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Kristen L. Kroll, Ph.D.
Developmental, Regenerative and Stem Cell Biology Program
Molecular Cell Biology Program
320 McDonnell Medical Sciences Building
epigenetic, neurobiology, embryonic stem cell, neural stem cell, transcription, gene regulatory network, cancer, cell cycle, chromatin, embryo
Transcriptional and epigenetic regulation of neural development and its dysregulation in neurodevelopmental disorders
Our research focuses on defining gene regulatory networks (GRNs) that control neural cell specification, neurogenesis, and the generation of specific neuronal cell types. We are particularly interested in understanding how epigenetic regulation modulates these networks and how their dysregulation contributes to neurodevelopmental disorders, including inherited epilepsies, autism spectrum disorder, and neural tube defects. This work uses directed differentiation of human pluripotent stem cells (embryonic stem cells and induced pluripotent stem cells), mouse models, and a wide range of cellular, molecular, and genomic approaches, to define roles for transcriptional and epigenetic regulation in shaping developmental transitions.
Gene regulatory networks in neural cell fate acquisition (Figure 1)
Neural cell fate acquisition is mediated by transcription factors expressed in nascent neuroectoderm, including Geminin and members of the Zic transcription factor family. Recently, we identified chromatin association profiles for Geminin and Zic1 during neural fate acquisition at a genome-wide level (Sankar et al., 2016). We determined how Geminin deficiency affected histone acetylation at gene promoters during this process. We integrated these data to determine that Geminin associates with and promotes histone acetylation at neurodevelopmental genes, while Geminin and Zic1 bind a shared gene subset. Geminin- and Zic1-associated genes exhibit embryonic nervous system-enriched expression and encode other regulators of neural development. Both Geminin and Zic1-associated peaks are enriched for Zic1 consensus binding motifs, while Zic1-bound peaks are also enriched for Sox3 motifs, suggesting co-regulatory potential. Accordingly, we found that Geminin and Zic1 could cooperatively activate the expression of several shared targets encoding transcription factors that control neurogenesis, neural plate patterning, and neuronal differentiation. We used these data to construct gene regulatory networks underlying neural cell fate acquisition. Establishment of this molecular program in nascent neuroectoderm directly links early neural cell fate acquisition with regulatory control of later neurodevelopment.
We are characterizing how disruption of this network contributes to the etiology of neural tube defects (NTDs) and other neurodevelopmental disorders. Although NTDs are the second most common birth defect (~1:1000 fetuses and newborns), their causation is poorly understood. This is, in part, because NTD occurrence usually has a multi-factorial etiology, with both genetic and environmental susceptibility factors contributing to phenotypic manifestation and severity. Therefore, defining aspects of this regulatory network that are dysregulated to contribute to NTDs in multiple models may identify core processes that, when disrupted, increase NTD susceptibility. This work also defined novel epigenetic regulators with likely roles in neural development, some of which we are functionally assessing in ongoing work.
Modeling human cortical interneuron development and dysregulation in neurodevelopmental disorders (Figure 2)
Another major focus of our work is to identify transcriptional and epigenetic regulatory controls underlying the development of human cortical interneurons and to understand how their dysregulation contributes to neurodevelopmental disorders. The mammalian cerebral cortex consists of two major types of neurons, excitatory glutamatergic projection neurons that convey information to different regions of the brain and GABAergic interneurons that provide local inhibitory inputs to modulate responses of projection neurons and prevent over-excitation. Imbalances between excitatory and inhibitory neuronal activities can emerge during cortical development. These imbalances often reduce inhibitory signaling through dysfunctional specification, migration, differentiation, or survival of interneurons. Interneuron hypoplasia or dysfunction contributes to many neurological disorders, including epilepsy, schizophrenia, autism spectrum disorder, and intellectual disability syndromes.
We can now model gene regulatory networks that control human cortical interneuron specification and differentiation at a genome-wide level by generating mature interneurons from human pluripotent stem cells (embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs)). We are building gene regulatory networks underlying this process by integrating genome-wide binding profiles of key transcription factors and chromatin regulators, developmental transcriptome and epigenome changes, and effects of manipulating these activities. This work has revealed new regulators of interneuron development, including transcriptional and chromatin modifying activities and non-coding RNAs, enriched in and/or involved in interneuron specification and differentiation.
