Tarik F. Haydar

Mammalian neocortical development follows a precise spatiotemporal sequence to generate the organized structure responsible for higher-order cognition and behavior. Increasing evidence suggests that diversification of neural stem and progenitor cells during prenatal development is a key step in the emergence of the intricate circuitry and functional architecture of the cerebral cortex. This review discusses novel findings with an emphasis on mechanisms and consequences of cell lineage variation during normal and altered brain development, including focus on neurodevelopmental disorders such as autism spectrum disorders and Down syndrome.

Cognitive, social behavior, speech, and motor skills are known challenges for people with trisomy 21/Down syndrome (DS), but the precise mechanisms that lead to these impactful changes have not yet been described. Data from human and mouse model fetal brains indicate that alterations in prenatal neurogenesis might account for the neurological phenotypes that manifest after birth. Here, we evaluated key features of cortical neurogenesis in the humanized mouse model of DS (TcMAC21 of undetermined sex) to test whether and how the presence of the human HSA21q transchromosome impacts cortical development. Brain growth measurements throughout the second half of gestation and at several periods of postnatal development show overall that the TcMAC21 brain phenotype is less severe than in other DS mouse models that have less genetic similarity to humans with DS. However, despite the lack of gross changes in brain growth, we uncovered a significant temporally limited neurogenesis defect at midgestation that correlates with long-lasting effects on neuronal dispersion and neuronal function in the neocortex. Using Cre/Lox-mediated genetic fate mapping, we discovered a transient reduction in neocortical basal intermediate progenitor cells (bIPCs) and that bIPC neuronal progeny are underrepresented in the superficial layers of the neocortex. This change in neuronal production is associated with cortical activity changes after birth. Altogether, our data isolate the cell types associated with a very specific temporal change in cortical formation that, due to the high levels of excitability of bIPC-derived neurons, creates lasting effects on network activity and circuit development in trisomic brains.

Zika, a mosquito-borne flavivirus, has been found in 87 countries and territories. Global outbreaks peaked in 2016. Prenatal infection of Zika virus was found to be associated with microcephaly, arthrogryposis, intracranial calcifications, fetal growth restriction, and fetal demise. The most severely affected children were diagnosed with congenital Zika syndrome, which impacts thousands worldwide. With no approved treatment or preventative measures for Zika, future viral outbreaks have the potential to cause epidemic levels of prenatal brain injury, as seen over the past 70 years. Therefore, there is a great need for a reliable and clinically translational experimental system that mimics the human condition of prenatal Zika infection. To this end, we developed a humanized, immunocompetent mouse model system of virally induced brain injury from prenatal Zika infection, which ranges from mild to severe. Here, we describe the extent to which this system mirrors the human phenotypic spectrum. Using our thorough preclinical system, we find that prenatal Zika infection of mice impacts survival rate, anthropometric measurements, tissue formation, and neurological outcomes, all of which are typical of prenatal infection. Single-cell RNA sequencing of the Zika-infected cerebral cortex reveals severely disrupted transcriptome profiles and suggests that these injuries are a result of a depletion of neural stem cells. Current and future applications include the identification of genetic or environmental modifiers of brain injury, molecular or mechanistic studies of pathogenesis, and preclinical evaluation of future therapies.

Zika, a mosquito-borne flavivirus, has been found in 87 countries and territories. Global outbreaks peaked in 2016. Prenatal infection of Zika virus was found to be associated with microcephaly, arthrogryposis, intracranial calcifications, fetal growth restriction, and fetal demise. The most severely affected children were diagnosed with congenital Zika syndrome, which impacts thousands worldwide. With no approved treatment or preventative measures for Zika, future viral outbreaks have the potential to cause epidemic levels of prenatal brain injury, as seen over the past 70 years. Therefore, there is a great need for a reliable and clinically translational experimental system that mimics the human condition of prenatal Zika infection. To this end, we developed a humanized, immunocompetent mouse model system of virally induced brain injury from prenatal Zika infection, which ranges from mild to severe. Here, we describe the extent to which this system mirrors the human phenotypic spectrum. Using our thorough preclinical system, we find that prenatal Zika infection of mice impacts survival rate, anthropometric measurements, tissue formation, and neurological outcomes, all of which are typical of prenatal infection. Single-cell RNA sequencing of the Zika-infected cerebral cortex reveals severely disrupted transcriptome profiles and suggests that these injuries are a result of a depletion of neural stem cells. Current and future applications include the identification of genetic or environmental modifiers of brain injury, molecular or mechanistic studies of pathogenesis, and preclinical evaluation of future therapies.

