Rostral Diencephalon Research Articles

A high-resolution interactive atlas of the human brainstem using magnetic resonance imaging

Conventional atlases of the human brainstem are limited by the inflexible, sparsely-sampled, two-dimensional nature of histology, or the low spatial resolution of conventional magnetic resonance imaging (MRI). Postmortem high-resolution MRI circumvents the challenges associated with both modalities. A single human brainstem specimen extending from the rostral diencephalon through the caudal medulla was prepared for imaging after the brain was removed from a 65-year-old male within 24 h of death. The specimen was formalin-fixed for two weeks, then rehydrated and placed in a custom-made MRI compatible tube and immersed in liquid fluorocarbon. MRI was performed in a 7-Tesla scanner with 120 unique diffusion directions. Acquisition time for anatomic and diffusion images were 14 h and 208 h, respectively. Segmentation was performed manually. Deterministic fiber tractography was done using strategically chosen regions of interest and avoidance, with manual editing using expert knowledge of human neuroanatomy. Anatomic and diffusion images were rendered with isotropic resolutions of 50 μm and 200 μm, respectively. Ninety different structures were segmented and labeled, and 11 different fiber bundles were rendered with tractography. The complete atlas is available online for interactive use at https://www.civmvoxport.vm.duke.edu/voxbase/login.php?return_url=%2Fvoxbase%2F. This atlas presents multiple contrasting datasets and selected tract reconstruction with unprecedented resolution for MR imaging of the human brainstem. There are immediate applications in neuroanatomical education, with the potential to serve future applications for neuroanatomical research and enhanced neurosurgical planning through “safe” zones of entry into the human brainstem.

A Highly Conserved Shh Enhancer Coordinates Hypothalamic and Craniofacial Development.

Enhancers that are conserved deep in evolutionary time regulate characteristics held in common across taxonomic classes. Here, deletion of the highly conserved Shh enhancer SBE2 (Shh brain enhancer 2) in mouse markedly reduced Shh expression within the embryonic brain specifically in the rostral diencephalon; however, no abnormal anatomical phenotype was observed. Secondary enhancer activity was subsequently identified which likely mediates low levels of expression. In contrast, when crossing the SBE2 deletion with the Shh null allele, brain and craniofacial development were disrupted; thus, linking SBE2 regulated Shh expression to multiple defects and further enabling the study of the effects of differing levels of Shh on embryogenesis. Development of the hypothalamus, derived from the rostral diencephalon, was disrupted along both the anterior-posterior (AP) and the dorsal-ventral (DV) axes. Expression of DV patterning genes and subsequent neuronal population induction were particularly sensitive to Shh expression levels, demonstrating a novel morphogenic context for Shh. The role of SBE2, which is highlighted by DV gene expression, is to step-up expression of Shh above the minimal activity of the second enhancer, ensuring the necessary levels of Shh in a regional-specific manner. We also show that low Shh levels in the diencephalon disrupted neighbouring craniofacial development, including mediolateral patterning of the bones along the cranial floor and viscerocranium. Thus, SBE2 contributes to hypothalamic morphogenesis and ensures there is coordination with the formation of the adjacent midline cranial bones that subsequently protect the neural tissue.

A High-Resolution Interactive Atlas of the Human Brainstem Using Magnetic Resonance Histology

