Probst Bundles Research Articles

Agenesis of the Corpus Callosum Due to Defective Glial Wedge Formation in Lhx2 Mutant Mice

Establishment of the corpus callosum involves coordination between callosal projection neurons and multiple midline structures, including the glial wedge (GW) rostrally and hippocampal commissure caudally. GW defects have been associated with agenesis of the corpus callosum (ACC). Here we show that conditional Lhx2 inactivation in cortical radial glia using Emx1-Cre or Nestin-Cre drivers results in ACC. The ACC phenotype was characterized by aberrant ventrally projecting callosal axons rather than Probst bundles, and was 100% penetrant on 2 different mouse strain backgrounds. Lhx2 inactivation in postmitotic cortical neurons using Nex-Cre mice did not result in ACC, suggesting that the mutant phenotype was not autonomous to the callosal projection neurons. Instead, ACC was associated with an absent hippocampal commissure and a markedly reduced to absent GW. Expression studies demonstrated strong Lhx2 expression in the normal GW and in its radial glial progenitors, with absence of Lhx2 resulting in normal Emx1 and Sox2 expression, but premature exit from the cell cycle based on EdU-Ki67 double labeling. These studies define essential roles for Lhx2 in GW, hippocampal commissure, and corpus callosum formation, and suggest that defects in radial GW progenitors can give rise to ACC.

Heparan Sulfotransferases Hs6st1 and Hs2st Keep Erk in Check for Mouse Corpus Callosum Development

The corpus callosum (CC) connects the left and right cerebral hemispheres in mammals and its development requires intercellular communication at the telencephalic midline mediated by signaling proteins. Heparan sulfate (HS) is a sulfated polysaccharide that decorates cell surface and extracellular matrix proteins and regulates the biological activity of numerous signaling proteins via sugar-protein interactions. HS is subject to regulated enzymatic sulfation and desulfation and an attractive, although not proven, hypothesis is that the biological activity of HS is regulated by a sugar sulfate code. Mutant mouse embryos lacking the heparan sulfotransferases Hs2st or Hs6st1 have severe CC phenotypes and form Probst bundles of noncrossing axons flanking large tangles of midline glial processes. Here, we identify a precocious accumulation of Sox9-expressing glial cells in the indusium griseum region and a corresponding depletion at the glial wedge associated with the formation of Probst bundles along the rostrocaudal axis in both mutants. Molecularly, we found a surprising hyperactivation of Erk signaling in Hs2st(-/-) (2-fold) and Hs6st1(-/-) (6-fold) embryonic telencephalon that was most striking at the midline, where Erk signaling is lowest in wild-types, and a 2-fold increase in Fgf8 protein levels in Hs6st1(-/-) embryos that could underpin Erk hyperactivation and excessive glial movement to the indusium griseum. The tightly linked Hs6st1(-/-) CC glial and axonal phenotypes can be rescued by genetic or pharmacological suppression of Fgf8/Erk axis components. Overall, our data fit a model in which Hs2st and Hs6st1 normally generate conditions conducive to CC development by generating an HS-containing environment that keeps Erk signaling in check.

Neuronal Basic Helix–Loop–Helix Proteins Neurod2/6 Regulate Cortical Commissure Formation before Midline Interactions

Establishment of long-range fiber tracts by neocortical projection neurons is fundamental for higher brain functions. The molecular control of axon tract formation, however, is still poorly understood. Here, we have identified basic helix-loop-helix (bHLH) transcription factors Neurod2 and Neurod6 as key regulators of fasciculation and targeted axogenesis in the mouse neocortex. In Neurod2/6 double-mutant mice, callosal axons lack expression of the cell adhesion molecule Contactin2, defasciculate in the subventricular zone, and fail to grow toward the midline without forming Probst bundles. Instead, mutant axons overexpress Robo1 and follow random trajectories into the ipsilateral cortex. In contrast to long-range axogenesis, generation and maintenance of pyramidal neurons and initial axon outgrowth are grossly normal, suggesting that these processes are under distinct transcriptional control. Our findings define a new stage in corpus callosum development and demonstrate that neocortical projection neurons require transcriptional specification by neuronal bHLH proteins to execute an intrinsic program of remote connectivity.