Background: Prenatally transmitted viruses can cause severe damage to the developing brain. There is unexplained variability in prenatal brain injury and postnatal neurodevelopmental outcomes, suggesting disease modifiers. Of note, prenatal Zika infection can cause a spectrum of neurodevelopmental disorders, including congenital Zika syndrome. Currently, there is no preventative treatment or cure. The Prenatal Infection and Neurodevelopmental Genetics (PING) Consortium aims to identify modulators of brain injury and adverse neurodevelopmental outcomes for Zika and other prenatal viral infections. Methods: The Consortium pools information from eight multi-site studies conducted at 23 research centers in six countries to build a growing clinical and genomic repository, which is being mined for modifiers of virally induced brain injury. Partners include Children’s National Hospital (USA), Instituto Nacional de Salud (Colombia), the Natural History of Zika Virus Infection in Gestation program (Brazil), Zika Instituto Fernandes Figueira (Brazil), the Centers for Disease Control and Prevention, and the National Institutes of Health. Results: We have enrolled 4102 mothers and 3877 infants with 3063 biological samples and clinical data covering over 80 phenotypic fields and 5000 variables. Thus far, we have performed whole exome sequencing on 1226 participants. Conclusion: Here, we present the Consortium’s formation and overarching study design. Impact: The PING Consortium brings together investigators and institutions to determine the causes of virally induced brain injury and neurological deficits. The clinical and genomic repository, with data from over 8000 patients, will serve as a foundation for a variety of basic and clinical studies.

Summary: Sample multiplexing is a common approach to reduce experimental cost and technical batch effect. Here, we present a protocol that for the first time allows the pooling of single nuclei from multiple biological samples prior to performing simultaneous single nuclei RNA-seq and ATAC-seq, which we term Mu ltiplexed Mu ltiome (MuMu). We describe steps for assembling the custom Tn5 transposome, performing the transposition reaction, nuclei pooling, sequencing library preparation, and sequencing data pre-processing. This protocol will greatly reduce the cost of sn-Multiome. Graphical

Background: Prenatally transmitted viruses can cause severe damage to the developing brain. There is unexplained variability in prenatal brain injury and postnatal neurodevelopmental outcomes, suggesting disease modifiers. Discordant outcomes among dizygotic twins could be explained by genetic susceptibly or protection. Among several well-recognized threats to the developing brain, Zika is a mosquito-borne, positive-stranded RNA virus that was originally isolated in Uganda and spread to cause epidemics in Africa, Asia, and the Americas. In the Americas, the virus caused congenital Zika syndrome and a multitude of neurodevelopmental disorders. As of now, there is no preventative treatment or cure for the adverse outcomes caused by prenatal Zika infection. The Prenatal Infection and Neurodevelopmental Genetics (PING) Consortium was initiated in 2016 to identify factors modulating prenatal brain injury and postnatal neurodevelopmental outcomes for Zika and other prenatal viral infections. Methods: The Consortium has pooled information from eight multi-site studies conducted at 23 research centers in six countries to build a growing clinical and genomic data repository. This repository is being mined to search for modifiers of virally induced brain injury and developmental outcomes. Multilateral partnerships include commitments with Children’s National Hospital (USA), Instituto Nacional de Salud (Colombia), the Natural History of Zika Virus Infection in Gestation program (Brazil), and Zika Instituto Fernandes Figueira (Brazil), in addition to the Centers for Disease Control and Prevention and the National Institutes of Health. Discussion: Our goal in bringing together these sets of patient data was to test the hypothesis that personal and populational genetic differences affect the severity of brain injury after a prenatal viral infection and modify neurodevelopmental outcomes. We have enrolled 4,102 mothers and 3,877 infants with 3,063 biological samples and clinical data covering over 80 phenotypic fields and 5,000 variables. There were several notable challenges in bringing together cohorts enrolled in different studies, including variability in the timepoints evaluated and the collected clinical data and biospecimens. Thus far, we have performed whole exome sequencing on 1,226 participants. Here, we present the Consortium’s formation and the overarching study design. We began our investigation with prenatal Zika infection with the goal of applying this knowledge to other prenatal infections and exposures that can affect brain development.

