Background
Acknowledgements
ADDDDDDDDDReferences Funding Sources
1Department of Pediatrics, Section of Neurology and Developmental Neuroscience, Baylor College of Medicine, Houston, TX, USA, 2Jan and Dan Duncan Neurological Research Institute, Texas Children’s Hospital, Houston, TX, USA, 3Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX, USA, 4Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA,
5Department of Neuroscience, Baylor College of Medicine, Houston, TX, USA, 6McNair Medical Institute, The Robert and Janice McNair Foundation, Houston, TX, USA
Neuroanatomical Consequences of Ebf3 Haploinsufficiency on Brain Size and MorphologyJohn Hayes1,2, Darrion Nguyen1,2, Sahana Murthy1,2, Denise Lanza4, Jonathon Romero3, Robia Pautler3, Jason Heaney4, Hsiao-Tuan Chao1,2,4,5,6
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 dysfunction1,2,3. EBF3 is a Collier/Olf/EBF transcription factor that regulates neuronal development and cell type specification4,5. 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 (Ebf3null/+) and conditional Ebf3flox/+
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 Patients1
Development of Ebf3Null Mouse Allele
MRI 3D Reconstruction and Volumetric Analysis Ebf3 Conditional Knockout Mating Scheme6,7,8 Atoh1-Cre Conditional Heterozygous Knockout of Ebf3 Hydrocephalous in a Subset of Ptf1aCre/+;Ebf3Flox/+ Mice
Hydrocephalous in a Subset of Ebf3Null/+ Mice
Development of Ebf3Flox Mouse Allele
Validation of Ebf3Flox Allele by Germline DeletionPtf1a-Cre Conditional Heterozygous Knockout of Ebf3
Lobule 6/7 Invagination Scoring
Conclusions
Future Directions
Ebf3 haploinsufficient mice display decreased brain volume and abnormal cerebellar morphologyHeterozygous germline deletion of Ebf3 by Sox2-Cre recapitulates
constitutive heterozygous knockout phenotypesConditional heterozygous knockout of Ebf3 in excitatory granule cells
of the cerebellum by Atoh1-Cre causes mild cerebellar abnormalitiesConditional heterozygous knockout of Ebf3 in inhibitory neurons of the
cerebellum by Ptf1a-Cre causes moderate decrease in cerebellar volume and cerebellar abnormalitiesEbf3 haploinsufficiency perturbs cerebellar invagination, with lobule
IV/V, lobule VI, and lobule VII displaying the greatest abnormalityLobules 6/7 are known to be important in cognitive and social
behavior9 providing a possible link to these HADDS phenotypes
Examine the effects of cerebellum specific heterozygous knockout of Ebf3 by EN1-CreQuantify the expression level of Ebf3 in heterozygous conditional
knockout mice by Western Blot and qPCRIdentify the impact of Ebf3 on cell proliferation and cell death in
developing cerebellumElucidate the cognitive and behavioral effects of Ebf3
haploinsufficiency in excitatory and inhibitory cerebellar neuronsDetermine the viability of Ebf3 homozygous conditional knockout
mice with Atoh1-Cre, Ptf1a-Cre, and EN1-Cre
AcknowledgementsIDDRC 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
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