Browsing by Author "Silver, Debra L"

During development, cell-cell recognition events mediate crucial steps in the formation of organized cellular patterns critical for tissue function. In the nervous system, cell recognition cues guide migrating neurons during development to appropriate terminal locations and sculpt their characteristic sizes, shapes, and circuit connectivity. The retina contains a multitude of neuron types; however, neurons of the same cell type (homotypic) are patterned into evenly spaced arrangements known as “mosaics” across the retina surface. Disrupting mosaic formation impairs visual function, so it is important to understand the precise cellular and molecular mechanisms that allow homotypic neurons to recognize and adjust their proximity to neighbors. To understand this process, we studied two populations of interneurons, the OFF and ON starbursts amacrine cells (SACs), which require the cell-surface receptor MEGF10 to establish their mosaics. We find that SACs in Megf10 mutants still make lateral movements in the plane of the retina, but fail to recognize their proximity to homotypic neighbors. Using transgenic tools to visualize SACs early in development, we identify a transient developmental phase where SAC dendrite territories are bounded by homotypic somata, a relationship which is lost in SACs lacking MEGF10. Further, we determine that MEGF10 utilizes distinct signal transduction pathways in neurons from those identified in non-neuronal cells. Lastly, we demonstrate that specific amino acids within the intracellular domain of MEGF10 are required to recapitulate a cellular recognition-like event in a heterologous cell system. These findings support a model whereby MEGF10 signals in SACs by a distinct mechanism to mediate dendrite-soma interactions necessary to pattern the organization of retinal neurons.

Radial glial cells (RGCs) are essential for the generation and organization of neurons in the cerebral cortex. RGCs have an elongated bipolar morphology with basal and apical endfeet that reside in distinct niches. Yet, how this subcellular compartmentalization of RGCs controls cortical development is largely unknown. Here, we employ in vivo proximity labeling, in the mouse, using unfused BirA to generate the first subcellular proteome of RGCs and uncover new principles governing local control of cortical development. We discover a cohort of proteins that are significantly enriched in RGC basal endfeet, with MYH9 and MYH10 among the most abundant. Myh9 and Myh10 transcripts also localize to endfeet with distinct temporal dynamics. Although they each encode isoforms of non-muscle myosin II heavy chain, Myh9 and Myh10 have drastically different requirements for RGC integrity. Myh9 loss from RGCs decreases branching complexity and causes endfoot protrusion through the basement membrane. In contrast, Myh10 controls endfoot adhesion, as mutants have unattached apical and basal endfeet. Finally, we show that Myh9- and Myh10-mediated regulation of RGC complexity and endfoot position non-cell autonomously controls interneuron number and organization in the marginal zone. The first part of this study demonstrates the utility of in vivo proximity labeling for dissecting local control of complex systems, and reveals new mechanisms for dictating RGC integrity and cortical architecture. In the second portion of this work, we have developed a method for purification of endfeet from the embryonic mouse brain and employed it to discover the first global transcriptome of RGC endfeet. Analysis at E15.5 revealed that the network of localized mRNAs is much more extensive than previously appreciated. There are over 3,000 transcripts localized to RGC endfeet and 870 of them are highly enriched in the endfeet compared to the cell body. These data uncovered hundreds of new genes in endfeet and also reinforced our previous findings that cytoskeletal regulators and ECM components are especially important in endfeet. Exploration of the newly discovered localized transcripts will provide valuable insights into additional RGC functions and allow us to assess potential signaling interactions between endfeet and surrounding cells. We also propose a method for subcellular gene knockdown in which we can modulate mRNA levels of a gene of interest in the cell body and endfeet independently in vivo. Through these studies we have discovered vital roles for subcellular gene regulation in RGCs and developed tools to facilitate future studies.

Microcephaly patients are born with a brain size >3 standard deviations below normal and have mild to severe cognitive deficits. 12 microcephaly-linked genes identified in human genetics studies encode microtubule/centrosome-associated proteins and mutations in these genes are strongly tied to disrupted mitotic processes in neural stem cells during cortical development. Yet, how perturbed neural stem cell mitosis kinetics affects cell fate following neural stem cell division is not well understood. Our lab recently discovered prolonging mitosis of mouse neural progenitors, either ex vivo or in vitro, alters fate decisions forcing early increased neurogenic divisions at the expense of maintaining the stem cell pool. Yet, the consequences of prolonged mitosis in vivo, and directly in human stem cells, remain unexplored. Additionally, how prolonged mitosis mechanistically affects cell stress response and cell fate decisions during development is not well-studied.

