Magnetic Resonance in Medicine 70:1707–1717 (2013) MRI Analysis of Cerebellar and Vestibular Developmental Phenotypes in Gbx2 Conditional Knockout Mice Kamila U. Szulc,1,2 Brian J. Nieman,3 Edward J. Houston,1 Benjamin B. Bartelle,1,4 Jason P. Lerch,3 Alexandra L. Joyner,5 and Daniel H. Turnbull1,2,4,6,7* Purpose: Our aim in this study was to apply three-dimensional mouse models of neurodevelopmental diseases. Magn Reson MRI methods to analyze early postnatal morphological pheno- Med 70:1707–1717, 2013. VC 2013 Wiley Periodicals, Inc. types in a Gbx2 conditional knockout (Gbx2-CKO) mouse that has variable midline deletions in the central cerebellum, remi- Key words: brain development; cerebellum; gastrulation brain niscent of many human cerebellar hypoplasia syndromes. homeobox 2 gene (Gbx2); manganese-enhanced MRI Methods: In vivo three-dimensional manganese-enhanced (MEMRI); mid-hindbrain; vestibulo-cochlear organ MRI at 100-mm isotropic resolution was used to visualize mouse brains between postnatal days 3 and 11, when cere- bellum morphology undergoes dramatic changes. Deforma- Advances in the field of mouse genetics have been criti- tion-based morphometry and volumetric analysis of cal in elucidating the roles of different genes in mamma- manganese-enhanced MRI images were used to, respectively, lian brain development and neurodevelopmental dis- detect and quantify morphological phenotypes in Gbx2-CKO eases (1–3). Specifically, genetic defects are commonly mice. Ex vivo micro-MRI was performed after perfusion-fixa- associated with congenital brain malformations, which tion with supplemented gadolinium for higher resolution (50- can be mimicked in mutant mice to better understand mm) analysis. the molecular and cellular basis of developmental brain Results: In vivo manganese-enhanced MRI and deformation- based morphometry correctly identified known cerebellar disorders. The early postnatal period is a time of signifi- defects in Gbx2-CKO mice, and novel phenotypes were dis- cant brain growth and patterning. Indeed, many mouse covered in the deep cerebellar nuclei and the vestibulo-cere- mutant brain phenotypes are first manifested during bellum, both validated using histology. Ex vivo micro-MRI early postnatal stages, especially in the cerebellum, revealed subtle phenotypes in both the vestibulo-cerebellum which acquires its complex foliation pattern during the and the vestibulo-cochlear organ, providing an interesting first 2 weeks after birth. It is therefore important to de- example of complementary phenotypes in a sensory organ velop and validate three-dimensional (3D) neuroimaging and its associated brain region. approaches for quantitative analyses of neonatal mouse Conclusion: These results show the potential of three-dimen- brain structures, enabling effective mutant phenotype sional MRI for detecting and analyzing developmental defects in analysis (4–6). The cerebellum (derived from anterior hindbrain) and midbrain are regulated during embryogenesis from a common mid-hindbrain (MHB) organizing center—a tis- 1Kimmel Center for Biology and Medicine at the Skirball Institute of sue that sends molecular signals that instruct the fates of Biomolecular Medicine, New York University School of Medicine, New York, the surrounding midbrain and anterior hindbrain (cere- New York, USA. 2Biomedical Imaging Program, New York University School of Medicine, bellum) cells. The MHB region has been the focus of New York, New York, USA. numerous previous studies, with the result that many 3Mouse Imaging Centre, Hospital for Sick Children, Toronto, Ontario, genes have been identified that are required for normal Canada. MHB development, and many mouse mutants are avail- 4Molecular Biophysics Graduate Program, New York University School of Medicine, New York, New York, USA. able that display a wide range of MHB defects (7–10). 5Developmental Biology Program, Sloan-Kettering Institute, New York, New These mouse mutants are considered relevant to several York, USA. human neurodevelopmental diseases, including Joubert, 6Department of Radiology, New York University School of Medicine, New Dandy-Walker, and other cerebellar hypoplasia syn- York, New York, USA. dromes, as well as diseases with more subtle anatomical 7Department of Pathology, New York University School of Medicine, New York, New York, USA. features such as schizophrenia and autism (11–14). Im- Additional Supporting Information may be found in the online version of portant for the current studies, normal development of this article. the MHB region depends on the gastrulation brain Grant sponsor: NIH; Grand number: RO1 N5038461. homeobox 2 (Gbx2) gene that is involved in determining *Correspondence to: Daniel H. Turnbull, Ph.D., Skirball Institute of