Biology subjects

Kroll, K. L.

Publications and source records attributed to Kroll, K. L..

11 recordsLinked to original sources

bioRxiv2026

Intellectual disability risk gene RFX4 regulates cortical neurogenesis by restraining neuronal differentiation

Despite the recent identification of RFX4 as a neurodevelopmental disorder risk gene, its role in cortical development remained unclear. Here, we identified both shared and lineage-specific RFX4 requirements for human cortical development using new human stem cell models of deficiency and pathogenic mutation. We found that RFX4 restrains neurogenesis by acting cooperatively with NOTCH signaling, specifically repressing pro-neuronal and synaptic gene expression in neural progenitors. We also determined that genome-wide binding of RFX3, another neurodevelopmental disorder risk gene, depends upon RFX4 to regulate synaptic gene expression. Furthermore, we identified lineage-specific functions for RFX4 in regulating proliferation during cortical inhibitory neuron development. Ultimately, we demonstrated that RFX4 deficiency persistently dysregulates neuronal gene expression through neuronal differentiation and disrupts cortical neuron stratification in organoid models. These consequences were absent in neurons generated by direct differentiation, confirming that neuronal phenotypes resulted from unconstrained neurogenesis. Finally, we modeled pathogenic missense mutation of the RFX4 DNA-binding domain. While this mutation strongly reduced DNA binding, it dysregulated synaptic gene expression distinctly from our deficiency models, supporting pathogenic mechanisms distinct from haploinsufficiency. Together, this work identified both shared and lineage-specific requirements for RFX4 during cortical development, building a necessary foundation for elucidating the etiology of RFX4-associated disorders.

developmental biology↗

bioRxiv2025

A single binge ethanol exposure is apoptotic within hours across neurodevelopment and partially regulated by the Myt1l gene.

Ethanol rapidly produces widespread neuronal apoptosis during early development, but this susceptibility declines as the brain matures. In previous research, we found Myt1l (a proneuronal transcription factor) mutations can cause precocious differentiation, neuronal immaturity, and transcriptomic alterations, including many in apoptotic regulators. Therefore, we used a recently developed Myt1l haploinsufficient mouse model to examine this genes effects on ethanol-induced apoptosis across different developmental stages. We discovered that haploinsufficiency can moderately influence vulnerability to ethanol in a complex, age- and cell type-specific manner: apoptosis was reduced on P7, increased P21, but unaffected on P60. Remarkably, we also discovered the previously unrecognized ability of a single binge of ethanol to rapidly increase apoptosis within six hours in early adolescent and adult wild-type mice occurring in microglia and the newborn granule neurons in the hippocampus. This suggests apoptosis is an underappreciated contributor to ethanols neuropathology at older ages and, translated to human use, occurs far more frequently than previously recognized.

neuroscience↗

bioRxiv2025

TBRS-associated DNMT3A mutations disrupt cortical interneuron differentiation and neuronal networks

Pathogenic mutations in DNMT3A cause Tatton-Brown-Rahman Syndrome (TBRS), a disorder characterized by somatic overgrowth of multiple tissues including the brain and intellectual disability (OGID). Here, we investigated TBRS etiology using new human pluripotent stem cell models, modeling varying levels of TBRS-associated loss of DNMT3A function. We identified lineage-specific overgrowth in TBRS ventral forebrain medial ganglionic eminence (MGE)-like progenitors, due in part to increased signaling through the PIK3/AKT/mTOR pathway that could be modulated to ameliorate this phenotype. By contrast, reduced DNA methylation during MGE-like progenitor differentiation into GABAergic interneurons caused premature expression of neuronal and synaptic genes, triggering precocious neuronal maturation. As a result, TBRS GABAergic neurons exhibited sufficient hyperactivity to alter the development and structure of neuronal networks, likely contributing to the intellectual disability and autism spectrum disorder common to TBRS patients. Together, this work elucidates new roles for DNMT3A-mediated gene repression in human cortical development, identifying critical requirements for regulating GABAergic neuron production and neuronal network function. These findings also support potential relationships between pathogenic mechanisms underlying TBRS and other OGIDs, including PIK3CA-related overgrowth syndrome and Weaver Syndrome, thus providing a foundation for future studies to identify common paradigms to treat these related disorders.

neuroscience↗

bioRxiv2024

A survey of hypothalamic phenotypes identifies molecular and behavioral consequences of MYT1L haploinsufficiency in male and female mice.