Altered Functional and Structural Connectivity in a Schizophrenia Patient With Complete Agenesis of the Corpus Callosum

Altered Functional and Structural Connectivity in a Schizophrenia Patient With Complete Agenesis of the Corpus Callosum

The structural connectome of the human brain in agenesis of the corpus callosum

Adopting a network perspective, the structural connectome reveals the large-scale white matter connectivity of the human brain, yielding insights into cerebral organization otherwise inaccessible to researchers and clinicians. Connectomics has great potential for elucidating abnormal connectivity in congenital brain malformations, especially axonal pathfinding disorders. Agenesis of the corpus callosum (AgCC) is one of the most common brain malformations and can also be considered a prototypical genetic disorder of axonal guidance in humans. In this exploratory study, the structural connectome of AgCC is mapped and compared to that of the normal human brain. Multiple levels of granularity of the AgCC connectome are investigated, including summary network metrics, modularity analysis, and network consistency measures, with comparison to the normal structural connectome after simulated removal of all callosal connections ("virtual callostomy"). These investigations reveal four major findings. First, global connectivity is abnormally reduced in AgCC, but local connectivity is increased. Second, the network topology of AgCC is more variable than that of the normal human connectome, contradicting the predictions of the virtual callostomy model. Third, modularity analysis reveals that many of the tracts that comprise the structural core of the cerebral cortex have relatively weak connectivity in AgCC, especially the cingulate bundles bilaterally. Finally, virtual lesions of the Probst bundles in the AgCC connectome demonstrate that there is consistency across subjects in many of the connections generated by these ectopic white matter tracts, and that they are a mixture of cortical and subcortical fibers. These results go beyond prior diffusion tractography studies to provide a systems-level perspective on anomalous connectivity in AgCC. Furthermore, this work offers a proof of principle for the utility of the connectome framework in neurodevelopmental disorders.

Lissencephaly with marked ventricular dilation, agenesis of corpus callosum, and cerebellar hypoplasia caused by TUBA1A mutation

We described the clinical course and pathological findings in a child with TUBA1A mutation. MRI revealed marked ventricular dilation with thin cortex, poorly differentiated basal ganglia, agenesis of corpus callosum, cerebellar hypoplasia with preserved vermis at 2months of age. No gain of developmental milestones was observed until she died with respiratory failure at 23months of age. A de novo missense mutation of c.1096G>A (G366R) was identified in TUBA1A gene. Pathological findings included a lack in lamination in the cerebral cortex, absent corpus callosum without Probst bundle, blurred demarcation among the striatum, internal capsule and globus pallidus in association with irregular running of myelinated fibers, cerebellar hypoplasia with irregular undulation in the dentate nucleus and inferior olivary nucleus, absent olfactory bulbs and tracts, and pyramidal tract hypoplasia. These findings are consistent with previous reports and will be a clue to diagnosis of TUBA1A mutation.

Novel Proximal 14q Deletion: Clinical and Diffusion Tensor Imaging Tractography Findings in a Patient with Lissencephaly, Agenesis of the Corpus Callosum, and Septo-Optic Dysplasia

We report the unique CNS findings in a patient with a proximal chromosome 14q interstitial deletion. Conventional MR imaging allowed the clear delineation of agenesis of the corpus callosum, SOD, and diffuse lissencephaly. DTI tractography played a significant role in the evaluation of the proximal 14q deletion-associated abnormalities, delineating the extent of the dysmorphic connections of the Probst bundles and clarifying that apparent areas of heterotopias were the corticospinal tracts.