Through in vivo pharmacological approaches, and in vitro culture of human neural progenitors, I provide evidence that prolonged mitosis in vivo directly alters cell fate, and that this consequence of prolonged mitosis is conserved from mice to humans. I find prolonged mitosis of neural stem cells in vivo results in increased phosphorylation of H2AX in mitosis, and increased pATR in a subset of newborn cells. P53 is then activated in a subset of daughter cells and upregulates downstream target genes. Within approximately the first cell cycle, prolonged mitosis results in an increase in neurogenic fates in the daughter cell population at the expense of progenitor renewal. Conditional loss of P53 rescues these effects on cell fate, while loss of BAX does not. Additionally, I find that time to cell death occurs on a log-normal distribution within the population. These experiments suggest that identifying the factors sensitive to prolonged prometaphase/mitosis arrest that transduce P53 activating signals is critical for our understanding of microcephaly etiologies. Together, data presented in this thesis suggest prolonged mitosis directly alters cell fate in vivo during cortical development and in human neural stem cells, that response to prolonged mitosis is relatively specific, involving P53 signaling, and that prolonged mitosis is a main contributing factor to microcephaly as a result of mitotic gene disruption.

The neocortex expanded spectacularly during human origins. That expansion is thought to form the foundation for our cognitive faculties underlying abstract reasoning and socialization. The human neocortex differs from that of other great apes in several notable regards including altered cell cycle, prolonged corticogenesis, and massively increased size. However, despite decades of effort, little progress has been made in uncovering the genetic contributions that underlie these differences that distinguish our species from closely related primate, such as chimpanzees. A subset of highly conserved non-coding regions that show rapid sequence changes along the human lineage are candidate loci for the development and evolution of uniquely human traits. Several studies have identified human-accelerated enhancers, but none have linked an expression difference to a organismal traits, such as brain sizes. Here we report the discovery of a human-accelerated regulatory enhancer (HARE5) near the Wnt receptor FRIZZLED-8 (FZD8). Using a variety of approaches, we demonstrate dramatic differences in human and chimpanzee HARE5 activity, with human HARE5 driving significantly strong expression. We show that HARE5 likely regulates FZD8 and that expression differences influence cell cycle kinetics, cortical layers, and brain size. At present, this would provide the first evidence of a human-chimpanzee genetic difference influencing the evolution of brain size.

The six-layered neuron structure in the cerebral cortex is the foundation for human mental abilities. In the developing cerebral cortex, neural stem cells undergo proliferation and differentiate into intermediate progenitors and neurons, a process known as embryonic neurogenesis. Disrupted embryonic neurogenesis is the root cause of a wide range of neurodevelopmental disorders, including microcephaly and intellectual disabilities. Multiple layers of regulatory networks have been identified and extensively studied over the past decades to understand this complex but extremely crucial process of brain development. In recent years, post-transcriptional RNA regulation through RNA binding proteins has emerged as a critical regulatory nexus in embryonic neurogenesis. The exon junction complex (EJC) is a highly conserved RNA binding complex composed of four core proteins, Magoh, Rbm8a, Eif4a3, and Casc3. The EJC plays a major role in regulating RNA splicing, nuclear export, subcellular localization, translation, and nonsense mediated RNA decay. Human genetic studies have associated individual EJC components with various developmental disorders. We showed previously that haploinsufficiency of Magoh causes microcephaly and disrupted neural stem cell differentiation in mouse. However, it is unclear if other EJC core components are also required for embryonic neurogenesis. More importantly, the molecular mechanism through which the EJC regulates embryonic neurogenesis remains largely unknown. Here, we demonstrated with genetically modified mouse models that both Rbm8a and Eif4a3 are required for proper embryonic neurogenesis and the formation of a normal brain. Using transcriptome and proteomic analysis, we showed that the EJC posttranscriptionally regulates genes involved in the p53 pathway, splicing and translation regulation, as well as ribosomal biogenesis. This is the first in vivo evidence suggesting that the etiology of EJC associated neurodevelopmental diseases can be ribosomopathies. We also showed that, different from other EJC core components, depletion of Casc3 only led to mild neurogenesis defects in the mouse model. However, our data suggested that Casc3 is required for embryo viability, development progression, and is potentially a regulator of cardiac development. Together, data presented in this thesis suggests that the EJC is crucial for embryonic neurogenesis and that the EJC and its peripheral factors may regulate development in a tissue-specific manner.

The cerebral cortex is an anatomically complex brain structure that controls our higher cognitive functions such as abstract thought and language. The cortex is largely shaped during embryonic development when radial glial progenitors divide and differentiate to produce neurons. Neurons are organized into 6-layers through migration guided by the structural support of radial glial cells. Disruptions in progenitor proliferation or neuronal migration underly diverse neurodevelopmental disorders with life-long impacts on cognitive, psychiatric, and motor functions. Developmental mechanisms that build the brain are regulated by precise gene-expression networks. Here, we uncover two novel layers of post-transcriptional mRNA regulation in the developing cortex. First, we used model models to describe a widespread phenomenon of mRNA localization to the distal structures—the basal process and endfeet—of radial glial progenitors. With live imaging approaches, we detected active mRNA transport to and local translation within radial glial endfeet. Transcriptomic and proteomic analyses revealed that endfeet contain cytoskeletal, signaling, and proteostasis factors that may be locally and dynamically controlled. The second line of investigation focused on the RNA-helicase DDX3X which is frequently mutated in neurodevelopmental disorders. We discovered that Ddx3x is required both in progenitor differentiation and neuronal migration in the developing mouse cortex. DDX3X mutations varied from loss-of-function to missense alleles, and a subset of missense variants caused severe cortical malformations in patients. Biochemical and cell biological assays revealed that dominant missense mutations reduced DDX3X helicase activity and induced formation of RNA-protein aggregates associated with impaired translation, uncovering novel pathologies underlying developmental disorders. Together, these studies extend our understanding of post-transcriptional regulation in brain development and neurodevelopmental disorders.