The transcription factor MYT1L supports proper neuronal differentiation and maturation during brain development. MYT1L haploinsufficiency results in a neurodevelopmental disorder characterized by intellectual disability, developmental delay, autism, behavioral disruptions, aggression, obesity and epilepsy. While MYT1L is expressed throughout the brain, how it supports proper neuronal function in distinct regions has not been assessed. Some features of MYT1L Neurodevelopmental Syndrome suggest disruption of hypothalamic function, such as obesity and endocrine issues, and previous research showed changes in hypothalamic neuropeptide expression following knockdown in zebrafish. Here, we leveraged our heterozygous Myt1l mutant, previously shown to recapitulate aspects of the human syndrome such as hyperactivity, social challenges, and obesity, to examine the impact of MYT1L loss on hypothalamic function. Examining the molecular profile of the MYT1L haploinsufficient hypothalamus revealed a similar scale of disruption to previously studied brain regions, yet with region-specific roles for MYT1L, including regulation of neuropeptide systems. Alterations in oxytocin and arginine vasopressin cell numbers were also found. Behaviors studied included maternal care, social group hierarchies, and aggression, all of which were unchanged. Feeding and metabolic markers were also largely unchanged in MYT1L haploinsufficient mice, yet an interaction was observed between diet and MYT1L genotype on weight gain. Our findings here suggest that gross endocrine function was not altered by MYT1L haploinsufficiency, and that key sex-specific behaviors related to proper hypothalamic function remain intact. Further study is needed to understand the functional impact of the altered hypothalamic molecular profile and changes in neuropeptide cell numbers that result from MYT1L haploinsufficiency.

animal behavior and cognition↗

bioRxiv2024

Lifespan in rodents with MYT1L heterozygous mutation

MYT1L syndrome is a newly recognized disorder characterized by intellectual disability, speech and motor delay, neuroendocrine disruptions, ADHD, and autism. In order to study this gene and its association with these phenotypes, our lab recently created a Myt1l heterozygous mutant mouse inspired by a clinically relevant mutation. This model recapitulates several of the physical and neurologic abnormalities seen in humans with MYT1L syndrome, such as weight gain, microcephaly, and behavioral disruptions. The majority of patients with this syndrome are young, and little is known about the impact of age on health and mortality in these patients. Using a Myt1l mutant mouse, we examined the impact of Myt1l mutation on body weights, lifespan, and histopathology findings of mice at the end of life. This cohort of heterozygous mice demonstrated increased body weight across the lifespan, however there was no significant difference in lifespan, apparent cause of death, or end of life histopathological findings between Myt1l heterozygous and wildtype mice. These findings suggest while Myt1l heterozygous mutation may influence overall brain development, it does not strongly impact other organ systems in the body over time.

genetics↗

bioRxiv2024

Autism and Intellectual Disability-Associated MYT1L Mutation Alters Human Cortical Interneuron Differentiation, Maturation, and Physiology

MYT1L is a neuronal transcription factor highly expressed in the developing and adult brain. While pathogenic MYT1L mutation causes neurodevelopmental disorders, these have not been characterized in human models of neurodevelopment. Here, we defined the consequences of pathogenic MYT1L mutation in human pluripotent stem cell-derived cortical interneurons. During differentiation, mutation reduced MYT1L expression and increased progenitor cell cycle exit and neuronal differentiation and synapse-related gene expression, morphological complexity, and synaptic puncta formation. Conversely, interneuron maturation was compromised, while variant neurons exhibited altered sodium and potassium channel activity and reduced function in electrophysiological analyses. CRISPRi-based knockdown similarly impaired interneuron differentiation and maturation, supporting loss of function-based effects. We further defined MYT1L genome-wide occupancy in interneurons and related this to the transcriptomic dysregulation resulting from MYT1L mutation, to identify direct targets that could mediate these phenotypic consequences. Together, this work delineates contributors to the etiology of neurodevelopmental disorders resulting from MYT1L mutation.

developmental biology↗

bioRxiv2022

LILAC: Enhanced actin imaging with an optogenetic Lifeact

We have designed an improved Lifeact variant that binds to actin under the control of light using the LOV2 protein. Light control enables one to subtract the pre-illumination signal of the unbound label, yielding an enhanced view of F-actin dynamics in cells. Furthermore, the tool eliminates actin network perturbations and cell sickness caused by Lifeact overexpression.