RA-GEF-1 (Rapgef2) is essential for proper development of the midline commissures

The cerebral hemispheres are directly connected by three major interhemispheric fibers: the corpus callosum, the anterior commissure, and the hippocampal commissure. RA-GEF-1 (also termed Rapgef2) is a guanine nucleotide exchange factor responsible for sustained activation of Rap1. We previously reported anatomical defects of the major forebrain commissures in the adult dorsal telencephalon-specific RA-GEF-1 conditional knockout (cKO) mice. In this study, we use neuroanatomical tracing and immunohistochemistry to study the formation of the commissural fibers during early postnatal development. DiI anterograde tracing reveals the inability of the callosal axons to cross the midline in cKO mice, thereby forming Probst bundles on the ipsilateral side, which is associated with the absence of the indusium griseum glia and the glial sling at the cortical midline. Wheat germ agglutinin-conjugated horseradish peroxidase retrograde tracing verifies the agenesis of the anterior commissure in cKO mice, and DiI anterograde tracing confirms the deviation of the fibers from their original tract. As for the hippocampal commissure, agenesis and hypoplasia are observed in its dorsal and ventral parts, respectively. These results indicate the essential role of RA-GEF-1 in the proper formation of the cerebral midline commissures.

Appropriate Bmp7 levels are required for the differentiation of midline guidepost cells involved in corpus callosum formation

Guidepost cells are essential structures for the establishment of major axonal tracts. How these structures are specified and acquire their axon guidance properties is still poorly understood. Here, we show that in mouse embryos appropriate levels of Bone Morphogenetic Protein 7 (Bmp7), a member of the TGF-β superfamily of secreted proteins, are required for the correct development of the glial wedge, the indusium griseum, and the subcallosal sling, three groups of cells that act as guidepost cells for growing callosal axons. Bmp7 is expressed in the region occupied by these structures and its genetic inactivation in mouse embryos caused a marked reduction and disorganization of these cell populations. On the contrary, infusion of recombinant Bmp7 in the developing forebrain induced their premature differentiation. In both cases, changes were associated with the disruption of callosal axon growth and, in most animals fibers did not cross the midline forming typical Probst bundles. Addition of Bmp7 to cortical explants did not modify the extent of their outgrowth nor their directionality, when explants were exposed to a focalized source of the protein. Together, these results indicate that Bmp7 is indirectly required for corpus callosum formation by controlling the timely differentiation of its guidepost cells.

A novel locus on proximal chromosome 18 associated with agenesis of the corpus callosum in mice

Agenesis of the corpus callosum (ACC) is a congenital abnormality of the brain structure. We have produced transgenic mice expressing both reverse tetracycline-controlled transactivator (rtTA) and transcriptional silencer (tTS) ubiquitously. Although the transgene products do not affect development of the mouse brain, one of the founder lines, TAS, showed ACC, suggesting transgenic disruption of endogenous gene(s). To identify the causative gene and its role in ACC, we performed pathological investigations of the brain and chromosomal mapping of foreign genes in TAS mice. Sixty-two percent of the heterozygous TAS mice showed ACC accompanied with formation of Probst bundles, as seen in human. Complete penetrance of ACC was observed in homozygous TAS mice. Furthermore, homozygous TAS fetuses revealed that ACC is a congenital anomaly. Moreover, axons of the corpus callosum were not repelled by the midline glial structures in TAS mice. These findings suggested that the causative gene for ACC is involved in critical steps in corpus callosum development. Multiple FISH analyses were performed to determine the site of transgene insertion. On 1-color FISH analyses, rtTA and tTS were detected on the A/B region of chromosome 18, suggesting cointegration of the transgenes. On 2-color FISH analyses, tTS signal was observed in a region from 9.3 to 16.9 Mb on chromosome 18. The TAS mice may serve as a useful model to identify a novel gene regulating corpus callosum development and to gain a new insight into molecular genetics of ACC.

“MULTICYSTIC DYSPLASTIC KIDNEY (POTTER TYPE II SYNDROME) AND AGENESIS OF CORPUS CALLOSUM (ACC) IN TWO CONSECUTIVE PREGNANCIES: A POSSIBLE TERATOGENIC EFFECT OF ELECTROMAGNETIC EXPOSURE IN UTERO”

