Cerebral organoids as tools to identify the developmental roots of autism.

Some autism spectrum disorders (ASD) likely arise as a result of abnormalities during early embryonic development of the brain. Studying human embryonic brain development directly is challenging, mainly due to ethical and practical constraints. However, the recent development of cerebral organoids provides a powerful tool for studying both normal human embryonic brain development and, potentially, the origins of neurodevelopmental disorders including ASD. Substantial evidence now indicates that cerebral organoids can mimic normal embryonic brain development and neural cells found in organoids closely resemble their in vivo counterparts. However, with prolonged culture, significant differences begin to arise. We suggest that cerebral organoids, in their current form, are most suitable to model earlier neurodevelopmental events and processes such as neurogenesis and cortical lamination. Processes implicated in ASDs which occur at later stages of development, such as synaptogenesis and neural circuit formation, may also be modeled using organoids. The accuracy of such models will benefit from continuous improvements to protocols for organoid differentiation.

Gliogenesis in the outer subventricular zone promotes enlargement and gyrification of the primate cerebrum.

The primate cerebrum is characterized by a large expansion of cortical surface area, the formation of convolutions, and extraordinarily voluminous subcortical white matter. It was recently proposed that this expansion is primarily driven by increased production of superficial neurons in the dramatically enlarged outer subventricular zone (oSVZ). Here, we examined the development of the parietal cerebrum in macaque monkey and found that, indeed, the oSVZ initially adds neurons to the superficial layers II and III, increasing their thickness. However, as the oSVZ grows in size, its output changes to production of astrocytes and oligodendrocytes, which in primates outnumber cerebral neurons by a factor of three. After the completion of neurogenesis around embryonic day (E) 90, when the cerebrum is still lissencephalic, the oSVZ enlarges and contains Pax6+/Hopx+ outer (basal) radial glial cells producing astrocytes and oligodendrocytes until after E125. Our data indicate that oSVZ gliogenesis, rather than neurogenesis, correlates with rapid enlargement of the cerebrum and development of convolutions, which occur concomitantly with the formation of cortical connections via the underlying white matter, in addition to neuronal growth, elaboration of dendrites, and amplification of neuropil in the cortex, which are primary factors in the formation of cerebral convolutions in primates.

Developmental profiles of SUMOylation pathway proteins in rat cerebrum and cerebellum.

Protein SUMOylation regulates multiple processes involved in the differentiation and maturation of cells and tissues during development. Despite this, relatively little is known about the spatial and temporal regulation of proteins that mediate SUMOylation and deSUMOylation in the CNS. Here we monitor the expression of key SUMO pathway proteins and levels of substrate protein SUMOylation in the forebrain and cerebellum of Wistar rats during development. Overall, the SUMOylation machinery is more highly-expressed at E18 and decreases thereafter, as previously described. All of the proteins investigated are less abundant in adult than in embryonic brain. Furthermore, we show for first time that the profiles differ between cerebellum and cerebrum, indicating differential regional regulation of some of the proteins analysed. These data provide further basic observation that may open a new perspective of research about the role of SUMOylation in the development of different brain regions.

Fetuses with Isolated Congenital Heart Defects Show Normal Cerebral and Extracerebral Fluid Volume Growth: A 3D Sonographic Study in the Second and Third Trimester.

OBJECTIVE: The aim of our study is to explore whether the cerebral growth is delayed in fetuses with congenital heart defects (CHD) in the second and early third trimester. METHODS: A prospective cohort study was conducted in 77 CHD cases, with 75 healthy controls. 3D cerebral volume acquisition was performed sequentially. The volumes of the fetal hemicerebrum and extracerebral fluid were compared by linear regression analysis, and the Sylvian fissure was measured. RESULTS: Between 19 and 32 weeks of gestation, 158 measurements in cases and 183 measurements in controls were performed (mean 2.2/subject). The volume growth of the hemicerebrum (R2 = 0.95 vs. 0.95; p = 0.9) and the extracerebral fluid (R2 = 0.84 vs. 0.82, p = 0.9) were similar. Fetuses with abnormal oxygen delivery to the brain have a slightly smaller brain at 20 weeks of gestation (p = 0.02), but this difference disappeared with advancing gestation. CHD cases demonstrated a slightly shallower Sylvian fissure (mean ratio 0.146 vs. 0.153; p = 0.004). CONCLUSIONS: Our study shows no differences in cerebral growth, studied in an unselected cohort, with successive cases of isolated CHD. Even in the severest CHD cases, cerebral size is similar in the early third trimester. The cause and meaning of a shallower Sylvian fissure is unclear; possibly, it is a marker for delayed cerebral maturation or it might be an expression of decreasing amount of extracerebral fluid.