Abstract INTRODUCTION Traditional atlases of the human brainstem are limited by the inflexible, sparsely-sampled, two-dimensional nature of histology or the low spatial resolution of magnetic resonance imaging (MRI). Magnetic resonance histology (MRH) uses postmortem high-resolution MRI to circumvent the challenges associated with both modalities. METHODS A human brainstem specimen extending from the rostral diencephalon through the caudal medulla was removed from a 65-year-old male within 24 hours of death. The specimen was formalin-fixed for two weeks, then rehydrated and placed in a custom-made MRI compatible tube and immersed in buffered liquid fluorocarbon. MRI was performed in a 7-Tesla machine with 120 unique diffusion directions. Acquisition time for anatomic and diffusion images were 14 hours and 208 hours, respectively. Segmentation was performed manually. Deterministic fiber tractography was done using strategically chosen regions of interest and avoidance, with manual editing using expert knowledge of human neuroanatomy. RESULTS Anatomic and diffusion images were rendered with isotropic resolutions of 50 μm and 200 μm, respectively. Spatial resolution was high enough to visualize individual fasciculi of the descending corticospinal tracts intercalated between the transverse pontocerebellar fibers. Ninety different structures were segmented and 11 different fiber bundles were rendered with tractography. Angular resolution was high enough to visualize crossing fibers, such as those of the superior cerebellar peduncle. Both gray and white matter can be visualized in 3D simultaneously, such as the subthalamic nuclei and corticospinal tracts, as may be used in deep brain stimulation. CONCLUSION We used MRH to enable unprecedented resolution in digital imaging of the human brainstem and adjacent diencephalic structures, and we then performed comprehensive segmentation and tractography to render an interactive, three-dimensional atlas of both gray and white matter. This atlas has immediate applications in neuroanatomical study and education, with the potential for future neurosurgical applications in enhancing neurosurgical planning through “safe” zones of entry into the human brainstem. We are currently building the computer infrastructure to make this atlas publicly-available.

Ultrasonographic examination of the normal caprine neonatal brain.

Ultrasonography is a safe, rapid, and non-invasive diagnostic tool that has been previously used for imaging infants and canine neonatal brains. The purpose of the present study was to describe the ultrasonographic appearance of the brain in clinically normal caprine neonates. Ultrasonographic examination was done on 12 day-old goat kids, transverse and sagittal transcranial scans were obtained through the frontal bone. Three image planes were recorded through transverse scans including plane I (level of the caudate nucleus), plane II (level of the rostral diencephalon) and plane III (level of the caudal diencephalon). Parallel post mortem examinations were done for two kids that died a day following examination due to accidental trauma by the dam. Reliable and repeatable ultrasonographic images of the goat kid’s brain were described based on the gross post mortem findings. The head of the caudate nucleus was taken as an anatomical landmark in the plane I where it appeared as a curved hyperechoic structure. In plane II, the longitudinal fissure with its characteristic umbrella-like structure was taken as a landmark, while in plane III, the laterally located hyperechoic hippocampus was taken as a landmark. Normal ultrasonographic examination of the caprine neonatal brain represented the basis for diagnosing congenital brain lesions as well as intracranial hemorrhage.

Cytomegalovirus Infection: The Neurodevelopmental Peptide Signatures.

HCMV infection may cause neurodevelopmental disorders, including intellectual disability, hearing loss, cortical malformations, and calcifications. Theorizing about the still unknown molecular basis of HCMV-related diseases, this study analyzes the peptide sharing between HCMV, strains AD169 and Merlin, and human proteins, searching for shared sequences that might lead to crossreactive autoimmune injuries in the brain during immune responses following HCMV infection. HCMV proteins were analyzed for peptides shared with the human proteome using the Pir Peptide Match resource. Numerous HCMV peptides (ranging from 9 to 13 mer in length) are disseminated through hundreds of human proteins. The peptide sharing mostly involves crucial neurodevelopmental antigens such as PITX3, implicated in the differentiation of meso-diencephalic dopaminergic neurons; SIX3, which controls proper anterioposterior patterning of the diencephalon and formation of the rostral diencephalon during forebrain development; and ZIC2, which plays a fundamental role in the early stage of organogenesis of the central nervous system. This study describes a HCMV vs human peptide overlap that may represent a crossreactive platform linking the pathologic sequelae of HCMV infection to the immune anti-HCMV response. The data could inform development of effective and safe immune therapeutic/preventive approaches against HCMV infections.

