xcore: an R package for inference of gene expression regulators.

BACKGROUND: Elucidating the Transcription Factors (TFs) that drive the gene expression changes in a given experiment is a common question asked by researchers. The existing methods rely on the predicted Transcription Factor Binding Site (TFBS) to model the changes in the motif activity. Such methods only work for TFs that have a motif and assume the TF binding profile is the same in all cell types. RESULTS: Given the wealth of the ChIP-seq data available for a wide range of the TFs in various cell types, we propose that gene expression modeling can be done using ChIP-seq "signatures" directly, effectively skipping the motif finding and TFBS prediction steps. We present xcore, an R package that allows TF activity modeling based on ChIP-seq signatures and the user's gene expression data. We also provide xcoredata a companion data package that provides a collection of preprocessed ChIP-seq signatures. We demonstrate that xcore leads to biologically relevant predictions using transforming growth factor beta induced epithelial-mesenchymal transition time-courses, rinderpest infection time-courses, and embryonic stem cells differentiated to cardiomyocytes time-course profiled with Cap Analysis Gene Expression. CONCLUSIONS: xcore provides a simple analytical framework for gene expression modeling using linear models that can be easily incorporated into differential expression analysis pipelines. Taking advantage of public ChIP-seq databases, xcore can identify meaningful molecular signatures and relevant ChIP-seq experiments.

Dynamics of cardiomyocyte transcriptome and chromatin landscape demarcates key events of heart development.

Organogenesis involves dynamic regulation of gene transcription and complex multipathway interactions. Despite our knowledge of key factors regulating various steps of heart morphogenesis, considerable challenges in understanding its mechanism still exist because little is known about their downstream targets and interactive regulatory network. To better understand transcriptional regulatory mechanism driving heart development and the consequences of its disruption in vivo, we performed time-series analyses of the transcriptome and genome-wide chromatin accessibility in isolated cardiomyocytes (CMs) from wild-type zebrafish embryos at developmental stages corresponding to heart tube morphogenesis, looping, and maturation. We identified genetic regulatory modules driving crucial events of heart development that contained key cardiac TFs and are associated with open chromatin regions enriched for DNA sequence motifs belonging to the family of the corresponding TFs. Loss of function of cardiac TFs Gata5, Tbx5a, and Hand2 affected the cardiac regulatory networks and caused global changes in chromatin accessibility profile, indicating their role in heart development. Among regions with differential chromatin accessibility in mutants were highly conserved noncoding elements that represent putative enhancers driving heart development. The most prominent gene expression changes, which correlated with chromatin accessibility modifications within their proximal promoter regions, occurred between heart tube morphogenesis and looping, and were associated with metabolic shift and hematopoietic/cardiac fate switch during CM maturation. Our results revealed the dynamic regulatory landscape throughout heart development and identified interactive molecular networks driving key events of heart morphogenesis.

Genomic and physiological analyses of the zebrafish atrioventricular canal reveal molecular building blocks of the secondary pacemaker region.

The atrioventricular canal (AVC) is the site where key structures responsible for functional division between heart regions are established, most importantly, the atrioventricular (AV) conduction system and cardiac valves. To elucidate the mechanism underlying AVC development and function, we utilized transgenic zebrafish line sqet31Et expressing EGFP in the AVC to isolate this cell population and profile its transcriptome at 48 and 72 hpf. The zebrafish AVC transcriptome exhibits hallmarks of mammalian AV node, including the expression of genes implicated in its development and those encoding connexins forming low conductance gap junctions. Transcriptome analysis uncovered protein-coding and noncoding transcripts enriched in AVC, which have not been previously associated with this structure, as well as dynamic expression of epithelial-to-mesenchymal transition markers and components of TGF-ß, Notch, and Wnt signaling pathways likely reflecting ongoing AVC and valve development. Using transgenic line Tg(myl7:mermaid) encoding voltage-sensitive fluorescent protein, we show that abolishing the pacemaker-containing sinoatrial ring (SAR) through Isl1 loss of function resulted in spontaneous activation in the AVC region, suggesting that it possesses inherent automaticity although insufficient to replace the SAR. The SAR and AVC transcriptomes express partially overlapping species of ion channels and gap junction proteins, reflecting their distinct roles. Besides identifying conserved aspects between zebrafish and mammalian conduction systems, our results established molecular hallmarks of the developing AVC which underlies its role in structural and electrophysiological separation between heart chambers. This data constitutes a valuable resource for studying AVC development and function, and identification of novel candidate genes implicated in these processes.