Cerebral Diffusivity Changes in Fetuses with Chiari II Malformation.

OBJECTIVES: Chiari II malformation is a congenital abnormality characterized by a small posterior fossa with downward displacement of the hindbrain into the foramen magnum. Diffusion-weighted imaging (DWI) can be used to quantitatively characterize brain injury and physiological maturation. We aim to evaluate DWI parameters of the infra- and supratentorial brain in fetuses with Chiari II malformation. METHODS: MRI and US studies of 26 fetuses with Chiari II malformation and 15 controls were evaluated for the presence/severity of hydrocephalus, myelomeningocele level, and brain apparent diffusion coefficient (ADC) values. Cerebral flow impedance parameters (resistance and pulsatile indexes, resistance index [RI], and pulsatility index [PI] respectively) from concurrently preformed fetal US were also evaluated. RESULTS: Of the Chiari II group, 16 fetuses had hydrocephalus. There was increasing severity of lateral ventriculomegaly in the high compared to low spinal dysraphism subgroups. There were significant lower ADC values in the frontal (p = 0.01) and temporal lobes (p = 0.05) in Chiari II group compared with normal, regardless of the presence or severity of hydrocephalus. Fetuses with Chiari II showed significantly lower RI and PI values. CONCLUSIONS: Abnormal ADC values indicate supratentorial microstructural changes in fetuses with Chiari II malformation. Further investigation of the role of diffusion imaging metrics in evaluating abnormal brain development, parenchymal damage and efficacy of fetal surgery is needed.

Clinical outcomes following prenatal diagnosis of asymmetric ventriculomegaly, interhemispheric cyst, and callosal dysgenesis (AVID).

OBJECTIVES: When identified prenatally, the imaging triad of asymmetric ventriculomegaly, interhemispheric cyst, and dysgenesis of the corpus callosum (AVID) can indicate a more serious congenital brain anomaly. In this follow-up series of 15 fetuses, we present the neurodevelopmental outcomes of a single institution cohort of children diagnosed prenatally with AVID. METHODS: Our fetal ultrasound database was queried for cases of AVID between 2000 and 2016. All available fetal MR imaging studies were reviewed for the presence of (a) interhemispheric cysts or ventricular diverticula and (b) dysgenesis or agenesis of the corpus callosum. Clinical records were reviewed for perinatal management, postnatal surgical management, and neurodevelopmental outcomes. RESULTS: Fifteen prenatal cases of AVID were identified. Twelve were live-born and three pregnancies were terminated. Of the 12 patients, 11 underwent neurosurgical intervention. Of the eight patients surviving past infancy, seven of eight have moderate to severe neurodevelopmental delays or disabilities, encompassing both motor and language skills, and all have variable visual abnormalities. CONCLUSION: In our cohort of 15 prenatally diagnosed fetuses with AVID, eight survived past infancy and all have neurodevelopmental disabilities, including motor and language deficits, a wide range of visual defects, craniofacial abnormalities, and medical comorbidities.

Spatiotemporal Proteomic Profiling of Human Cerebral Development.