biophysics↗

bioRxiv2022

Structure-informed microbial population genetics elucidate selective pressures that shape protein evolution

Comprehensive sampling of natural genetic diversity with metagenomics enables highly resolved insights into the interplay between ecology and evolution. However, intra-population genomic variation represents the outcome of both stochastic and selective forces, making it difficult to identify whether variants are maintained by adaptive, neutral, or purifying processes. This is partly due to the reliance on gene sequences to interpret variants, which disregards the physical properties of three-dimensional gene products that define the functional landscape on which selection acts. Here we describe an approach to analyze genetic variation in the context of predicted protein structures, and apply it to study a marine microbial population within the SAR11 subclade 1a.3.V, which dominates low-latitude surface oceans. Our analyses reveal a tight association between the patterns of nonsynonymous polymorphism, selective pressures, and structural properties of proteins such as per-site relative solvent accessibility and distance to ligands, which explain up to 59% of genetic variance in some genes. In glutamine synthetase, a central gene in nitrogen metabolism, we observe decreased occurrence of nonsynonymous variants from ligand binding sites as a function of nitrate concentrations in the environment, revealing genetic targets of distinct evolutionary pressures maintained by nutrient availability. Our data also reveals that rare codons are purified from ligand binding sites when genes are under high selection, demonstrating the utility of structure-aware analyses to study the variants that likely impact translational processes. Overall, our work yields insights into the governing principles of evolution that shape the genetic diversity landscape within a globally abundant population, and makes available a software framework for structure-aware investigations of microbial population genetics. SignificanceIncreasing availability of metagenomes offers new opportunities to study evolution, but the equal treatment of all variants limits insights into drivers of sequence diversity. By capitalizing on recent advances in protein structure prediction capabilities, our study examines subtle evolutionary dynamics of a microbial population that dominates surface oceans through the lens of structural biology. We demonstrate the utility of structure-informed metrics to understand the distribution of nonsynonymous polymorphism, establish insights into the impact of changing nutrient availability on protein evolution, and show that even synonymous variants are scrutinized strictly to maximize translational efficiency when selection is high. Overall, our work illustrates new opportunities for discovery at the intersection between metagenomics and structural bioinformatics, and offers an interactive and scalable software platform to visualize and analyze genetic variants in the context of predicted protein structures and ligand-binding sites.

microbiology↗

bioRxiv2020

Alterations in neuronal physiology, development, and function associated with a common duplication of chromosome 15 involving CHRNA7

BackgroundCopy number variants at chromosome 15q13.3 contribute to liability for multiple intellectual and developmental disabilities including Autism Spectrum Disorder (ASD). Individuals with duplications of this interval, which includes the gene CHRNA7, have multiple psychiatric disorders with widely variable penetrance. However, the basis of such differential affectation remains uncharacterized. MethodsInduced pluripotent stem cell (iPSC) models were generated from two first degree relatives with the same 15q13.3 duplication, a boy with distinct features of autism and emotional dysregulation (the affected proband, AP) and his clinically unaffected mother (the UM). These models were compared to unrelated control subjects lacking this duplication (UC, male and female). iPSC-derived neural progenitors and cortical neuroids consisting of cortical excitatory and inhibitory neurons were used to model potential contributors to neuropsychiatric impairment. ResultsThe AP-derived model uniquely exhibited disruptions of cellular physiology and neurodevelopment not observed in either the UM or the unrelated male and female controls. These included enhanced neural progenitor proliferation but impaired neuronal differentiation, maturation, and migration, and increased endoplasmic reticulum (ER) stress. Both the AP models neuronal migration deficit and elevated ER stress could be selectively rescued by different pharmacologic agents. Neuronal gene expression was also specifically dysregulated in the AP, including reduced expression of genes related to behavior, psychological disorders, neuritogenesis, neuronal migration, and WNT, axonal guidance, and GABA receptor signaling. Interestingly, the UM model exhibited upregulated expression of genes in many of these same pathways, by comparison with both the AP and UC models, suggesting that cell intrinsic molecular compensation could have contributed to the lack of neurodevelopmental phenotypes in the UM model. However, by contrast with the AP-specific neurodevelopmental phenotypes, both the AP- and UM-derived neurons exhibited shared alterations of neuronal function, including increased action potential firing and elevated cholinergic activity, consistent with increased homomeric CHRNA7 channel activity. ConclusionTogether, these data define both affectation-specific phenotypes seen only in the AP, as well as abnormalities observed in both individuals with CHRNA7 duplication, the AP and UM, but not in UC-derived neurons. This is, to our knowledge, the first study to use a human stem cell-based platform to study the basis of variable affectation in cases of 15q13.3 duplication at the cellular, molecular, and functional levels. This work suggests potential approaches for suppressing abnormal neurodevelopment or physiology that may contribute to severity of affectation. Some of these AP-specific neurodevelopmental anomalies, or the functional anomalies observed in both 15q13.3 duplication carriers (the AP and UM), could also contribute to the variable phenotypic penetrance seen in other individuals with 15q13.3 duplication.