Multi-omics analyses of early liver injury reveals cell-type-specific transcriptional and epigenomic shift.

BACKGROUND: Liver fibrosis is a wound-healing response to tissue injury and inflammation hallmarked by the extracellular matrix (ECM) protein deposition in the liver parenchyma and tissue remodelling. Different cell types of the liver are known to play distinct roles in liver injury response. Hepatocytes and liver endothelial cells receive molecular signals indicating tissue injury and activate hepatic stellate cells which produce ECM proteins upon their activation. Despite the growing knowledge on the molecular mechanism underlying hepatic fibrosis in general, the cell-type-specific gene regulatory network associated with the initial response to hepatotoxic injury is still poorly characterized. RESULTS: In this study, we used thioacetamide (TAA) to induce hepatic injury in adult zebrafish. We isolated three major liver cell types - hepatocytes, endothelial cells and hepatic stellate cells - and identified cell-type-specific chromatin accessibility and transcriptional changes in an early stage of liver injury. We found that TAA induced transcriptional shifts in all three cell types hallmarked by significant alterations in the expression of genes related to fatty acid and carbohydrate metabolism, as well as immune response-associated and vascular-specific genes. Interestingly, liver endothelial cells exhibit the most pronounced response to liver injury at the transcriptome and chromatin level, hallmarked by the loss of their angiogenic phenotype. CONCLUSION: Our results uncovered cell-type-specific transcriptome and epigenome responses to early stage liver injury, which provide valuable insights into understanding the molecular mechanism implicated in the early response of the liver to pro-fibrotic signals.

Transcriptome profile of the sinoatrial ring reveals conserved and novel genetic programs of the zebrafish pacemaker.

BACKGROUND: Sinoatrial Node (SAN) is part of the cardiac conduction system, which controls the rhythmic contraction of the vertebrate heart. The SAN consists of a specialized pacemaker cell population that has the potential to generate electrical impulses. Although the SAN pacemaker has been extensively studied in mammalian and teleost models, including the zebrafish, their molecular nature remains inadequately comprehended. RESULTS: To characterize the molecular profile of the zebrafish sinoatrial ring (SAR) and elucidate the mechanism of pacemaker function, we utilized the transgenic line sqet33mi59BEt to isolate cells of the SAR of developing zebrafish embryos and profiled their transcriptome. Our analyses identified novel candidate genes and well-known conserved signaling pathways involved in pacemaker development. We show that, compared to the rest of the heart, the zebrafish SAR overexpresses several mammalian SAN pacemaker signature genes, which include hcn4 as well as those encoding calcium- and potassium-gated channels. Moreover, genes encoding components of the BMP and Wnt signaling pathways, as well as members of the Tbx family, which have previously been implicated in pacemaker development, were also overexpressed in the SAR. Among SAR-overexpressed genes, 24 had human homologues implicated in 104 different ClinVar phenotype entries related to various forms of congenital heart diseases, which suggest the relevance of our transcriptomics resource to studying human heart conditions. Finally, functional analyses of three SAR-overexpressed genes, pard6a, prom2, and atp1a1a.2, uncovered their novel role in heart development and physiology. CONCLUSION: Our results established conserved aspects between zebrafish and mammalian pacemaker function and revealed novel factors implicated in maintaining cardiac rhythm. The transcriptome data generated in this study represents a unique and valuable resource for the study of pacemaker function and associated heart diseases.

Cytoplasmic polyadenylation-mediated translational control of maternal mRNAs directs maternal-to-zygotic transition.

In the earliest stages of animal development following fertilization, maternally deposited mRNAs direct biological processes to the point of zygotic genome activation (ZGA). These maternal mRNAs undergo cytoplasmic polyadenylation (CPA), suggesting translational control of their activation. To elucidate the biological role of CPA during embryogenesis, we performed genome-wide polysome profiling at several stages of zebrafish development. Our analysis revealed a correlation between CPA and polysome-association dynamics, demonstrating a coupling of translation to the CPA of maternal mRNAs. Pan-embryonic CPA inhibition disrupted the maternal-to-zygotic transition (MZT), causing a failure of developmental progression beyond the mid-blastula transition and changes in global gene expression that indicated a failure of ZGA and maternal mRNA clearance. Among the genes that were differentially expressed were those encoding chromatin modifiers and key transcription factors involved in ZGA, including nanog, pou5f3 and sox19b, which have distinct CPA dynamics. Our results establish the necessity of CPA for ensuring progression of the MZT. The RNA-seq data generated in this study represent a valuable zebrafish resource for the discovery of novel elements of the early embryonic transcriptome.

