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