After transplantation into the neonatal or adult nervous system, cortical interneurons have a striking ability to integrate into neural circuits and to provide inhibitory neuronal function in many locations, including their normal targets in the forebrain (hippocampus, cortex, striatum) as well as upon transplantation into heterologous locations such as the spinal cord. Because of this, transplanted mouse interneurons acquired from the medial ganglionic eminence have shown therapeutic utility in treating disease in mouse models of epilepsy, Parkinson’s disease, chronic pain, schizophrenia and anxiety. We are currently applying this approach with in vitro differentiated human interneurons, to assess their capacity to engraft and migrate to target locations in the murine brain, and to suppress seizures in a murine model of temporal lobe epilepsy. These approaches can be used to determine how interneuron differentiation or maturation state, or subtype identity, contributes to capacity to engraft, migrate and ameliorate disease phenotypes.
We have also defined genes with interneuron-enriched expression whose mutation contributes to human neurodevelopmental disorders, including inherited epilepsies and autism spectrum disorder. In collaboration with the WUSM Intellectual and Developmental Disability Research Center, we are deriving iPSCs from epilepsy and autism patients with mutations in some of these genes and are performing directed differentiation into cortical excitatory and inhibitory neurons and cerebral organoids. These are subjected to a battery of assays to define developmental, cellular, molecular, and functional abnormalities that contribute to the disorder. We can use these models to assess the relative contributions of genetic background versus pathogenic mutations, by comparisons of isogenic neurons with versus without engineered correction of mutations. We are also using these models to assess how differential expressivity of a disorder among family members carrying a known pathogenic mutation manifests in cellular, molecular, and functional differences between neurons from these family members.
Sankar S, Yellajoshyula D, Zhang B, Teets B, Rockweiler N, and Kroll KL. (2016) Gene regulatory networks in neural cell fate acquisition from genome-wide chromatin association of Geminin and Zic1. Scientific Reports 6:37412. doi: 10.1038/srep37412.
Dandulakis MG, Meganathan K, Kroll KL, Bonni A, Constantino JN. (2016) Complexities of X chromosome inactivation status in female human induced pluripotent stem cells. Journal of Neurodevelopmental Disorders 8:22. doi: 10.1186/s11689-016-9155-8
Corley M, Kroll KL. (2015) The roles and regulation of Polycomb complexes in neural development. Cell Tissue Res. Jan;359(1):65-85. doi: 10.1007/s00441-014-2011-9. [In the Special Issue: How to build a neuron – the problem of generic neuronal differentiation.]
Patterson ES, Waller LE, Kroll KL. (2014) Geminin loss causes neural tube defects through disrupted progenitor specification and neuronal differentiation. Dev Biol. Sep 1;393(1):44-56. doi: 10.1016/j.ydbio.2014.06.021
Caronna, E, Patterson, ES, Hummert P, and Kroll, KL. (2013) Geminin restrains mesendodermal fate acquisition of mouse embryonic stem cells and is associated with antagonism of Wnt signaling and enhanced Polycomb-mediated repression. Stem Cells. Aug;31(8):1477-87. doi: 10.1002/stem.1410.
Siles, L, Lim, J, Sanchez-Tillo, E, Lim, JW, Darling, DS, Kroll, KL, and Postigo, A (2013) ZEB1 imposes a temporary stage-dependent inhibition of muscle gene expression and differentiation via CtBP-mediated transcriptional repression. Molecular and Cellular Biology, 33(7): 1368-82.
Yellajoshyula, D., Lim, J., Thompson, D.M., Witt, J.S., Patterson, E.S., and Kroll, K.L. (2012) Geminin regulates the transcriptional and epigenetic status of neuronal fate promoting genes during mammalian neurogenesis. Mol Cell Biol. 2012 Nov;32(22):4549-60. doi: 10.1128/MCB.00737-12.
Sengupta R, Dubuc, A., Ward, S., Yang, L., Northcott, P., Woerner, B., Kroll, K., Luo, J., Taylor, M., Wechsler-Reya, R., and Rubin, J. (2012) Cxcr4 Activation Defines a New Subgroup of Sonic Hedgehog-Driven Medulloblastoma. Cancer Research 72(1): 122-32. PMID: 22052462
Yellajoshyula D, Patterson ES, Elitt MS Kroll KL. (2011) Geminin promotes neural fate acquisition of embryonic stem cells by maintaining chromatin in an accessible and hyperacetylated state. Proc. Natl. Acad. of Sciences 108(8):3294-9. PMID: 21300881. PMCID: PMC3044367
Lim JW, Hummert P, Mills JC Kroll KL. (2011) Geminin cooperates with Polycomb to restrain multi-lineage commitment in the early embryo. Development. 138(1):33-44. PMID: 21098561.