Fish-Ing for Enhancers in the Heart.

Precise control of gene expression is crucial to ensure proper development and biological functioning of an organism. Enhancers are non-coding DNA elements which play an essential role in regulating gene expression. They contain specific sequence motifs serving as binding sites for transcription factors which interact with the basal transcription machinery at their target genes. Heart development is regulated by intricate gene regulatory network ensuring precise spatiotemporal gene expression program. Mutations affecting enhancers have been shown to result in devastating forms of congenital heart defect. Therefore, identifying enhancers implicated in heart biology and understanding their mechanism is key to improve diagnosis and therapeutic options. Despite their crucial role, enhancers are poorly studied, mainly due to a lack of reliable way to identify them and determine their function. Nevertheless, recent technological advances have allowed rapid progress in enhancer discovery. Model organisms such as the zebrafish have contributed significant insights into the genetics of heart development through enabling functional analyses of genes and their regulatory elements in vivo. Here, we summarize the current state of knowledge on heart enhancers gained through studies in model organisms, discuss various approaches to discover and study their function, and finally suggest methods that could further advance research in this field.

A novel conserved enhancer at zebrafish zic3 and zic6 loci drives neural expression.

BACKGROUND: Identifying enhancers and deciphering their putative roles represent a major step to better understand the mechanism of metazoan gene regulation, development, and the role of regulatory elements in disease. Comparative genomics and transgenic assays have been used with some success to identify critical regions that are involved in regulating the spatiotemporal expression of genes during embryogenesis. RESULTS: We identified two novel tetrapod-teleost conserved noncoding elements within the vicinity of the zic3 and zic6 loci in the zebrafish genome and demonstrated their ability to drive tissue-specific expression in a transgenic zebrafish assay. The syntenic analysis and robust green fluorescent expression in the developing habenula in the stable transgenic line were correlated with known sites of endogenous zic3 and zic6 expression. CONCLUSION: This transgenic line that expresses green fluorescent protein in the habenula is a valuable resource for studying a specific population of cells in the zebrafish central nervous system. Our observations indicate that a genomic sequence that is conserved between humans and zebrafish acts as an enhancer that likely controls zic3 and zic6 expression.

Personalized genome sequencing coupled with iPSC technology identifies GTDC1 as a gene involved in neurodevelopmental disorders.

The cellular and molecular mechanisms underlying neurodevelopmental conditions such as autism spectrum disorders have been studied intensively for decades. The ability to generate patient-specific induced pluripotent stem cells (iPSCs) now offers a novel strategy for modelling human diseases. Recent studies have reported the derivation of iPSCs from patients with neurological disorders. The key challenge remains the demonstration of disease-related phenotypes and the ability to model the disease. Here we report a case study with signs of neurodevelopmental disorders (NDDs) harbouring chromosomal rearrangements that were sequenced using long-insert DNA paired-end tag (DNA-PET) sequencing approach. We identified the disruption of a specific gene, GTDC1. By deriving iPSCs from this patient and differentiating them into neural progenitor cells (NPCs) and neurons we dissected the disease process at the cellular level and observed defects in both NPCs and neuronal cells. We also showed that disruption of GTDC1 expression in wild type human NPCs and neurons showed a similar phenotype as patient's iPSCs. Finally, we utilized a zebrafish model to demonstrate a role for GTDC1 in the development of the central nervous system. Our findings highlight the importance of combining sequencing technologies with the iPSC technology for NDDs modelling that could be applied for personalized medicine.

Zebrafish Zic Genes Mediate Developmental Signaling.