Mass spectrometry (MS) analysis of human post-mortem central nervous system (CNS) tissue and induced pluripotent stem cell (iPSC)-based directed differentiations offer complementary avenues to define protein signatures of neurodevelopment. Methodological improvements of formalin-fixed, paraffin-embedded (FFPE) protein isolation now enable widespread proteomic analysis of well-annotated archival tissue samples in the context of development and disease. Here, we utilize a shotgun label-free quantification (LFQ) MS method to profile magnetically enriched human cortical neurons and neural progenitor cells (NPCs) derived from iPSCs. We use these signatures to help define spatiotemporal protein dynamics of developing human FFPE cerebral regions. We show that the use of high resolution Q Exactive mass spectrometers now allow simultaneous quantification of >2700 proteins in a single LFQ experiment and provide sufficient coverage to define novel biomarkers and signatures of NPC maintenance and differentiation. Importantly, we show that this abbreviated strategy allows efficient recovery of novel cytoplasmic, membrane-specific and synaptic proteins that are shared between both in vivo and in vitro neuronal differentiation. This study highlights the discovery potential of non-comprehensive high-throughput proteomic profiling of unfractionated clinically well-annotated FFPE human tissue from a diverse array of development and diseased states.

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.

Astroglial-Mediated Remodeling of the Interhemispheric Midline Is Required for the Formation of the Corpus Callosum.

The corpus callosum is the major axon tract that connects and integrates neural activity between the two cerebral hemispheres. Although ∼1:4,000 children are born with developmental absence of the corpus callosum, the primary etiology of this condition remains unknown. Here, we demonstrate that midline crossing of callosal axons is dependent upon the prior remodeling and degradation of the intervening interhemispheric fissure. This remodeling event is initiated by astroglia on either side of the interhemispheric fissure, which intercalate with one another and degrade the intervening leptomeninges. Callosal axons then preferentially extend over these specialized astroglial cells to cross the midline. A key regulatory step in interhemispheric remodeling is the differentiation of these astroglia from radial glia, which is initiated by Fgf8 signaling to downstream Nfi transcription factors. Crucially, our findings from human neuroimaging studies reveal that developmental defects in interhemispheric remodeling are likely to be a primary etiology underlying human callosal agenesis.

Zika Virus Depletes Neural Progenitors in Human Cerebral Organoids through Activation of the Innate Immune Receptor TLR3.

Emerging evidence from the current outbreak of Zika virus (ZIKV) indicates a strong causal link between Zika and microcephaly. To investigate how ZIKV infection leads to microcephaly, we used human embryonic stem cell-derived cerebral organoids to recapitulate early stage, first trimester fetal brain development. Here we show that a prototype strain of ZIKV, MR766, efficiently infects organoids and causes a decrease in overall organoid size that correlates with the kinetics of viral copy number. The innate immune receptor Toll-like-Receptor 3 (TLR3) was upregulated after ZIKV infection of human organoids and mouse neurospheres and TLR3 inhibition reduced the phenotypic effects of ZIKV infection. Pathway analysis of gene expression changes during TLR3 activation highlighted 41 genes also related to neuronal development, suggesting a mechanistic connection to disrupted neurogenesis. Together, therefore, our findings identify a link between ZIKV-mediated TLR3 activation, perturbed cell fate, and a reduction in organoid volume reminiscent of microcephaly.

Bioeffects of Diagnostic Dynamic 3-Dimensional (4-Dimensional) Ultrasound on Ultrastructure of Cerebral Cells of Fetal Mice in Late Pregnancy.

The aim of this study was to study the bioeffects of diagnostic dynamic 3-dimensional ultrasound (4D) on ultrastructure of cerebral cells of fetal mice in late pregnancy. Thirty pregnant mice carrying 18th embryonic day fetuses were randomly allocated into 6 groups, namely, control group, sham-exposed group, 5 minute-exposed group, 10 minute-exposed group, 20 minute-exposed group, and 30 minute-exposed groups (5 in each group). In exposure groups, mice were put under the dynamic 3D ultrasound system's probe for 5 to 30 minutes. Mice in sham-exposed group did not receive ultrasound wave. At 24th hour after birth, 10 pups of each group were randomly selected (2 in each litter) and euthanized by decapitation, and the brains were immediately removed. Right parietal lobes were taken as specimen. The specimens were firstly fixed with glutaraldehyde and secondly with osmic acid, then sections were made and observed under the transmission electron microscope. There were no obvious abnormal ultrastructure changes in control group, sham-exposed group, and 5 minute-exposed group under transmission electron microscope. Ten minute-exposed group showed some enlarged mitochondria, broken crista, vacuolated endoplasmic resticulums, and a few apoptosis cells. More abnormal organelles and apoptosis cells were observed in 20 minute-exposed and 30 minute-exposed groups (P < 0.05). Dynamic 3D (4D) ultrasound exposure for more than 10 minutes may result in abnormal neuronal ultrastructure changes and apoptosis cells in fetal mouse cerebrum.