Agenesis of the corpus callosum is found in about 5 per 1,000 births and it is due to maldevelopment or, secondary, to destructive lesions. Multicystic dysplastic kidneys is a consequence of either developmental failure of the mesonephric blastema to form nephrons or to early urinary obstruction due to urethral or ureteric atresia and can be found in about 1 per 1,000 live births. A case of fetal multicystic dysplastic kidney disease (Potter type II syndrome) and complete agenesis of the corpus callosum demonstrated by the presence of Probst bundles associated with colpocephaly occurring in the same mother in her two consecutive pregnancies is reported. Data regarding possible teratogenetic effect due to electromagnetic exposure in utero have also been investigated and raised suspicionus as a potential risk factor. In cases of suspected second trimester ultrasound diagnosis of agenesis of corpus callosum (ACC), the following clinical management should be recommended: fetal karyotype; a second level scan with differentiation between underlying conditions such as hydrocephalus and holoprosencephaly; antenatal MRI to enhance the diagnostic accuracy of possible associated neuronal migration (when possible); and direct demonstration of the presence of the Probst bundles to neurohistology.

Diffusion tensor magnetic resonance imaging and tract-tracing analysis of Probst bundle structure in Netrin1- and DCC-deficient mice.

In many cases of callosal dysgenesis in both human patients and mouse models, misguided fibers from the cortex form abnormal bilateral, barrel-shaped structures known as Probst bundles. Because little is known about how axons are arranged within these anomalous fiber bundles, understanding this arrangement may provide structural and molecular insights into how axons behave when they are misguided in vivo. Previous studies described these bundles as longitudinal swirls of axons that fail to cross the midline (Ozaki et al., 1987). However, recent studies on human acallosal patients using diffusion tensor magnetic resonance imaging (DTMRI) technology suggest that axons project in an anteroposterior direction within the Probst bundle (Lee et al., 2004; Tovar-Moll et al., 2007). This led us to ask the question, is DTMRI an accurate method for analyzing axonal tracts in regions of high axon overlap and disorganization, or is our current perception of axon arrangement within these bundles inaccurate? Using DTMRI, immunohistochemistry, and carbocyanine dye tract-tracing studies, we analyzed the Probst bundles in both Netrin1 and deleted in colorectal cancer (DCC) mutant mice. Our findings indicate that DTMRI can accurately demonstrate fiber tract orientation and morphology where axons are in ordered arrays such as in the dorsal part of the bundle. In ventral areas, where the axons are disorganized, no coordinated diffusion is apparent via DTMRI. In these regions, a higher-resolution approach such as tract tracing is required. We conclude that in DCC and Netrin1 mutant mice, guidance mechanisms remain in the dorsal part of the tract but are lost ventrally.

Spinophilin Facilitates Dephosphorylation of Doublecortin by PP1 to Mediate Microtubule Bundling at the Axonal Wrist

The axonal shafts of neurons contain bundled microtubules, whereas extending growth cones contain unbundled microtubule filaments, suggesting that localized activation of microtubule-associated proteins (MAP) at the transition zone may bundle these filaments during axonal growth. Dephosphorylation is thought to lead to MAP activation, but specific molecular pathways have remained elusive. We find that Spinophilin, a Protein-phosphatase 1 (PP1) targeting protein, is responsible for the dephosphorylation of the MAP Doublecortin (Dcx) Ser 297 selectively at the "wrist" of growing axons, leading to activation. Loss of activity at the "wrist" is evident as an impaired microtubule cytoskeleton along the shaft. These findings suggest that spatially restricted adaptor-specific MAP reactivation through dephosphorylation is important in organization of the neuronal cytoskeleton.

Arrangement of fiber tracts forming probst bundle in complete callosal agenesis: report of two cases with an evaluation by diffusion tensor tractography

We report two patients with complete callosal agenesis in whom Probst bundles in both hemispheres could be depicted by diffusion tensor tractography (DTT). While one patient had no associated telencephalic anomaly other than callosal agenesis, the other had cortical dysplasia in the right frontal lobe. Although Probst bundles in the three normal hemispheres were well developed, that in the hemisphere which was affected by cortical dysplasia was small and poorly developed. DTT also showed that the fibers from the frontal pole ran more on the inner side of the Probst bundle than those from a more caudal region of the frontal lobe. Furthermore, fibers from the orbital gyri ran along the outermost side of Probst bundle. The arrangement of these fiber tracts in Probst bundle may reflect the developmental process of callosal fibers in their normal formation.

