CST.
Section snippets
CST trajectory in the adult brain revealed by PKCγ staining
To reinvestigate the CST defects in adult Sema6A mutants, we first performed section immunostaining for protein kinase C gamma (PKCγ), a postnatal CST marker (Ding et al., 2005, Joshi et al., 2008, Mori et al., 1990). In the wild-type brain, PKCγ was highly expressed in the cerebral cortex, hippocampus, thalamus, and cerebellum, whereas the staining was so scarce in the midbrain/hindbrain region that the PKCγ-positive CST fibers were easily tracked (Fig. 1, Fig. 2). The CST fibers extended in
Discussion
In this study, we performed 3D analysis of the CST trajectory to revisit the CST defects of Sema6A mutant mice. Our results clearly show that the CST defects persist into adulthood and are much more complex than was previously thought (Faulkner et al., 2008, Rünker et al., 2008). The defects at the midbrain–hindbrain boundary and in the pons are somewhat symmetric and similar among individuals, whereas those in the caudal medulla are highly variable. This may be related to the fact that proper
Knockout mice
The Sema6A KO mice were described previously (Rünker et al., 2008). All animal experiments were approved by and performed according to the guidelines of the Animal Care and Use Committees of the University of Tsukuba and the National Institute of Neuroscience, National Center of Neurology and Psychiatry.
Immunohistochemistry
Adult mice were transcardially perfused with 4% paraformaldehyde in phosphate-buffered saline (PBS) under deep anesthesia with diethyl ether. After the brains had been removed, they were
Author contributions
T.O., K.K.-M., and M.M. designed the research and performed the experiments. T.O. carried out the image analysis and 3D reconstruction. F.S. prepared the fixed mouse brains. K.J.M. generated the Sema6A knockout mice. T.O., K.K.-M., K.J.M., and M.M. wrote the manuscript. All the authors read and approved the final manuscript.
Funding
This work was supported by Kakenhi grants (grant numbers 22123006, 25293065) from the Japanese Ministry of Education, Culture, Sports, Science, and Technology (MEXT) and from the Japan Society for the Promotion of Science (JSPS) to M.M.
Competing interests
The authors have no competing interests to declare.
Acknowledgements
We thank T. Shiga, H. Ichijo, T. Masuda, and F. Miyamasu for critical reading of the mansucript and H. Yuyama, S. Imaizumi, and Y. Iwahashi for their experimental support.
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