Using Xenopus laevis to investigate developmental mechanisms underlying human neurodevelopmental disorders and intellectual disabilities:

Thesis advisor: Laura Anne LoweryThesis advisor: Sarah McMenaminDevelopment of the central nervous system (CNS) is a complex process that requires the proper function of many genes in order for neurons to proliferate and divide, differentiate, and subsequently migrate long distances to form connections with one another. Abnormalities in any one of these cellular processes can lead to detrimental developmental defects. Growing evidence suggests that genetic mutations caused by rare copy number variants (CNVs) are associated with neurodevelopmental disorders including intellectual disabilities (ID), Autism spectrum disorder (ASD), and schizophrenia. Additionally, these pathogenic CNVs are characterized by extensive phenotypic heterogeneity, as affected individuals often present with microcephaly, craniofacial and heart defects, growth retardation, and seizures. Despite their strong association as risk factors towards neurodevelopmental disorders, the developmental role of individual CNV-affected genes and disrupted cellular mechanisms underlying these mutations remains poorly understood. Moreover, it is unclear as to how the affected genes both individually and combinatorially contribute to the phenotypes associated with pathogenic CNVs. Thus, in this thesis, we explore the functional basis of phenotypic variability of pathogenic CNVs linked to neurodevelopmental disorders. In particular, we focus on the 3q29 deletion and 16p12.1 deletion, to provide insight towards the convergent cellular, molecular, and developmental mechanisms associated with decreased dosage of the affected gene homologs using two complementary model systems, Xenopus laevis and Drosophila melanogaster. First, we examine the role of individual homologs of several CNV-affected genes at chromosome 3q29 and their interactions towards cellular processes underlying the deletion. We find that multiple 3q29-affected genes, including NCBP2, DLG1, FBXO45, PIGZ, and BDH1, contribute to disruptions in apoptosis and cell cycle pathways, leading to neuronal and developmental defects. We then expand further upon this work by discerning the individual contribution of four CNV-affected genes at chromosome 16p12.1, POLR3E, MOSMO, UQCRC2, and CDR2, towards neurodevelopment and craniofacial morphogenesis. We demonstrate that several of these genes affect multiple phenotypic domains during neurodevelopment leading to brain size alterations, abnormal neuronal morphology, and cellular proliferation defects. We then explore their functions during vertebrate craniofacial morphogenesis and demonstrate that some 16p12.1-affected genes are enriched in migratory neural crest, and contribute to early craniofacial patterning and formation of cartilaginous tissue structures. Together, these data are the first to suggest that signature neurodevelopmental phenotypes demonstrated in the 3q29 deletion and 16p12.1 deletion may stem from convergent cellular mechanisms including aberrations in neuronal proliferation, apoptosis and cell cycle regulation, and neural crest cell development.Thesis (PhD) — Boston College, 2020.Submitted to: Boston College. Graduate School of Arts and Sciences.Discipline: Biology

The Role of the Microtubule Cytoskeleton in Neurodevelopmental Disorders

Neurons depend on the highly dynamic microtubule (MT) cytoskeleton for many different processes during early embryonic development including cell division and migration, intracellular trafficking and signal transduction, as well as proper axon guidance and synapse formation. The coordination and support from MTs is crucial for newly formed neurons to migrate appropriately in order to establish neural connections. Once connections are made, MTs provide structural integrity and support to maintain neural connectivity throughout development. Abnormalities in neural migration and connectivity due to genetic mutations of MT-associated proteins can lead to detrimental developmental defects. Growing evidence suggests that these mutations are associated with many different neurodevelopmental disorders, including intellectual disabilities (ID) and autism spectrum disorders (ASD). In this review article, we highlight the crucial role of the MT cytoskeleton in the context of neurodevelopment and summarize genetic mutations of various MT related proteins that may underlie or contribute to neurodevelopmental disorders

NCBP2 modulates neurodevelopmental defects of the 3q29 deletion in Drosophila and Xenopus laevis models.

The 1.6 Mbp deletion on chromosome 3q29 is associated with a range of neurodevelopmental disorders, including schizophrenia, autism, microcephaly, and intellectual disability. Despite its importance towards neurodevelopment, the role of individual genes, genetic interactions, and disrupted biological mechanisms underlying the deletion have not been thoroughly characterized. Here, we used quantitative methods to assay Drosophila melanogaster and Xenopus laevis models with tissue-specific individual and pairwise knockdown of 14 homologs of genes within the 3q29 region. We identified developmental, cellular, and neuronal phenotypes for multiple homologs of 3q29 genes, potentially due to altered apoptosis and cell cycle mechanisms during development. Using the fly eye, we screened for 314 pairwise knockdowns of homologs of 3q29 genes and identified 44 interactions between pairs of homologs and 34 interactions with other neurodevelopmental genes. Interestingly, NCBP2 homologs in Drosophila (Cbp20) and X. laevis (ncbp2) enhanced the phenotypes of homologs of the other 3q29 genes, leading to significant increases in apoptosis that disrupted cellular organization and brain morphology. These cellular and neuronal defects were rescued with overexpression of the apoptosis inhibitors Diap1 and xiap in both models, suggesting that apoptosis is one of several potential biological mechanisms disrupted by the deletion. NCBP2 was also highly connected to other 3q29 genes in a human brain-specific interaction network, providing support for the relevance of our results towards the human deletion. Overall, our study suggests that NCBP2-mediated genetic interactions within the 3q29 region disrupt apoptosis and cell cycle mechanisms during development

Functional assessment of the "two-hit" model for neurodevelopmental defects in Drosophila and X. laevis.

We previously identified a deletion on chromosome 16p12.1 that is mostly inherited and associated with multiple neurodevelopmental outcomes, where severely affected probands carried an excess of rare pathogenic variants compared to mildly affected carrier parents. We hypothesized that the 16p12.1 deletion sensitizes the genome for disease, while "second-hits" in the genetic background modulate the phenotypic trajectory. To test this model, we examined how neurodevelopmental defects conferred by knockdown of individual 16p12.1 homologs are modulated by simultaneous knockdown of homologs of "second-hit" genes in Drosophila melanogaster and Xenopus laevis. We observed that knockdown of 16p12.1 homologs affect multiple phenotypic domains, leading to delayed developmental timing, seizure susceptibility, brain alterations, abnormal dendrite and axonal morphology, and cellular proliferation defects. Compared to genes within the 16p11.2 deletion, which has higher de novo occurrence, 16p12.1 homologs were less likely to interact with each other in Drosophila models or a human brain-specific interaction network, suggesting that interactions with "second-hit" genes may confer higher impact towards neurodevelopmental phenotypes. Assessment of 212 pairwise interactions in Drosophila between 16p12.1 homologs and 76 homologs of patient-specific "second-hit" genes (such as ARID1B and CACNA1A), genes within neurodevelopmental pathways (such as PTEN and UBE3A), and transcriptomic targets (such as DSCAM and TRRAP) identified genetic interactions in 63% of the tested pairs. In 11 out of 15 families, patient-specific "second-hits" enhanced or suppressed the phenotypic effects of one or many 16p12.1 homologs in 32/96 pairwise combinations tested. In fact, homologs of SETD5 synergistically interacted with homologs of MOSMO in both Drosophila and X. laevis, leading to modified cellular and brain phenotypes, as well as axon outgrowth defects that were not observed with knockdown of either individual homolog. Our results suggest that several 16p12.1 genes sensitize the genome towards neurodevelopmental defects, and complex interactions with "second-hit" genes determine the ultimate phenotypic manifestation