A Novel Monoclonal Antibody Against Neuroepithelial and Ependymal Cells and Characteristics of Its Positive Cells in Neurospheres.

There are still few useful cell membrane surface antigens suitable for identification and isolation of neural stem cells (NSCs). We generated a novel monoclonal antibody (mAb), designated as mAb against immature neural cell antigens (INCA mAb), which reacted with the areas around a lateral ventricle of a fetal cerebrum. INCA mAb specifically reacted with neuroepithelial cells in fetal cerebrums and ependymal cells in adult cerebrums. The recognition molecules were O-linked 40 and 42 kDa glycoproteins on the cell membrane surface (gp40 INCA and gp42 INCA). Based on expression pattern analysis of the recognition molecules in developing cerebrums, it was concluded that gp42 INCA was a stage-specific antigen expressed on undifferentiated neuroepithelial cells, while gp40 INCA was a cell lineage-specific antigen expressed at the stages of differentiation from neuroepithelial cells to ependymal cells. A flow cytometric analysis showed that fetal and young adult neurospheres were divided into INCA mAb(-) CD133 polyclonal antibody (pAb)(-), INCA mAb(+) CD133 pAb(-), and INCA mAb(+) CD133 pAb(+) cell populations based on the reactivity against INCA mAb and CD133 pAb. The proportion of cells having the neurosphere formation capability in the INCA mAb(+) CD133 pAb(+) cell population was significantly larger than that of undivided neurospheres. Neurospheres formed by clonal expansion of INCA mAb(+) CD133 pAb(+) cells gave rise to neurons and glial cells. INCA mAb will be a useful immunological probe in the study of NSCs.

NMR Based Cerebrum Metabonomic Analysis Reveals Simultaneous Interconnected Changes during Chick Embryo Incubation.

To find out if content changes of the major functional cerebrum metabolites are interconnected and formed a network during the brain development, we obtained high-resolution magic-angle-spinning (HR-MAS) 1H NMR spectra of cerebrum tissues of chick embryo aged from incubation day 10 to 20, and postnatal day 1, and analyzed the data with principal component analysis (PCA). Within the examined time window, 26 biological important molecules were identified and 12 of them changed their relative concentration significantly in a time-dependent manner. These metabolites are generally belonged to three categories, neurotransmitters, nutrition sources, and neuronal or glial markers. The relative concentration changes of the metabolites were interconnected among/between the categories, and, more interestingly, associated with the number and size of Nissl-positive neurons. These results provided valuable biochemical and neurochemical information to understand the development of the embryonic brain.

Sonographic evaluation of fetal cerebral structures correlated with histological aspects.

Prenatal development of the human brain from undifferentiated neuroepithelium, crosses numerous steps towards primordial organization and subsequent cytoarchitectural layering, ascending and progressive from the lower cortical layers to the superior ones. Our study represents a systematic, comparative assessment of imaging studies and the histological evaluation of the prenatal development of the human brain. We evaluated 232 cases using 3D ultrasound. Histological study was performed on 17 cases aged between 8 and 32 weeks pregnancy and compared with imaging results. For the ultrasound study, we chose five anatomical landmarks: the choroid plexus, thalamus, cerebellum, hippocampus and island (Sylvian fissure). The histological study was performed on dissected brain specimens preserved in formaldehyde and was followed by immunohistochemical determination in order to complete the picture of the morphological evolution of the structures evaluated. We analyzed the accuracy of the description of marker elements (choroid plexus, thalamus, cerebellum, hippocampus and Sylvian fissure) in three-dimensional ultrasound evaluation. This showed a good correlation with the morphological evaluation as well as with the dimensional descriptions from the literature. Histological and immunohistochemical assessment helped complete the picture of the central nervous system development. Highlighting fetal cerebral structures by three-dimensional ultrasound, together with morphological examination helped us create a dynamic array of the central nervous system development.