Anomalies of the Corpus Callosum: An MR Analysis of the Phenotypic Spectrum of Associated Malformations

We sought to categorize the structural brain anomalies associated with abnormalities of the corpus callosum and anterior and hippocampal commissures in a large cohort. Brain MR images of adult and pediatric patients from our institution and from a national support organization (the ACC Network) were retrospectively evaluated for the type and severity of commissural anomalies and the presence and type of other structural abnormalities. Of 142 cases that were reviewed, 82 patients had agenesis of the corpus callosum (ACC), while 60 had hypogenesis of the corpus callosum (HCC). Of the overall cohort, almost all had reduced white matter volume outside the commissures, the majority had malformations of cortical development (most commonly heterotopia or abnormal sulcation), many had noncallosal midline anomalies (including abnormal anterior or hippocampal commissures and interhemispheric cysts and lipomas), and several patients had abnormalities of the cerebellum or brainstem. Sixty-six patients had Probst bundles, which were more common in patients with ACC than in those with HCC. Probst bundles were present in all four patients who had ACC or HCC but no other midline, cortical, or posterior fossa anomalies. Isolated commissural anomalies were rare in the populations of patients examined. Most cases of ACC and HCC were associated with complex telencephalic, diencephalic, or rhombencephalic malformations. Reduced cerebral hemispheric white matter volume and malformations of cortical development were seen in more than half of the patients, suggesting that many commissural anomalies are part of an overall cerebral dysgenesis. ACC and HCC appear to lie along a dysgenetic spectrum, as opposed to representing distinct disorders.

The Wnt Receptor Ryk Is Required for Wnt5a-Mediated Axon Guidance on the Contralateral Side of the Corpus Callosum

Ryk (receptor related to tyrosine kinase) has been shown to be a novel Wnt receptor in both Caenorhabditis elegans and Drosophila melanogaster. Recently, Ryk-Wnt interactions were shown to guide corticospinal axons down the embryonic mouse spinal cord. Here we show that, in Ryk-deficient mice, cortical axons project aberrantly across the major forebrain commissure, the corpus callosum. Many mouse mutants have been described in which loss-of-function mutations result in the inability of callosal axons to cross the midline, thereby forming Probst bundles on the ipsilateral side. In contrast, loss of Ryk does not interfere with the ability of callosal axons to cross the midline but impedes their escape from the midline into the contralateral side. Therefore, Ryk(-/-) mice display a novel callosal guidance phenotype. We also show that Wnt5a acts as a chemorepulsive ligand for Ryk, driving callosal axons toward the contralateral hemisphere after crossing the midline. In addition, whereas callosal axons do cross the midline in Ryk(-/-) embryos, they are defasciculated on the ipsilateral side, indicating that Ryk also promotes fasciculation of axons before midline crossing. In summary, this study expands the emerging role for Wnts in axon guidance and identifies Ryk as a key guidance receptor in the establishment of the corpus callosum. Our analysis of Ryk function further advances our understanding of the molecular mechanisms underlying the formation of this important commissure.

Neuroplasticity in Human Callosal Dysgenesis: A Diffusion Tensor Imaging Study

Callosal dysgenesis (CD) is observed in many neurodevelopmental conditions, but its subjacent mechanisms are unknown, despite extensive research on animals. Here we employ magnetic resonance diffusion tensor imaging and tractography in human CD to reveal the aberrant circuitry of these brains. We searched particularly for evidence of plasticity. Four main findings are described--1) in the presence of a callosal remnant or a hypoplastic corpus callosum (CC), fibers therein largely connect the expected neocortical regions; 2) callosal remnants and hypoplastic CCs display a fiber topography similar to normal; 3) at least 2 long abnormal tracts are formed in patients with defective CC: the well-known Probst bundle (PB) and a so far unknown sigmoid, asymmetrical aberrant bundle connecting the frontal lobe with the contralateral occipitoparietal cortex; and 4) whereas the PB is topographically organized and has an ipsilateral U-connectivity, the sigmoid bundle is a long, heterotopic commissural tract. These observations suggest that when the developing human brain is confronted with factors that hamper CC fibers to cross the midline, some properties of the miswired fibers are maintained (such as side-by-side topography), whereas others are dramatically changed, leading to the formation of grossly abnormal white matter tracts.

