Conserved Interhemispheric Morphogenesis in Amniotes Preceded the Evolution of the Corpus Callosum.

Judgment of abnormalities in fetal cortical axon development is more sensitive when a good standard of normal ontogeny is established. The recent availability of postmortem tract tracing methods has greatly improved the observation of axon extension and growth cone morphology in mouse fetuses, which allows much stronger statements about the timing of crucial steps in the formation of the corpus callosum in particular. The first outgrowth and crossing of midplane by axons of the corpus callosum (CC) were examined in 153 normal mouse embryos and fetuses of the hybrid cross B6D2F2/J with carbocyanine dyes applied to brains fixed by perfusion. In most brains a crystal of DiI was inserted into either frontal, parietal, temporal, or occipital cortex in one hemisphere, and a crystal of DiA was placed into a different site in the opposite hemisphere. Although dye diffusion obscured the emergence of axons, linear regression analysis revealed that the first callosal axons emerged from their cortical cells of origin at about 0.4 g body weight or 15.5 days after conception for all four sites. Subsequent axon growth rate was substantially faster for those from frontal cortex (3.2 mm/day) than occipital cortex (1.8 mm/day). Axons from frontal cortex crossed the cerebral midplane first (0.69 g, E16.3), followed by those from parietal (0.74 g), temporal (0.77 g) and occipital cortex (0.92 g, E16.9). Prior to crossing midplane, the pioneering CC axons were usually 200 microns or less in advance of the main bundle, but when they crossed midplane and encountered CC axons growing from homotopic sites in the opposite hemisphere, the pioneering axons were often 0.5 to 2.5 mm ahead of the main bundle. Growth cones were usually large and complex until they had crossed midplane and were thereafter smaller with simple and flat morphologies. The topography of axons in the CC at midplane was organized according to cortical region of origin from the very beginning, when the CC was only a small cap over the hippocampal commissure and dorsal septum. The quantitative results provide a convenient standard for normal callosal development in mice and should facilitate comparative studies.

Defects in neural guidepost structures and failure to remove leptomeningeal cells from the septal midline behind the interhemispheric fusion defects in Netrin1 deficient mice

The forebrain hemispheres are predominantly separated during embryogenesis by the interhemispheric fissure (IHF). Radial astroglia remodel the IHF to form a continuous substrate between the hemispheres for midline crossing of the corpus callosum (CC) and hippocampal commissure (HC). Deleted in colorectal carcinoma (DCC) and netrin 1 (NTN1) are molecules that have an evolutionarily conserved function in commissural axon guidance. The CC and HC are absent in Dcc and Ntn1 knockout mice, while other commissures are only partially affected, suggesting an additional aetiology in forebrain commissure formation. Here, we find that these molecules play a critical role in regulating astroglial development and IHF remodelling during CC and HC formation. Human subjects with DCC mutations display disrupted IHF remodelling associated with CC and HC malformations. Thus, axon guidance molecules such as DCC and NTN1 first regulate the formation of a midline substrate for dorsal commissures prior to their role in regulating axonal growth and guidance across it.

The forebrain hemispheres are predominantly separated during embryogenesis by the interhemispheric fissure (IHF). Radial astroglia remodel the IHF to form a continuous substrate between the hemispheres for midline crossing of the corpus callosum (CC) and hippocampal commissure (HC). Deleted in colorectal carcinoma (DCC) and netrin 1 (NTN1) are molecules that have an evolutionarily conserved function in commissural axon guidance. The CC and HC are absent in Dcc and Ntn1 knockout mice, while other commissures are only partially affected, suggesting an additional aetiology in forebrain commissure formation. Here, we find that these molecules play a critical role in regulating astroglial development and IHF remodelling during CC and HC formation. Human subjects with DCC mutations display disrupted IHF remodelling associated with CC and HC malformations. Thus, axon guidance molecules such as DCC and NTN1 first regulate the formation of a midline substrate for dorsal commissures prior to their role in regulating axonal growth and guidance across it.