The exon junction complex (EJC) is comprised of three core components: MAGOH, RBM8A, and EIF4A3. The EJC is canonically known to regulate many aspects of RNA metabolism as well as function in mitosis. Previous work on the EJC has primarily focused on functions for the EJC as a complex, and thus independent roles for EJC components are lacking. It was also recently discovered that EIF4A3 is the causative gene in Richieri-Costa-Pereira Syndrome (RCPS), a craniofacial disease primarily characterized by a severely undersized mandible.

We used two systems to examine EIF4A3 function. First, HeLa cells allowed for dissection of EJC complex requirements. We depleted EIF4A3, MAGOH, or RBM8A and saw that MAGOH and RBM8A protein levels are interdependent, while EIF4A3 levels are independent. We next used point mutant constructs that disrupt EJC core formation to assay EJC complex requirements during mitosis. Constructs that disrupt MAGOH-RBM8A from interacting with EIF4A3 were able to rescue prometaphase arrest, suggesting they may regulate mitosis independently. Further, localization studies show that during mitosis MAGOH and RBM8A localize pericentrosomally whereas EIF4A3 is more expanded across microtubules. Biochemistry studies reveal that EIF4A3 is able to bind to microtubules in the absence of other EJC components or RNA. We also found that overexpression of EIF4A3 results in telophase arrest, suggesting that EIF4A3 dosage is important throughout mitosis.

We next used mouse models to examine the developmental requirements of Eif4a3 both ubiquitously and in the neural crest. We show that heterozygous loss of Eif4a3 at early embryonic ages results in disrupted mandibular arch fusion. These defects later manifest as severe craniofacial abnormalities and loss of adult mandibular structures. Examination of the skeletons of these embryos shows premature ossification of the clavicle. Parallel studies in patient-derived iPSCs show that neural crest cells are less able to migrate and when pushed down an osteogenic lineage, they prematurely differentiate into bone. The craniofacial phenotypes seen in Eif4a3 mutant mice are also distinct from other EJC mutants.

From these data we conclude that EIF4A3 has EJC-independent functions in mitosis, microtubule interaction, and neural crest development. Future studies that disentangle EJC-dependent and independent functions will allow for a more thorough understanding of how these proteins work at the molecular level and in human disease.

Mutations in components of the exon junction complex (EJC) are associated with neurodevelopment and disease. In particular, reduced levels of the RNA helicase EIF4A3 cause Richieri-Costa-Pereira Syndrome (RCPS) and CNVs are linked to intellectual disability. Consistent with this, Eif4a3 haploinsufficient mice are microcephalic. Altogether, this implicates EIF4A3 in cortical development; however, the underlying mechanisms are poorly understood. Here, we use mouse and human models to demonstrate that EIF4A3 promotes cortical development by controlling progenitor mitosis, cell fate, and survival. Eif4a3 haploinsufficiency in mice causes extensive cell death and impairs neurogenesis. Using Eif4a3;p53 compound mice, we show that apoptosis is most impactful for early neurogenesis, while additional p53-independent mechanisms contribute to later stages. Live imaging of mouse and human neural progenitors reveals Eif4a3 controls mitosis length, which influences progeny fate and viability. These phenotypes are conserved as cortical organoids derived from RCPS iPSCs exhibit aberrant neurogenesis. Finally, using rescue experiments we show that EIF4A3 controls neuron generation via the EJC. Altogether, our study demonstrates that EIF4A3 mediates neurogenesis by controlling mitosis duration and cell survival, implicating new mechanisms underlying EJC-mediated disorders.Next, we focus on the function of EIF4A3 in neurons. We unexpectedly discovered that that Eif4a3 – but not Magoh or Rbm8a – is required for neuronal maturation and development of the axonal tract using genetic mouse models. Here we use neuronal cultures, super resolution imaging, and biochemical assays and show that EIF4A3 controls neurite outgrowth in an EJC-independent manner and binds directly to microtubules. Additionally, we perform quantitative proteomics to ask whether other interactors of EIF4A3 vary across progenitors and neurons in the developing brain, finding an enrichment of cell cycle regulators during early neurogenesis and cytoskeletal regulators in later neurogenesis. Altogether, these data argue that EIF4A3 has cell-type specific functions and controls brain development through multiple mechanisms.