The use of modern ultrasound tridimensional techniques for the evaluation of fetal cerebral midline structures- a practical approach.

Fetal central nervous system midline structures represent an essential landmark for the confirmation of normality or for the identification of severe pathology. The ultrasound examination of the fetal brain using modern 3D techniques allows the creation of high sensitivity reconstructions. The facility of 3D volume acquisition permits the identification of corpus callosum, median septum, cavum septi pellucid and cerebellar vermis even in difficult cases. The examination should rely on both static (3D) and dynamic acquisition (4D). The use of a practical ultrasound protocol in clinical settings ensures the visualization of the midline cerebral structures in the vast majorities of fetuses. In selected cases MRI can be performed.

[Reactivity of microvessels in the cerebral hemispheres and the skeletal muscles in chicken during second half of embryogenesis].

At Leghorn hens in the second half of embryogenesis and in 4-day-old chicks are studied reaction volume flow velocity (VF) in the superficial layers of the cerebral hemispheres and in skeletal muscle (Lazer Doppler FIowmetry) after the local influence of norepinephrine and sodium nitroprusside. It is shown that the response to these substances begins to manifest itself in the hemisphere in the last quarter of embryogenesis and authentically expressed by the end of it and in the chickens. It is noted that the response to these substances skeletal muscle VF (according to the new and previously published data on gastrocnemius and pectoral muscle) is also clearly manifested by the end of embryogenesis.

Cerebral angiogenesis during development: who is conducting the orchestra?

Blood vessels provide the brain with the oxygen and the nutrients it requires to develop and function. Endothelial cells (ECs) are the principal cell type forming the vascular system and driving its development and remodeling. All vessels are lined by a single EC layer. Larger blood vessels are additionally enveloped by vascular smooth muscle cells (VSMCs) and pericytes, which increase their stability and regulate their perfusion and form the blood-brain barrier (BBB). The development of the vascular system occurs by two processes: (1) vasculogenesis, the de novo assembly of the first blood vessels, and (2) angiogenesis, the creation of new blood vessels from preexisting ones by sprouting from or by division of the original vessel. The walls of maturing vessels produce a basal lamina and recruit pericytes and vascular smooth muscle cells for structural support. Whereas the process of vasculogenesis seems to be genetically programmed, angiogenesis is induced mainly by hypoxia in development and disease. Both processes and the subsequent vessel maturation are further orchestrated by a complex interplay of inhibiting and stimulating growth factors and their respective receptors, many of which are hypoxia-inducible. This chapter intends to give an overview about the array of factors directing the development and maintenance of the brain vasculature and their interdependent actions.

Development of the fetal cerebral cortex in the second trimester: assessment with 7T postmortem MR imaging.

BACKGROUND AND PURPOSE: Few investigators have analyzed the fetal cerebral cortex with MR imaging of high magnetic strength. Our purpose was to document the sulcal development and obtain quantitative measurements of the fetal brain in the second trimester. MATERIALS AND METHODS: The brains of 69 fetal specimens, with GA 12-22 weeks, were first scanned on a 7T MR imaging scanner. Then the sequential development of the different fissures and sulci was analyzed, and quantitative measurements of the cerebral cortex were obtained. RESULTS: A new chronology of sulcal development during 12-22 weeks GA was summarized. Before 12 weeks, few sulci were present; by 16 weeks, many sulci were present. The 16th week could be considered the most intensive time point for sulcal emergence. Most sulci, except for the postcentral sulcus and intraparietal sulcus, were present by 22 weeks GA. Measurements of the fetal brains, each with different growth rates, linearly increased with GA, but no sexual dimorphisms or cerebral asymmetries were detected. CONCLUSIONS: The second trimester is the most important phase, during which most sulci are present and can be clearly shown on 7T postmortem MR imaging. It is apparent that the specific time during which neuropathologic features of sulci appear, previously thought to be well understood, should be redefined. Quantitative data provide assistance in the precise understanding of the immature brain. The present results are valuable in anatomic education, research, and assessment of normal brain development in the uterus.