The Dynamics of Axon Bifurcation Development in the Cerebral Cortex of Typical and Acallosal Mice

Gli3 is required in Emx1+ progenitors for the development of the corpus callosum

The formation of the corpus callosum in the postnatal period is crucial for normal neurological function, and clinical genetic studies have identified an association of 6q24-25 microdeletion in this process. However, the mechanisms underlying corpus callosum formation and its critical gene(s) are not fully understood or identified. In this study, we examined the roles of AKAP12 in postnatal corpus callosum formation by focusing on the development of glial cells, because AKAP12 is coded on 6q25.1 and has recently been shown to play roles in the regulations of glial function. In mice, the levels of AKAP12 expression was confirmed to be larger in the corpus callosum compared to the cortex, and AKAP12 levels decreased with age both in the corpus callosum and cortex regions. In addition, astrocytes expressed AKAP12 in the corpus callosum after birth, but oligodendrocyte precursor cells (OPCs), another major type of glial cell in the developing corpus callosum, did not. Furthermore, compared to wild types, Akap12 knockout mice showed smaller numbers of both astrocytes and OPCs, along with slower development of corpus callosum after birth. These findings suggest that AKAP12 signaling may be required for postnatal glial formation in the corpus callosum through cell- and non-cell autonomous mechanisms.

A precise description of the timing and route traveled by axons traversing the telencephalic midline through the ventral hippocampal commissure (HC) is essential for understanding the role it plays in the formation of the corpus callosum (CC). A normal baseline of HC development was described in B6D2F2 hybrid mice and then compared with two inbred strains of mice displaying callosal agenesis, BALB/cWah1 (50% CC defect) and 129/J (70% CC defect), their F2 hybrid (C129F2—33% CC defect), and a recombinant inbred strain (Rl-1—100% CC defect) derived from pairs of C129F2 mice. Embryos weighing from 0.25 g to 0.70 g (E14.5–E17) were collected and fixed by perfusion. Axon tracts were labeled using crystals of the lipophilic dyes Dil and DiA inserted into the hippocampal fimbria and cerebral cortex. HC axons in B6D2F2 mice first cross the midline at about 0.350 g body weight (E14.8) by traveling over the dorsal septum and along the pia membrane lining the longitudinal fissure. Earlier crossing was prevented by the presence of a deep cleft formed by the longitudinal fissure extending down into the septal region. Subsequent axons fasciculated along existing axons, gradually building the dorsoventral height of the HC to about 200 μm by 0.600 g. The earliest callosal axons from frontal cortex crossed the midline at 0.620 g and were clearly seen fasciculating along and between existing hippocampal axons at the dorsal surface of the HC as they crossed. In the acallosal strains, HC formation was delayed by the continued presence of the cleft deep in the septal region. This delay in time of crossing was correlated with later CC defect expression. Initial HC crossing occurred at about 0.470 g (E16.25) in BALB mice and about 0.520 g (E16.5) in 129 mice. In the Rl-1 embryos, first HC crossing was estimated at about 0.750 g (E17.5), although several older embryos showed no crossing. These results show the importance of the HC for successful CC formation and suggest that absent CC arises as a consequence of a developmental defect which affects the formation of the hippocampal commissure prior to arrival of CC axons at midplane. Hippocampus 7:2–14, 1997. © 1997 Wiley-Liss, Inc.