Multiple Eph Receptors and B-Class Ephrins Regulate Midline Crossing of Corpus Callosum Fibers in the Developing Mouse Forebrain

Agenesis of the corpus callosum (CC) is a rare birth defect that occurs in isolated conditions and in combination with other developmental cerebral abnormalities. Recent identification of families of growth and guidance molecules has generated interest in the mechanisms that regulate callosal growth. One family, ephrins and Eph receptors, has been implicated in mediating midline pathfinding decisions; however, the complexity of these interactions has yet to be unraveled. Our studies shed light on which B-class ephrins and Eph receptors function to regulate CC midline growth and how these molecules interact with important guideposts during development. We show that multiple Eph receptors (B1, B2, B3, and A4) and B-class ephrins (B1, B2, and B3) are present and function in developing forebrain callosal fibers based on both spatial and temporal expression patterns and analysis of gene-targeted knock-out mice. Defects are most pronounced in the combination double knock-out mice, suggesting that compensatory mechanisms exist for several of these family members. Furthermore, these CC defects range from mild hypoplasia to complete agenesis and Probst's bundle formation. Further analysis revealed that Probst's bundle formation may reflect aberrant glial formations and/or altered sensitivity of CC axons to other guidance cues. Our results support a significant role for ephrins and Eph receptors in CC development and may provide insight to possible mechanisms involved in axon midline crossing and human disorder.

Regional differences in NREM sleep slow‐wave activity in mice with congenital callosal dysgenesis

Topographic differences in the sleep EEG have been repeatedly found in humans and rodents. A frontal predominance of EEG slow-wave activity (0.75-4 Hz; delta band) during non-rapid eye movement (NREM) sleep is particularly evident under conditions of increased sleep propensity. Local aspects of neuronal connectivity in the neocortex that are modified by specific neuronal stimulation may underlie these differences. To investigate the role of altered neuronal connectivity on anterior-posterior EEG topography, sleep was recorded in mice with congenital dysgenesis of the corpus callosum (B1 strain) during baseline and after 6 h sleep deprivation (SD). In these mice neuronal connections within a hemisphere are increased due to the longitudinal Probst bundle, a structure of re-routed callosal fibers. After SD the frequencies above 1.5 Hz within the delta band in NREM sleep were reduced in B1 mice compared with control C57BL/6 mice, a strain that has a normal corpus callosum, while power in the lowest frequency band (0.75-1.0 Hz) was enhanced in B1 mice. The differences between the strains subsided in the course of recovery. The redistribution of EEG power within the delta band in the frontal region in mice with a well developed Probst bundle, suggests a role of intracortical connectivity in local sleep regulation.

Axonal pathfinding mechanisms at the cortical midline and in the development of the corpus callosum.

The corpus callosum is a large fiber tract that connects neurons in the right and left cerebral hemispheres. Agenesis of the corpus callosum (ACC) is associated with a large number of human syndromes but little is known about why ACC occurs. In most cases of ACC, callosal axons are able to grow toward the midline but are unable to cross it, continuing to grow into large swirls of axons known as Probst bundles. This phenotype suggests that in some cases ACC may be due to defects in axonal guidance at the midline. General guidance mechanisms that influence the development of axons include chemoattraction and chemorepulsion, presented by either membrane-bound or diffusible molecules. These molecules are not only expressed by the final target but by intermediate targets along the pathway, and by pioneering axons that act as guides for later arriving axons. Midline glial populations are important intermediate targets for commissural axons in the spinal cord and brain, including the corpus callosum. The role of midline glial populations and pioneering axons in the formation of the corpus callosum are discussed. Finally the differential guidance of the ipsilaterally projecting perforating pathway and the contralaterally projecting corpus callosum is addressed. Development of the corpus callosum involves the coordination of a number of different guidance mechanisms and the probable involvement of a large number of molecules.