Background Acknowledgements ADDDDDDDDD References Funding Sources 1 Department of Pediatrics, Section of Neurology and Developmental Neuroscience, Baylor College of Medicine, Houston, TX, USA, 2 Jan and Dan Duncan Neurological Research Institute, Texas Children’s Hospital, Houston, TX, USA, 3 Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX, USA, 4 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA, 5 Department of Neuroscience, Baylor College of Medicine, Houston, TX, USA, 6 McNair Medical Institute, The Robert and Janice McNair Foundation, Houston, TX, USA Neuroanatomical Consequences of Ebf3 Haploinsufficiency on Brain Size and Morphology John Hayes 1,2 , Darrion Nguyen 1,2 , Sahana Murthy 1,2 , Denise Lanza 4 , Jonathon Romero 3 , Robia Pautler 3 , Jason Heaney 4 , Hsiao-Tuan Chao 1,2,4,5,6 Hypotonia, Ataxia, and Delayed Development syndrome (HADDS) (OMIM #617330) is a neurodevelopmental disorder due to heterozygous loss-of-function variants of the gene Early B-cell Factor 3 (EBF3) that causes the namesake features and a range of other symptoms including autistic features, variable cognition, genitourinary abnormalities, and cerebellar dysfunction 1,2,3 . EBF3 is a Collier/Olf/EBF transcription factor that regulates neuronal development and cell type specification 4,5 . We hypothesize that Ebf3 haploinsufficiency in mice would perturb cerebellar development and function, leading to the cardinal features of HADDS. To study the effects of EBF3 haploinsufficiency on cerebellar development, we developed novel constitutive heterozygous knock out (Ebf3 null/+ ) and conditional Ebf3 flox/+ mouse alleles by CRISPR/Cas9 deletion of exons two through four. We analyzed the effects of cell type specific deletion of Ebf3 in the cerebellum. (1) Chao et al., Am J Hum Genet., 2017, PMID 28017372 (2) Harms et al., Am J Hum Genet., 2017, PMID 28017373 (3) Sleven et al., Am J Hum Genet., 2017, PMID 28017370 (4) Liberg et al., Mol Cell Biol., 2002, PMID 12446759 (5) Pozzoli et al., Dev Biol., 2001, PMID 11336510 (6) Allen Institute for Brain Science, Mouse Brain Atlas, http://atlas.brain-map.org/atlas (7) https://www.genetargeting.com/conventional/conventional-vs-conditional- knockout/ (8) https://www.researchgate.net/figure/Schematic-illustration-of-the- Cre-LoxP-system-In-the-F0-generation-mouse-line-1_fig3_310514682 (9) D’Angelo et al., Front. Neural Circuits., 2013, PMID 23335884 • American Academy of Neurology • American Brain Foundation • CNCDP-K12 • BWF-CAMS • McNair Medical Institute at The Robert and Janice McNair Foundation • NIH DPS-EIA (DP5OD026428) • PERF-Elterman Abnormal Brain MRI in HADD Syndrome Patients 1 Development of Ebf3 Null Mouse Allele MRI 3D Reconstruction and Volumetric Analysis Ebf3 Conditional Knockout Mating Scheme 6,7,8 Atoh1-Cre Conditional Heterozygous Knockout of Ebf3 Hydrocephalous in a Subset of Ptf1a Cre/+ ;Ebf3 Flox/+ Mice Hydrocephalous in a Subset of Ebf3 Null/+ Mice Development of Ebf3 Flox Mouse Allele Validation of Ebf3 Flox Allele by Germline Deletion Ptf1a-Cre Conditional Heterozygous Knockout of Ebf3 Lobule 6/7 Invagination Scoring Conclusions Future Directions Ebf3 haploinsufficient mice display decreased brain volume and abnormal cerebellar morphology Heterozygous germline deletion of Ebf3 by Sox2-Cre recapitulates constitutive heterozygous knockout phenotypes Conditional heterozygous knockout of Ebf3 in excitatory granule cells of the cerebellum by Atoh1-Cre causes mild cerebellar abnormalities Conditional heterozygous knockout of Ebf3 in inhibitory neurons of the cerebellum by Ptf1a-Cre causes moderate decrease in cerebellar volume and cerebellar abnormalities Ebf3 haploinsufficiency perturbs cerebellar invagination, with lobule IV/V, lobule VI, and lobule VII displaying the greatest abnormality Lobules 6/7 are known to be important in cognitive and social behavior 9 providing a possible link to these HADDS phenotypes Examine the effects of cerebellum specific heterozygous knockout of Ebf3 by EN1-Cre Quantify the expression level of Ebf3 in heterozygous conditional knockout mice by Western Blot and qPCR Identify the impact of Ebf3 on cell proliferation and cell death in developing cerebellum Elucidate the cognitive and behavioral effects of Ebf3 haploinsufficiency in excitatory and inhibitory cerebellar neurons Determine the viability of Ebf3 homozygous conditional knockout mice with Atoh1-Cre, Ptf1a-Cre, and EN1-Cre Acknowledgements IDDRC Imaging Core: Dinghui Yu; BCM Embryonic Stem Cell: Jason Heaney, Denise Lanza; BCM Genetically Engineered Mouse Core: Lan Liao, Jianming Xu; SAIF Core: Jonathon Romero, Robia Pautler; Members of the Bellen, Xue, Silitoe, Zoghbi, and Chao labs