A precise description of the timing and route traveled by axons traversing the telencephalic midline through the ventral hippocampal commissure (HC) is essential for understanding the role it plays in the formation of the corpus callosum (CC). A normal baseline of HC development was described in B6D2F2 hybrid mice and then compared with two inbred strains of mice displaying callosal agenesis, BALB/cWah1 (50% CC defect) and 129/J (70% CC defect), their F2 hybrid (C129F2-33% CC defect), and a recombinant inbred strain (RI-1-100% CC defect) derived from pairs of C129F2 mice. Embryos weighing from 0.25 g to 0.70 g (E14.5-E17) were collected and fixed by perfusion. Axon tracts were labeled using crystals of the lipophilic dyes DiI and DiA inserted into the hippocampal fimbria and cerebral cortex. HC axons in B6D2F2 mice first cross the midline at about 0.350 g body weight (E14.8) by traveling over the dorsal septum and along the pia membrane lining the longitudinal fissure. Earlier crossing was prevented by the presence of a deep cleft formed by the longitudinal fissure extending down into the septal region. Subsequent axons fasciculated along existing axons, gradually building the dorsoventral height of the HC to about 200 microns by 0.600 g. The earliest callosal axons from frontal cortex crossed the midline at 0.620 g and were clearly seen fasciculating along and between existing hippocampal axons at the dorsal surface of the HC as they crossed. In the acallosal strains, HC formation was delayed by the continued presence of the cleft deep in the septal region. This delay in time of crossing was correlated with later CC defect expression. Initial HC crossing occurred at about 0.470 g (E16.25) in BALB mice and about 0.520 g (E16.5) in 129 mice. In the RI-1 embryos, first HC crossing was estimated at about 0.750 g (E17.5), although several older embryos showed no crossing. These results show the importance of the HC for successful CC formation and suggest that absent CC arises as a consequence of a developmental defect which affects the formation of the hippocampal commissure prior to arrival of CC axons at midplane.

Perinatal midline astrocyte development is impaired in fibroblast growth factor 8 hypomorphic mice

The corpus callosum (CC) is the largest fiber tract in the mammalian brain, linking the bilateral cerebral hemispheres. CC development depends on the proper balance of axon growth cone attractive and repellent cues leading axons to the midline and then directing them to the contralateral hemisphere. Imbalance of these cues results in CC agenesis or dysgenesis. Nogo receptors (NgR1, NgR2, and NgR3) are growth cone directive molecules known for inhibiting axon regeneration after injury. We report that mice lacking Nogo receptors (NgR123-null mice) display complete CC agenesis due to axon misdirection evidenced by ectopic axons including cortical Probst bundles. Because glia and glial-derived growth cone repellent factors (especially the diffusible factor Slit2) are required for CC development, their distribution was studied. Compared with wild-type mice, NgR123-null mice had a sharp increase in the glial marker glial fibrillary acidic protein (GFAP) and in Slit2 at the glial wedge and indusium griseum, midline structures required for CC formation. NgR123-null mice displayed reduced motor coordination and hyperactivity. These data are consistent with the hypotheses that Nogo receptors are membrane-bound growth cone repellent factors required for migration of axons across the midline at the CC, and that their absence results directly or indirectly in midline gliosis, increased Slit2, and complete CC agenesis. J. Comp. Neurol. 525:291-301, 2017. © 2016 Wiley Periodicals, Inc.

The corpus callosum (CC) plays a crucial role in interhemispheric communication. It has been shown that CC formation relies on the guidepost cells located in the midline region that include glutamatergic and GABAergic neurons as well as glial cells. However, the origin of these guidepost GABAergic neurons and their precise function in callosal axon pathfinding remain to be investigated. Here, we show that two distinct GABAergic neuronal subpopulations converge toward the midline prior to the arrival of callosal axons. Using in vivo and ex vivo fate mapping we show that CC GABAergic neurons originate in the caudal and medial ganglionic eminences (CGE and MGE) but not in the lateral ganglionic eminence (LGE). Time lapse imaging on organotypic slices and in vivo analyses further revealed that CC GABAergic neurons contribute to the normal navigation of callosal axons. The use of Nkx2.1 knockout (KO) mice confirmed a role of these neurons in the maintenance of proper behavior of callosal axons while growing through the CC. Indeed, using in vitro transplantation assays, we demonstrated that both MGE- and CGE-derived GABAergic neurons exert an attractive activity on callosal axons. Furthermore, by combining a sensitive RT-PCR technique with in situ hybridization, we demonstrate that CC neurons express multiple short and long range guidance cues. This study strongly suggests that MGE- and CGE-derived interneurons may guide CC axons by multiple guidance mechanisms and signaling pathways.