Specification of embryonic stem cell-derived tissues into eye fields by Wnt signaling using rostral diencephalic tissue-inducing culture

The eyes are subdivided from the rostral diencephalon in early development. How the neuroectoderm regulates this subdivision, however, is largely unknown. Taking advantage of embryonic stem cell (ESC) culture using a Rax reporter line to monitor rostral diencephalon formation, we found that ESC-derived tissues at day 7 grown in Glasgow Minimum Expression Media (GMEM) containing knockout serum replacement (KSR) exhibited higher levels of expression of axin2, a Wnt target gene, than those grown in chemically defined medium (CDM). Surprisingly, Wnt agonist facilitated eye field-like tissue specification in CDM. In contrast, the addition of Wnt antagonist diminished eye field tissue formation in GMEM+KSR. Furthermore, the morphological formation of the eye tissue anlage, including the optic vesicle, was accompanied by Wnt signaling activation. Additionally, using CDM culture, we developed an efficient method for generating Rax+/Chx10+ retinal progenitors, which could become fully stratified retina. Here we provide a new avenue for exploring the mechanisms of eye field specification in vitro.

Six3 dosage mediates the pathogenesis of holoprosencephaly.

Holoprosencephaly (HPE) is defined as the incomplete separation of the two cerebral hemispheres. The pathology of HPE is variable and, based on the severity of the defect, HPE is divided into alobar, semilobar, and lobar. Using a novel hypomorphic Six3 allele, we demonstrate in mice that variability in Six3 dosage results in different HPE phenotypes. Furthermore, we show that whereas the semilobar phenotype results from severe downregulation of Shh expression in the rostral diencephalon ventral midline, the alobar phenotype is caused by downregulation of Foxg1 expression in the anterior neural ectoderm. Consistent with these results, in vivo activation of the Shh signaling pathway rescued the semilobar phenotype but not the alobar phenotype. Our findings show that variations in Six3 dosage result in different forms of HPE.

Mice repeatedly exposed to Group-A β-Haemolytic Streptococcus show perseverative behaviors, impaired sensorimotor gating, and immune activation in rostral diencephalon.

Repeated exposure to Group-A β-Haemolytic Streptococcus (GAS) may constitute a vulnerability factor in the onset and course of pediatric motor disturbances. GAS infections/colonization can stimulate the production of antibodies, which may cross the blood brain barrier, target selected brain areas (e.g. basal ganglia), and exacerbate motor alterations. Here, we exposed developing SJL male mice to four injections with a GAS homogenate and evaluated the following domains: motor coordination; general locomotion; repetitive behaviors; perseverative responses; and sensorimotor gating (pre-pulse inhibition, PPI). To demonstrate that behavioral changes were associated with immune-mediated brain alterations, we analyzed, in selected brain areas, the presence of infiltrates and microglial activation (immunohistochemistry), monoamines (HPLC), and brain metabolites (in vivo Magnetic Resonance Spectroscopy). GAS-exposed mice showed increased repetitive and perseverative behaviors, impaired PPI, and reduced concentrations of serotonin in prefrontal cortex, a brain area linked to the behavioral domains investigated, wherein they also showed remarkable elevations in lactate. Active inflammatory processes were substantiated by the observation of infiltrates and microglial activation in the white matter of the anterior diencephalon. These data support the hypothesis that repeated GAS exposure may elicit inflammatory responses in brain areas involved in motor control and perseverative behavior, and result in phenotypic abnormalities.

The COUP-TFII/Neuropilin-2 is a molecular switch steering diencephalon-derived GABAergic neurons in the developing mouse brain

The preoptic area (POa) of the rostral diencephalon supplies the neocortex and the amygdala with GABAergic neurons in the developing mouse brain. However, the molecular mechanisms that determine the pathway and destinations of POa-derived neurons have not yet been identified. Here we show that Chicken ovalbumin upstream promoter transcription factor II (COUP-TFII)-induced expression of Neuropilin-2 (Nrp2) and its down-regulation control the destination of POa-derived GABAergic neurons. Initially, a majority of the POa-derived migrating neurons express COUP-TFII and form a caudal migratory stream toward the caudal subpallium. When a subpopulation of cells steers toward the neocortex, they exhibit decreased expression of COUP-TFII and Nrp2. The present findings show that suppression of COUP-TFII/Nrp2 changed the destination of the cells into the neocortex, whereas overexpression of COUP-TFII/Nrp2 caused cells to end up in the medial part of the amygdala. Taken together, these results reveal that COUP-TFII/Nrp2 is a molecular switch determining the pathway and destination of migrating GABAergic neurons born in the POa.