The introduction of genomics into the field of developmental biology led to a vast expansion of knowledge about developmental genes and signaling mechanisms they are involved in. Unlike mammals, the zebrafish features seven Zic genes. This provides an interesting insight into Zic gene evolution. In addition, an unprecedented bioimaging capability of semitransparent zebrafish embryos turns to be a crucial factor in medium- to large-scale analysis of the activity of potential regulatory elements. The Zic family of zinc finger proteins plays an important, relatively well-established, role in the regulation of stem cells and neural development and, in particular, during neural fate commitment and determination. At the same time, some Zic genes are expressed in mesodermal lineages, and their deficiency causes a number of developmental defects in axis formation, establishing body symmetry and cardiac morphogenesis. In stem cells, Zic genes are required to maintain pluripotency by binding to the proximal promoters of pluripotency genes (Oct4, Nanog, Sox2, etc.). During embryogenesis, the dynamic nature of Zic transcriptional regulation is manifested by the interaction of these factors with distal enhancers and other regulatory elements associated with the control of gene transcription and, in particular, with the Nodal and Wnt signaling pathways that play a role in establishing basic organization of the vertebrate body. Zic transcription factors may regulate development through acting alone as well as in combination with other transcription factors. This is achieved due to Zic binding to sites adjacent to the binding sites of other transcription factors, including Gli. This probably leads to the formation of multi-transcription factor complexes associated with enhancers.

Genome wide analysis reveals Zic3 interaction with distal regulatory elements of stage specific developmental genes in zebrafish.

Zic3 regulates early embryonic patterning in vertebrates. Loss of Zic3 function is known to disrupt gastrulation, left-right patterning, and neurogenesis. However, molecular events downstream of this transcription factor are poorly characterized. Here we use the zebrafish as a model to study the developmental role of Zic3 in vivo, by applying a combination of two powerful genomics approaches--ChIP-seq and microarray. Besides confirming direct regulation of previously implicated Zic3 targets of the Nodal and canonical Wnt pathways, analysis of gastrula stage embryos uncovered a number of novel candidate target genes, among which were members of the non-canonical Wnt pathway and the neural pre-pattern genes. A similar analysis in zic3-expressing cells obtained by FACS at segmentation stage revealed a dramatic shift in Zic3 binding site locations and identified an entirely distinct set of target genes associated with later developmental functions such as neural development. We demonstrate cis-regulation of several of these target genes by Zic3 using in vivo enhancer assay. Analysis of Zic3 binding sites revealed a distribution biased towards distal intergenic regions, indicative of a long distance regulatory mechanism; some of these binding sites are highly conserved during evolution and act as functional enhancers. This demonstrated that Zic3 regulation of developmental genes is achieved predominantly through long distance regulatory mechanism and revealed that developmental transitions could be accompanied by dramatic changes in regulatory landscape.

Changing Faces of Transcriptional Regulation Reflected by Zic3.

The advent of genomics in the study of developmental mechanisms has brought a trove of information on gene datasets and regulation during development, where the Zic family of zinc-finger proteins plays an important role. Genomic analysis of the modes of action of Zic3 in pluripotent cells demonstrated its requirement for maintenance of stem cells pluripotency upon binding to the proximal regulatory regions (promoters) of genes associated with cell pluripotency (Nanog, Sox2, Oct4, etc.) as well as cell cycle, proliferation, oncogenesis and early embryogenesis. In contrast, during gastrulation and neurulation Zic3 acts by binding the distal regulatory regions (enhancers, etc) associated with control of gene transcription in the Nodal and Wnt signaling pathways, including genes that act to break body symmetry. This illustrates a general role of Zic3 as a transcriptional regulator that acts not only alone, but in many instances in conjunction with other transcription factors. The latter is done by binding to adjacent sites in the context of multi-transcription factor complexes associated with regulatory elements.

Impaired development of neural-crest cell-derived organs and intellectual disability caused by MED13L haploinsufficiency.

MED13L is a component subunit of the Mediator complex, an important regulator of transcription that is highly conserved across eukaryotes. Here, we report MED13L disruption in a translocation t(12;19) breakpoint of a patient with Pierre-Robin syndrome, moderate intellectual disability, craniofacial anomalies, and muscular defects. The phenotype is similar to previously described patients with MED13L haploinsufficiency. Knockdown of MED13L orthologue in zebrafish, med13b, showed early defective migration of cranial neural crest cells (NCCs) that contributed to cartilage structure deformities in the later stage, recapitulating craniofacial anomalies seen in human patients. Notably, we observed abnormal distribution of developing neurons in different brain regions of med13b morphant embryos, which could be rescued upon introduction of full-length human MED13L mRNA. To compare with mammalian system, we suppressed MED13L expression by short-hairpin RNA in ES-derived human neural progenitors, and differentiated them into neurons. Transcriptome analysis revealed differential expression of components of Wnt and FGF signaling pathways in MED13L-deficient neurons. Our finding provides a novel insight into the mechanism of overlapping phenotypic outcome targeting NCCs derivatives organs in patients with MED13L haploinsufficiency, and emphasizes a clinically recognizable syndromic phenotype in these patients.