The human brain spends several years bootstrapping itself through intrinsic and extrinsic modulation, thus gradually developing both spatial organization and functions. Based on previous studies on developmental patterns and inter-individual variability of the corpus callosum (CC), we hypothesized that inherent variations of CC shape among infants emerge, depending on the position within the CC, along the developmental timeline. Here we used longitudinal magnetic resonance imaging data from infancy to toddlerhood and investigated the area, thickness, and shape of the midsagittal plane of the CC by applying multilevel modeling. The shape characteristics were extracted using the Procrustes method. We found nonlinearity, region-dependency, and inter-individual variability, as well as intra-individual consistencies, in CC development. Overall, the growth rate is faster in the first year than in the second year, and the trajectory differs between infants; the direction of CC formation in individual infants was determined within six months and maintained to two years. The anterior and posterior subregions increase in area and thickness faster than other subregions. Moreover, we clarified that the growth rate of the middle part of the CC is faster in the second year than in the first year in some individuals. Since the division of regions exhibiting different tendencies coincides with previously reported divisions based on the diameter of axons that make up the region, our results suggest that subregion-dependent individual variability occurs due to the increase in the diameter of the axon caliber, myelination partly due to experience and axon elimination during the early developmental period.

The corpus callosum (CC) is the major commissure that bridges the cerebral hemispheres. Agenesis of the CC is associated with human ciliopathies, but the origin of this default is unclear. Regulatory Factor X3 (RFX3) is a transcription factor involved in the control of ciliogenesis, and Rfx3–deficient mice show several hallmarks of ciliopathies including left–right asymmetry defects and hydrocephalus. Here we show that Rfx3–deficient mice suffer from CC agenesis associated with a marked disorganisation of guidepost neurons required for axon pathfinding across the midline. Using transplantation assays, we demonstrate that abnormalities of the mutant midline region are primarily responsible for the CC malformation. Conditional genetic inactivation shows that RFX3 is not required in guidepost cells for proper CC formation, but is required before E12.5 for proper patterning of the cortical septal boundary and hence accurate distribution of guidepost neurons at later stages. We observe focused but consistent ectopic expression of Fibroblast growth factor 8 (Fgf8) at the rostro commissural plate associated with a reduced ratio of GLIoma-associated oncogene family zinc finger 3 (GLI3) repressor to activator forms. We demonstrate on brain explant cultures that ectopic FGF8 reproduces the guidepost neuronal defects observed in Rfx3 mutants. This study unravels a crucial role of RFX3 during early brain development by indirectly regulating GLI3 activity, which leads to FGF8 upregulation and ultimately to disturbed distribution of guidepost neurons required for CC morphogenesis. Hence, the RFX3 mutant mouse model brings novel understandings of the mechanisms that underlie CC agenesis in ciliopathies.

The human Nogo-66 receptor 1 (NgR1) gene, also termed Nogo receptor 1 or reticulon 4 receptor (RTN4R) and located within 22q11.2, inhibits axonal growth and synaptic plasticity. Patients with the 22q11.2 deletion syndrome show multiple changes in brain morphology, with corpus callosum (CC) abnormalities being among the most prominent and frequently reported. Thus, we hypothesized that, in humans, NgR1 may be involved in CC formation. We focused on rs701428, a single nucleotide polymorphism of NgR1, which is associated with schizophrenia. We investigated the effects of the rs701428 genotype on CC structure in 50 healthy participants using magnetic resonance imaging. Polymorphism of rs701428 was associated with CC structural variation in healthy participants; specifically, minor A allele carriers had larger whole CC volumes and lower radial diffusivity in the central CC region compared with major G allele homozygous participants. Furthermore, we showed that the NgR1 3′ region, which contains rs701428, is a neuronal activity-dependent enhancer, and that the minor A allele of rs701428 is susceptible to regulation of enhancer activity by MYBL2. Our results suggest that NgR1 can influence the macro- and microstructure of the white matter of the human brain.