Genomic code for Sox2 binding uncovers its regulatory role in Six3 activation in the forebrain

The SRY-related HMG box transcription factor Sox2 plays critical roles throughout embryogenesis. Haploinsufficiency for SOX2 results in human developmental defects including anophthalmia, microphthalmia and septo-optic dysplasia, a congenital forebrain defect. To understand how Sox2 plays a role in neurogenesis, we combined genomic and in vivo transgenic approaches to characterize genomic regions occupied by Sox2 in the developing forebrain. Six3, a homeobox gene associated with holoprosencephaly, a forebrain midline defect, was identified as a Sox2 transcriptional target. This study shows that Sox2 directly regulates a previously unidentified long-range forebrain enhancer to activate Six3 expression in the rostral diencephalon. Further biochemical and genetic evidences indicated a direct regulatory link between Sox2 and Six3 during forebrain development, providing a better understanding of a common molecular mechanism underlying these forebrain defects.

Development of tyrosine hydroxylase‐immunoreactive cell populations and fiber pathways in the brain of the dogfish Scyliorhinus canicula: New perspectives on the evolution of the vertebrate catecholaminergic system

Developmental studies of the central catecholaminergic (CA) system are essential for understanding its evolution. To obtain knowledge about the CA system in chondrichthyans, an ancient gnathostome group, we used immunohistochemical techniques for detecting tyrosine hydroxylase (TH), the initial rate-limiting enzyme of the CA synthesis, to study: 1) the neuromery of developing TH-immunoreactive (ir) neuronal populations, 2) the development of TH-ir innervation, and 3) the organization of TH-ir cells and fibers in the brain of postembryonic stages of the shark Scyliorhinus canicula. The first TH-ir cells appeared in the hypothalamus and rostral diencephalon (suprachiasmatic, posterior recess and posterior tubercle nuclei at embryonic stage 26, and dorsomedial hypothalamus at stage 28); then in more caudal basal regions of the diencephalon and rostral mesencephalon (substantia nigra/ventral tegmental area); and later on in the anterior (locus coeruleus/nucleus subcoeruleus) and posterior (vagal lobe and reticular formation) rhombencephalon. The appearance of TH-ir cells in the telencephalon (pallium) was rather late (stage [S]31) with respect to the other TH-ir prosencephalic populations. The first TH-ir fibers arose from cells of the posterior tubercle (S30) and formed recognizable ascending (toward dorsal and rostral territories) and descending pathways at S31. When the second half of embryonic development started (S32), TH-ir fibers innervated most brain areas, and nearly all TH-ir cell groups of the postembryonic brain were already established. This study provides key information about the evolution of the developmental patterns of central CA systems in fishes and thus may help in understanding how the vertebrate CA systems have evolved.

A gene trap knockout of the Tiam-1 protein results in malformation of the early embryonic brain.

Tiam-1 has been implicated in the development of the central nervous system. However, the in vivo function of Tiam-1 has not been fully determined in the developing mouse brain. In this study, we generated Tiam-1 knockout mice using a Tiam-1 gene-trapped embryonic stem cell line. Insertion of a gene trap vector into a genomic site downstream of exon 5 resulted in a mutant allele encoding a truncated protein fused with the β-geo LacZ gene. Primary mouse embryonic fibroblasts lacking Tiam-1 revealed a significant decrease in Rac activity and cell proliferation. In addition, whole-mount embryonic LacZ expression analysis demonstrated that Tiam-1 is specifically expressed in regions of the developing brain, such as the caudal telencephalon and rostral diencephalon. More importantly, mouse embryos deficient in Tiam-1 gene expression displayed a severe defect in embryonic brain development, including neural tube closure defects or a dramatic decrease in brain size. These findings suggest that embryonic Tiam-1 expression plays a critical role during early brain development in mice.