Zebrafish mRNA sequencing deciphers novelties in transcriptome dynamics during maternal to zygotic transition.

Maternally deposited mRNAs direct early development before the initiation of zygotic transcription during mid-blastula transition (MBT). To study mechanisms regulating this developmental event in zebrafish, we applied mRNA deep sequencing technology and generated comprehensive information and valuable resources on transcriptome dynamics during early embryonic (egg to early gastrulation) stages. Genome-wide transcriptome analysis documented at least 8000 maternal genes and identified the earliest cohort of zygotic transcripts. We determined expression levels of maternal and zygotic transcripts with the highest resolution possible using mRNA-seq and clustered them based on their expression pattern. We unravel delayed polyadenylation in a large cohort of maternal transcripts prior to the MBT for the first time in zebrafish. Blocking polyadenylation of these transcripts confirms their role in regulating development from the MBT onward. Our study also identified a large number of novel transcribed regions in annotated and unannotated regions of the genome, which will facilitate reannotation of the zebrafish genome. We also identified splice variants with an estimated frequency of 50%-60%. Taken together, our data constitute a useful genomic information and valuable transcriptome resource for gene discovery and for understanding the mechanisms of early embryogenesis in zebrafish.

scRNA-seq reveals the diversity of the developing cardiac cell lineage and molecular players in heart rhythm regulation.

We utilized scRNA-seq to delineate the diversity of cell types in the zebrafish heart. Transcriptome profiling of over 50,000 cells at 48 and 72 hpf defined at least 18 discrete cell lineages of the developing heart. Utilizing well-established gene signatures, we identified a population of cells likely to be the primary pacemaker and characterized the transcriptome profile defining this critical cell type. Two previously uncharacterized genes, atp1b3b and colec10, were found to be enriched in the sinoatrial cardiomyocytes. CRISPR/Cas9-mediated knockout of these two genes significantly reduced heart rate, implicating their role in cardiac development and conduction. Additionally, we describe other cardiac cell lineages, including the endothelial and neural cells, providing their expression profiles as a resource. Our results established a detailed atlas of the developing heart, providing valuable insights into cellular and molecular mechanisms, and pinpointed potential new players in heart rhythm regulation.

Lack of Stim2 Affects Vision-Dependent Behavior and Sensitivity to Hypoxia.

Stromal interaction molecules (STIMs) are endoplasmic reticulum-resident proteins that regulate Ca2+ homeostasis and signaling by store-operated calcium entry (SOCE). The different properties and functions of STIM1 and STIM2 have been described mostly based on work in vitro. STIM2 knockout mice do not survive until adulthood. Therefore, we generated and characterized stim2a and stim2b double-knockout zebrafish. The (stim2a;stim2b)-/- zebrafish did not have any apparent morphological phenotype. However, RNA sequencing revealed 1424 differentially expressed genes. One of the most upregulated genes was annexin A3a, which is a marker of activated microglia. This corresponded well to an increase in Neutral Red staining in the in vivo imaging of the (stim2a;stim2b)-/- zebrafish brain. The lack of Stim2 decreased zebrafish survival under low oxygen conditions. Behavioral tests, such as the visual-motor response test and dark-light preference test, indicated that (stim2a;stim2b)-/- larvae might have problems with vision. This was consistent with the downregulation of many genes that are related to light perception. The periodic acid-Schiff staining of retina sections from adult zebrafish revealed alterations of the stratum pigmentosum, suggesting the involvement of a Stim2-dependent process in visual perception. Altogether, these data reveal new functions for Stim2 in the nervous system.

Profiling subcellular localization of nuclear-encoded mitochondrial gene products in zebrafish.