Langer, E.M., Feng, Y. Zhaoyuan, H., Rauscher, F.J., Kroll, K.L., and Longmore, G. D. (2008) Ajuba LIM proteins are Snail corepressors required for neural crest development. Developmental Cell 14(3): 424-36. PMID: 18331720. PMCID: PMC2279146
Seo S, Lim JW, Yellajoshyula D, Chang LW, Kroll KL. (2007) Neurogenin direct transcriptional targets and their regulatory enhancers. EMBO Journal 26(24): 5093-108. PMID: 18007592.
Kroll, K.L. (2007) Geminin in embryonic development: coordinating transcription and the cell cycle during differentiation. Frontiers in Bioscience 12, 1395-1409. PMID: 17127390
Taylor J, Wang T, and Kroll (2006) KL. Tcf- and Vent-binding sites regulate neural-specific geminin expression in the gastrula embryo. Developmental Biology 289(2): 494-506. PMID: 16337935
Seo S, Herr A, Lim J, Richardson G, and Kroll KL. (2005) Geminin regulates neuronal differentiation by antagonizing Brg1 activity. Genes and Development 19(14): 1723-1734. PMID: 16024661.
Seo, S., Cook, G.A, and Kroll, K.L. (2005) The SWI/SNF chromatin remodeling protein Brg1 is required for neurogenesis and mediates transactivation of Ngn and NeuroD. Development 132, 105-115. PMID: 1557641
Last Updated: 1/10/2017 8:54:27 AM
Figure 1. Genome-wide binding profiles for Geminin and Zic1 were used to define gene regulatory networks in neural cell fate acquisition. (A-D) Comparison of Gmnn-associated and Zic1-associated genes in NE. (A) A subset of genes is associated with both Gmnn and Zic1. p-value (Chi-square test with Yates’ correction) < 2.2X10-16. (B) z-scores for enrichment of expression in ES versus CNS tissues for all Zic and/or Gmnn associated genes. (C) Comparison of all Gmnn or Zic1 associated genes, subsets that undergo Gmnn-dependent acetylation, and transcription factors, with their relative enrichment of expression in embryonic CNS tissues. (D) GO enrichment analysis for associated genes with increased versus decreased expression in embryonic CNS, relative to ES cells. (E) Gene regulatory networks in neural cell fate acquisition. Gmnn- and Zic1-associated genes in NE that encode transcription factors and epigenetic regulatory activities were assessed for co-association with Sox2 and Sox3 in ES-derived NE (see text). The set of these genes that exhibit CNS-enriched expression (>two-fold greater expression in E14 CNS, relative to ES cells), and were associated with Gmnn and/or Zic1 plus Sox2 and/or Sox3 were used to build gene regulatory networks showing associations. Figure 2. Human cortical interneuron derivation and characterization. (A) Immunocytochemistry (ICC) for ventral telencephalic/MGE, neuron, cIN and alternate cell fate (RAX/DARPP32) markers, in day 35 hESC-derived interneurons (scale bar=100µm). (B) FACs for Nkx2-1 and (C) quantitation of % ICC+ cells. Inset: FACS analysis for the SST+ cortical interneuron subtype. (D) Synapsin-GFP expressing day 35 cIN (after PSA-NCAM+ selection; scale bar=100µM) were (E) transplanted into the cortex of P2 NOD/SCID mice (20 days post-transplantation shown; B’ box in B, scale bar=100µM) or (F) transplanted into the hippocampus (CA3) of NOD/SCID adult mice (1 month post-transplantation shown, C’ box in C; scale bar=50µM). (D-E) Representative action potential firing pattern (G) evoked by injection of a square pulse, (H) spontaneous. (I) Voltage-gated currents evoked by a step from -80 to +50 mV in the absence (black) or presence (red) of the sodium channel antagonist tetrodotoxin (TTX, 0.5 µM). (J) Spontaneous synaptic currents recorded at -20 and -80 mV. Exposure to the GABAA receptor antagonist bicuculline methiodide (200 µM), indicated by the open box above each trace, blocked spontaneous Inhibitory postsynaptic currents (IPSCs). IPSCs were inward at -80 mV and outward at -20 mV, consistent with the equilibrium potential for chloride (-54 mV) with our internal and external solutions. Work performed in collaboration with Jim Huettner’s laboratory