LRP2 Is an Auxiliary SHH Receptor Required to Condition the Forebrain Ventral Midline for Inductive Signals

Sonic hedgehog (SHH) is a regulator of forebrain development that acts through its receptor, patched 1. However, little is known about cellular mechanisms at neurulation, whereby SHH from the prechordal plate governs specification of the rostral diencephalon ventral midline (RDVM), a major forebrain organizer. We identified LRP2, a member of the LDL receptor gene family, as a component of the SHH signaling machinery in the RDVM. LRP2 acts as an apical SHH-binding protein that sequesters SHH in its target field and controls internalization and cellular trafficking of SHH/patched 1 complexes. Lack of LRP2 in mice and in cephalic explants results in failure to respond to SHH, despite functional expression of patched 1 and smoothened, whereas overexpression of LRP2 variants in cells increases SHH signaling capacity. Our data identify a critical role for LRP2 in SHH signaling and reveal the molecular mechanism underlying forebrain anomalies in mice and patients with Lrp2 defects.

Autotaxin controls caudal diencephalon‐mesencephalon development in the chick

The diencephalon is the embryonic anlagen of the higher integration centers of the brain. Recent studies have elucidated how the cells in the rostral diencephalon acquire their regional identities. However, the understanding of the mechanisms under which the caudal diencephalon is formed is still limited. Here we focus on the role of Autotaxin (ATX), a lysophospholipid-generating exoenzyme, whose mRNA is detected in the caudal diencephalon. RNA interference against ATX altered the expression pattern of Pax6-regualted genes, Tcf4, Lim1, and En1, implying that ATX is required for the maintenance of the regional identity of the caudal diencephalon and the diencephalon-mesencephalon boundary (DMB). Furthermore, ATX-RNAi inhibited neuroepithelial cell proliferation on both sides of the DMB. We propose a dual role of ATX in chick brain development, in which ATX not only contributes to the formation of caudal diencephalon as a short-range signal, but also regulates the growth of mesencephalon as a long-range signal.

Formation and patterning of the forebrain and olfactory system by zinc‐finger genes Fezf1 and Fezf2

The zinc finger genes Fezf1 (Fez) and Fezf2 (Fez-like, Fezl, Zfp312) were initially identified as anterior neuroectoderm-specific genes in Xenopus and zebrafish. They encode transcriptional regulators containing an Engrailed homology 1 (Eh1) repressor motif, which is known to interact with Groucho/TLE (Transducin-Like Enhancer of Split)-type transcriptional co-repressors. Both Fezf1 and Fezf2 are expressed in the prospective forebrain region during early embryogenesis, and they subsequently show both overlapping and distinct expression domains in the olfactory epithelium and forebrain. Loss-of-function studies in mouse and zebrafish revealed roles for Fezf1 and Fezf2 in the development of the olfactory system and forebrain. In mice, Fezf1, expressed in olfactory sensory neurons, is required for the axonal projection of olfactory sensory neurons, and controls the layer formation of the olfactory bulb in a non-cell autonomous manner. Fezf2 is involved in the differentiation of subplate neurons and the formation of the fimbria and fornix. Fezf2 is also essential for specification of the subcerebral projection neurons in the neocortex. Fezf1 and Fezf2 control the rostro-caudal patterning of the diencephalon by repressing the caudal diencephalon fate in the rostral diencephalon in mice and zebrafish. In zebrafish, fezf2 is also required for the development of monoaminergic (dopaminergic and serotonergic) neurons in the basal forebrain.

Genetic mapping of Foxb1‐cell lineage shows migration from caudal diencephalon to telencephalon and lateral hypothalamus

The hypothalamus is a brain region with vital functions, and alterations in its development can cause human disease. However, we still do not have a complete description of how this complex structure is put together during embryonic and early postnatal stages. Radially oriented, outside-in migration of cells is prevalent in the developing hypothalamus. In spite of this, cell contingents from outside the hypothalamus as well as tangential hypothalamic migrations also have an important role. Here we study migrations in the hypothalamic primordium by genetically labeling the Foxb1 diencephalic lineage. Foxb1 is a transcription factor gene expressed in the neuroepithelium of the developing neural tube with a rostral expression boundary between caudal and rostral diencephalon, and therefore appropriate for marking migrations from caudal levels into the hypothalamus. We have found a large, longitudinally oriented migration stream apparently originating in the thalamic region and following an axonal bundle to end in the anterior portion of the lateral hypothalamic area. Additionally, we have mapped a specific expansion of the neuroepithelium into the rostral diencephalon. The expanded neuroepithelium generates abundant neurons for the medial hypothalamus at the tuberal level. Finally, we have uncovered novel diencephalon-to-telencephalon migrations into septum, piriform cortex and amygdala.