Most mitochondrial proteins are encoded by nuclear genes, synthetized in the cytosol and targeted into the organelle. To characterize the spatial organization of mitochondrial gene products in zebrafish (Danio rerio), we sequenced RNA from different cellular fractions. Our results confirmed the presence of nuclear-encoded mRNAs in the mitochondrial fraction, which in unperturbed conditions, are mainly transcripts encoding large proteins with specific properties, like transmembrane domains. To further explore the principles of mitochondrial protein compartmentalization in zebrafish, we quantified the transcriptomic changes for each subcellular fraction triggered by the chchd4a -/- mutation, causing the disorders in the mitochondrial protein import. Our results indicate that the proteostatic stress further restricts the population of transcripts on the mitochondrial surface, allowing only the largest and the most evolutionary conserved proteins to be synthetized there. We also show that many nuclear-encoded mitochondrial transcripts translated by the cytosolic ribosomes stay resistant to the global translation shutdown. Thus, vertebrates, in contrast to yeast, are not likely to use localized translation to facilitate synthesis of mitochondrial proteins under proteostatic stress conditions.

The interaction of epithelial Ihha and mesenchymal Fgf10 in zebrafish esophageal and swimbladder development.

Developmental patterning and growth of the vertebrate digestive and respiratory tracts requires interactions between the epithelial endoderm and adjacent mesoderm. The esophagus is a specialized structure that connects the digestive and respiratory systems and its normal development is critical for both. Shh signaling from the epithelium regulates related aspects of mammalian and zebrafish digestive organ development and has a prominent effect on esophageal morphogenesis. The mechanisms underlying esophageal malformations, however, are poorly understood. Here, we show that zebrafish Ihha signaling from the epithelium acting in parallel, but independently of Shh, controls epithelial and mesenchymal cell proliferation and differentiation of smooth muscles and neurons in the gut and swimbladder. In zebrafish ihha mutants, the esophageal and swimbladder epithelium is dysmorphic, and expression of fgf10 in adjacent mesenchymal cells is affected. Analysis of the development of the esophagus and swimbladder in fgf10 mutant daedalus (dae) and compound dae/ihha mutants shows that the Ihha-Fgf10 regulatory interaction is realized through a signaling feedback loop between the Ihha-expressing epithelium and Fgf10-expressing mesenchyme. Disruption of this loop further affects the esophageal and swimbladder epithelium in ihha mutants, and Ihha acts in parallel to but independently of Shha in this process. These findings contribute to the understanding of epithelial-mesenchymal interactions and highlight an interaction between Hh and Fgf signaling pathways during esophagus and swimbladder development.

A Zebrafish/Drosophila Dual System Model for Investigating Human Microcephaly.

Microcephaly presents in neurodevelopmental disorders with multiple aetiologies, including bi-allelic mutation in TUBGCP2, a component of the biologically fundamental and conserved microtubule-nucleation complex, γ-TuRC. Elucidating underlying principles driving microcephaly requires clear phenotype recapitulation and assay reproducibility, areas where go-to experimental models fall short. We present an alternative simple vertebrate/invertebrate dual system to investigate fundamental TUBGCP2-related processes driving human microcephaly and associated developmental traits. We show that antisense morpholino knockdown (KD) of the Danio rerio homolog, tubgcp2, recapitulates human TUBGCP2-associated microcephaly. Co-injection of wild type mRNA pre-empts microcephaly in 55% of KD zebrafish larvae, confirming causality. Body shortening observed in morphants is also rescued. Mitotic marker (pH3) staining further reveals aberrantly accumulated dividing brain cells in microcephalic tubgcp2 KD morphants, indicating that tubgcp2 depletion disrupts normal mitosis and/or proliferation in zebrafish neural progenitor brain cells. Drosophila melanogaster double knockouts (KO) for TUBGCP2 homologs Grip84/cg7716 also develop microcephalic brains with general microsomia. Exacerbated Grip84/cg7716-linked developmental aberration versus single mutations strongly suggests interactive or coinciding gene functions. We infer that tubgcp2 and Grip84/cg7716 affect brain size similarly to TUBGCP2 and recapitulate both microcephaly and microcephaly-associated developmental impact, validating the zebrafish/fly research model for human microcephaly. Given the conserved cross-phyla homolog function, the data also strongly support mitotic and/or proliferative disruption linked to aberrant microtubule nucleation in progenitor brain cells as key mechanistic defects for human microcephaly.