Biomo- lecular Medicine, New York University School of Medicine, 540 First Ave- the position of the MHB region. Gbx2 null mutant mice nue, New York, NY 10016. E-mail: [email protected] die at birth with a major deletion of the anterior hind- Received 31 August 2012; revised 20 November 2012; accepted 21 brain, including the entire cerebellum, and a posterior November 2012. expansion of the midbrain (15). In contrast, Gbx2 condi- DOI 10.1002/mrm.24597 Published online 7 February 2013 in Wiley Online Library tional knockout (Gbx2-CKO) mice, in which Gbx2 (wileyonlinelibrary.com). expression is removed in the anterior hindbrain by VC 2013 Wiley Periodicals, Inc. 1707 1708 Szulc et al. embryonic day 9, are viable but smaller than wild-type Table 1 littermates. Interestingly, although Gbx2-CKO mice have Summary of Mice Used in the In Vivo MEMRI Studies a cerebellum, these mutants exhibit variable deletions of Number of datasets (P3–P11) the vermis, the central region of the cerebellum most per mouse affected in the majority of human hypoplasias (16). Genotype 12345 Total Previously, we demonstrated the feasibility of visualiz- WT 8223 419 ing early postnatal mouse cerebellum anatomy using 3D Gbx2-CKO 8223 318 in vivo manganese (Mn)-enhanced MRI (MEMRI), Number of mice including qualitative assessment of the variable deletions A combination of longitudinal and cross-sectional data was of the vermis in Gbx2-CKO mice during the second post- acquired from WT (N ¼ 19) and Gbx2-CKO (N ¼ 18) mice staged natal week (17). In this study, we further investigated between P3 and P11. Quantitative analyses were performed on Gbx2-CKO mice using 3D MEMRI imaging, analyzing data acquired from 15 WT and 15 Gbx2-CKO mice (text in bold), cerebellum and midbrain development in individual ani- where one to four image datasets were used for each mouse (see mals between postnatal day (P)3 and P11, with pheno- Supporting Information Table 1 for details). Additional longitudinal typic comparison to littermate controls. We applied de- studies (five datasets per mouse) were used for qualitative com- formation-based morphometry (DBM) approaches to parisons between WT (N ¼ 4) and Gbx2-CKO (N ¼ 3) mice. MEMRI images, which provided an unbiased method to detect and quantify defects in brain anatomy in Gbx2- CKO mice at early postnatal stages. Interestingly, DBM neonatal mouse was positioned in a custom-built holder analysis revealed novel phenotypes in both the deep cer- and anesthetized with 0.5–1% isoflurane in air delivered ebellar nuclei (DCN), the major output nuclei of the cere- via nosecone. Warm air was blown through the magnet bellar circuitry, as well as the flocculus-paraflocculus bore to maintain the mice at normal body temperature (Fl-Pfl), the vestibulo-cerebellum that is involved in con- during imaging. After MRI, neonatal mice recovered for trolling balance. These previously unreported defects 15–20 min in a separate cage before being returned to were both confirmed by histology. We further investi- their mothers and littermates. Tail polymerase chain gated the Gbx2-CKO vestibular phenotypes with ex vivo reaction was used to confirm the genotype of each mouse micro-MRI and revealed subtle changes in anatomy in being imaged, using previously reported primers and both the Fl-Pfl and the vestibulo-cochlear organ (VCO), conditions (16). the sensory organ that sends projections to this brain region. These phenotypes are difficult to appreciate without 3D imaging approaches and, taken together, our Imaging Methods results demonstrate the advantages of MRI approaches for 3D mutant phenotype analysis in the early postnatal MRI data were acquired using a 7-T micro-imaging sys- mouse brain. tem, 200-mm horizontal bore magnet (Magnex Scientific, Yarnton, UK) interfaced to a Bruker Biospec Avance II METHODS console (Bruker BioSpin MRI, Ettlingen, Germany) and equipped with actively shielded gradients (gradient Animals strength ¼ 750 mT/m; BGA9S, Bruker). A 25-mm (ID) All mice used in this study were maintained under pro- transmit/receive quadrature Litzcage coil (Doty Scien- tocols approved by the Institutional Animal Care and tific, Columbia, SC) was used to acquire the radiofre- Use Committee of New York University School of Medi- quency data. T1-weighted images were acquired using a cine. Contrast agent in the form of paramagnetic Mn2þ 3D gradient-echo pulse sequence (echo/repetition times, was delivered to neonates through lactation after mater- TE/TR ¼ 3.6/50 ms; flip angle ¼ 40 ; number of averages 3 nal intraperitoneal (IP) injection of MnCl2 solution (30 ¼ 2; field of view ¼ 2.56 Â 2.56 Â 2.56 cm ; and matrix mM solution in isotonic saline and 0.4 mmol/kg of body ¼ 256 Â 256 Â 256), achieving isotropic resolution of weight), equivalent