Haploinsufficiency of Six3 Fails to Activate Sonic hedgehog Expression in the Ventral Forebrain and Causes Holoprosencephaly

Holoprosencephaly (HPE), the most common forebrain malformation, is characterized by an incomplete separation of the cerebral hemispheres. Mutations in the homeobox gene SIX3 account for 1.3% of all cases of human HPE. Using zebrafish-based assays, we have now determined that HPE-associated Six3 mutant proteins function as hypomorphs. Haploinsufficiency of Six3 caused by deletion of one allele of Six3 or by replacement of wild-type Six3 with HPE-associated Six3 mutant alleles was sufficient to recapitulate in mouse models most of the phenotypic features of human HPE. We demonstrate that Shh is a direct target of Six3 in the rostral diencephalon ventral midline (RDVM). Reduced amounts of functional Six3 protein fail to activate Shh expression in the mutant RDVM and ultimately lead to HPE. These results identify Six3 as a direct regulator of Shh expression and reveal a crossregulatory loop between Shh and Six3 in the ventral forebrain.

Characterization of a distinct subpopulation of striatal projection neurons expressing the Dlx genes in the basal ganglia through the activity of the I56ii enhancer

Regulation of region-specific neuronal differentiation and migration in the embryonic forebrain is a complex mechanism that involves a variety of transcription factors such as the Dlx genes. At least four cis-acting regulatory elements (CREs) are responsible for the Dlx transcriptional regulation in the subcortical telencephalon and the rostral diencephalon. These include I12b and URE2 in the Dlx1/2 bigene cluster, and, I56i and I56ii in the Dlx5/6 cluster. We previously reported that URE2, I12b, and I56i, mark different progenitor cell populations in the ganglionic eminences as well as different subtypes of adult cortical interneurons. Here, we carried out a detailed spatial and temporal analysis of the I56ii CRE activity in the developing telencephalon between E10.5 and E15.5, and compared its activity with the other three Dlx CREs using lacZ reporter genes in transgenic mice. We show that I56ii marks distinct group(s) of neurons located in the superficial mantle of the LGE and MGE between E11.5 and E13.5. The I56ii-positive cells are Dlx- and GABA-immunoreactive. However, unlike the other CREs, I56ii does not label interneuron progenitors in the basal ganglia, nor tangentially migrating cells to the cortex at E13.5. Instead, I56ii-positive cells mark a subpopulation(s) of post-mitotic projection neurons that tangentially migrate from the LGE to the deep mantle of the MGE and reside between the subventricular zone and the globus pallidus during midgestation. The majority of these neurons express the striatal markers Meis2 and Islet1. Moreover, both Meis2 and Islet1 activate transcription of a reporter gene containing the I56ii sequence in co-transfection assays, indicating that these transcriptional factors may be potential upstream modulators of the Dlx genes in vivo.

Six3-promoted holoprosencephaly is caused by the absence of Shh expression in the rostral diencephalon ventral midline

Six3-promoted holoprosencephaly is caused by the absence of Shh expression in the rostral diencephalon ventral midline

Six3inactivation causes progressive caudalization and aberrant patterning of the mammalian diencephalon

The homeobox gene Six3 represses Wnt1 transcription. It is also required in the anterior neural plate for the development of the mammalian rostral forebrain. We have now determined that at the 15- to 17-somite stage, the prospective diencephalon is the most-anterior structure in the Six3-null brain, and Wnt1 expression is anteriorly expanded. Consequently, the brain caudalizes, and at the 22- to 24-somite stage, the prospective thalamic territory is the most-anterior structure. At around E11.0, the pretectum replaces this structure. Analysis of Six3;Wnt1 double-null mice revealed that Six3-mediated repression of Wnt1 is necessary for the formation of the rostral diencephalon and that Six3 activity is required for the formation of the telencephalon. These results provide insight into the mechanisms that establish anteroposterior identity in the developing mammalian brain.