developmental biology↗

bioRxiv2020

Geminin is required for Hox gene regulation to pattern the developing limb

Development of the complex structure of the vertebrate limb requires carefully orchestrated interactions between multiple regulatory pathways and proteins. Among these, precise regulation of 5 Hox transcription factor expression is essential for proper limb bud patterning and elaboration of distinct limb skeletal elements. Here, we identified Geminin (Gmnn) as a novel regulator of this process. A conditional model of Gmnn deficiency resulted in loss or severe reduction of forelimb skeletal elements, while both the forelimb autopod and hindlimb were unaffected. 5 Hox gene expression expanded into more proximal and anterior regions of the embryonic forelimb buds in this Gmnn-deficient model. A second conditional model of Gmnn deficiency instead caused a similar but less severe reduction of hindlimb skeletal elements and hindlimb polydactyly, while not affecting the forelimb. An ectopic posterior SHH signaling center was evident in the anterior hindlimb bud of Gmnn-deficient embryos in this model. This center ectopically expressed Hoxd13, the HOXD13 target Shh, and the SHH target Ptch1, while these mutant hindlimb buds also had reduced levels of the cleaved, repressor form of GLI3, a SHH pathway antagonist. Together, this work delineates a new role for Gmnn in modulating Hox expression to pattern the vertebrate limb. SummaryThis work identifies a new role for Geminin in mouse limb development. Geminin is a nuclear protein that regulates gene expression to control several other aspects of vertebrate development.

developmental biology↗

bioRxiv2019

Cellular and molecular characterization of multiplex autism in human induced pluripotent stem cell-derived neurons

BackgroundAutism spectrum disorder (ASD) is a neurodevelopmental disorder with pronounced heritability in the general population. This is largely attributable to effects of polygenic susceptibility, with inherited liability exhibiting distinct sex differences in phenotypic expression. Attempts to model ASD in human cellular systems have principally involved rare de novo mutations associated with ASD phenocopies. However, by definition, these models are not representative of polygenic liability, which accounts for the vast share of population-attributable risk.\n\nMethodsHere, we performed what is, to our knowledge, the first attempt to model multiplex autism using patient-derived induced pluripotent stem cells (iPSCs) in a family manifesting incremental degrees of phenotypic expression of inherited liability (absent, intermediate, severe). The family members share an inherited variant of unknown significance in GPD2, a gene that was previously associated with developmental disability but here is insufficient by itself to cause ASD. iPSCs from three first-degree relatives and an unrelated control were differentiated into both cortical excitatory (cExN) and cortical inhibitory (cIN) neurons, and cellular phenotyping and transcriptomic analysis were conducted.\n\nResultscExN neurospheres from the two affected individuals were reduced in size, compared to those derived from unaffected related and unrelated individuals. This reduction was, at least in part, due to increased apoptosis of cells from affected individuals upon initiation of cExN neural induction. Likewise, cIN neural progenitor cells from affected individuals exhibited increased apoptosis, compared to both unaffected individuals. Transcriptomic analysis of both cExN and cIN neural progenitor cells revealed distinct molecular signatures associated with affectation, including misregulation of suites of genes associated with neural development, neuronal function, and behavior, as well as altered expression of ASD risk-associated genes.\n\nConclusionsWe have provided evidence of morphological, physiological, and transcriptomic signatures of polygenic liability to ASD from an analysis of cellular models derived from a multiplex autism family. ASD is commonly inherited on the basis of additive genetic liability. Therefore, identifying convergent cellular and molecular phenotypes resulting from polygenic and monogenic susceptibility may provide a critical bridge for determining which of the disparate effects of rare highly deleterious mutations might also apply to common autistic syndromes.

neuroscience↗