The intellectual disability (ID) in Down syndrome (DS) is thought to result from a variety of developmental deficits such as alterations in neural progenitor division, neurogenesis, gliogenesis, cortical architecture, and reduced cortical volume. However, the molecular processes underlying these neurodevelopmental changes are still elusive, preventing an understanding of the mechanistic basis of ID in DS. In this study, we used a pair of isogenic (trisomic and euploid) induced pluripotent stem cell (iPSC) lines to generate cortical spheroids (CS) that model the impact of trisomy 21 on brain development. Cortical spheroids contain neurons, astrocytes, and oligodendrocytes and they are widely used to approximate early neurodevelopment. Using single cell RNA sequencing (scRNA-seq), we uncovered cell type-specific transcriptomic changes in the trisomic CS. In particular, we found that excitatory neuron populations were most affected and that a specific population of cells with a transcriptomic profile resembling layer IV cortical neurons displayed the most profound divergence in developmental trajectory between trisomic and euploid genotypes. We also identified candidate genes potentially driving the developmental asynchrony between trisomic and euploid excitatory neurons. Direct comparison between the current isogenic CS scRNA-seq data and previously published datasets revealed several recurring differentially expressed genes between DS and control samples. Altogether, our study highlights the power and importance of cell type-specific analyses within a defined genetic background, coupled with broader examination of mixed samples, to comprehensively evaluate cellular phenotypes in the context of DS.

Great strides have been made over the past 30 years in understanding the neurodevelopmental changes underlying the intellectual disability (ID) in Down syndrome (DS). Detailed studies of human tissue coupled with findings from rodent and induced pluripotent stem cells (iPSCs) model systems have uncovered the changes in neurogenesis, synaptic connectivity, and myelination that drive the anatomical and physiological changes resulting in the disability. However, there remain significant conflicting data between human studies and the models. To fully understand the development of ID in DS, these inconsistencies need to be reconciled. Here, we review the well documented neurodevelopmental phenotypes found in individuals with DS and examine the degree to which widely used models recapitulate these phenotypes. Resolving these areas of discord will further research on the molecular underpinnings and identify potential treatments to improve the independence and quality of life of people with DS.

The intellectual disability (ID) in Down syndrome (DS) is thought to result from a variety of developmental deficits such as alterations in neural progenitor division, neurogenesis, gliogenesis, cortical architecture, and reduced cortical volume. However, the molecular processes underlying these neurodevelopmental changes are still elusive, preventing an understanding of the mechanistic basis of ID in DS. In this study, we used a pair of isogenic (trisomic and euploid) induced pluripotent stem cell (iPSC) lines to generate cortical spheroids (CS) that model the impact of trisomy 21 on brain development. CS contain neurons, astrocytes, and oligodendrocytes and they are widely used to approximate early neurodevelopment. Using single cell RNA sequencing (scRNA-seq), we uncovered cell type-specific transcriptomic changes in the trisomic CS. In particular, we found that excitatory neuron populations were most affected and that a specific population of cells with a transcriptomic profile resembling layer IV cortical neurons displayed the most profound divergence in developmental trajectory between trisomic and euploid genotypes. We also identified candidate genes potentially driving the developmental asynchrony between trisomic and euploid excitatory neurons. Direct comparison between the current isogenic CS scRNA-seq data and previously published datasets revealed several recurring differentially expressed genes between DS and control samples. Altogether, our study highlights the power and importance of cell type-specific analyses within a defined genetic background, coupled with broader examination of mixed samples, to comprehensively evaluate cellular phenotypes in the context of DS.