Clinical data of the proband and his family.

A Brain MRI of proband performed at aged 5 months (T1-weighted image: i – sagittal, ii – coronal, iii/iv – axial) demonstrating prominent hypoplasia of all cerebellar structures, hypoplastic pons, generalized mild cerebral atrophy, thin corpus callosum, and delayed myelination. B Brain MRI of proband’s brother aged 4 years, 5 months. (T1-weighted image: i – sagittal, ii – coronal) demonstrating profound hypoplastic cerebellum including the vermis and the pons with a thin corpus callosum. Axial T2/FLAIR- weighted image (iii/iv) shows brain atrophy, diffuse diminished white matter, very small basal ganglia with high T2/FLAIR signal signifying damage/gliosis, with preserved thalamus structure. C Inter-ictal EEG recording from the proband demonstrating dysrhythmia and multifocal epileptiform activity. D Chromatograms of the proband and his parents. Top – the heterozygous parents, bottom – the homozygous proband. E Pedigree of affected family. Solid black squares indicate affected individuals, wt – wild type, m - c.416T > C MED29 variant.

Zebrafish knockdown modeling and rescue.

A In situ hybridization using antibodies for MED17 and MED29 in a zebrafish embryo model, demonstrating restricted brain expression pattern for both. AS – antisense, S – sense. B Immunohistochemistry staining of the zebrafish MED17, MED29, and P53 morphants using PValb7 antibodies directed at the GABAergic Purkinje cerebellar cells and Vglut1a antibodies directed at the Glutamatergic Granule cerebellar cells. Left panels – coronal sections, right panels – sagittal sections. WT – wild type. The arrows signify the location of the midbrain-hindbrain boundary (MHB) from which the cerebellar structures develop. MED17 and MED29 Morphants exhibit prominent reduced staining of GABAergic Purkinje neurons, but a much lesser reduction in staining of glutamatergic granule neurons. C Quantitative analysis of the relative expression GABAergic Purkinje cerebellar cells (on the left) and Glutamatergic Granule cerebellar cells (on the right) in the wt, and MED29 and MED17 morphants, by measuring mRNA levels of prototypical genes by rtPCR. A significant reduction of the expression of genes typical to Purkinje cells is seen in both MED29 and MED17 morphants, however, there is no reduction in the expression of genes typical to Granules cells. D Examples of immunohistochemistry with PValb7 staining, demonstrating normal cerebellar expression in the un-injected (UI) control larvae (left image), absent cerebellar expression in the MED29 morphant (second from left), and varying degrees of increasing cerebellar expression designated as weak and strong (two panels on the right) following rescue with a plasmid containing human wt MED29. E Quantitative analysis of the relative expression of pvalb7 gene typical for GABAergic Purkinje cerebellar cells (measured by rtPCR), show normal levels in the wt control larva (left), significantly decreased levels in the MED29 morphant (middle), and an increase in expression in the MED29 morphant following rescue with a human wt MED29, to levels that are not significantly different from the control wt. F Statistical analysis of the proportion of larvae demonstrating a positive touch response in the MED29 morphants (top bar) and MED29 morphants following rescue with plasmids containing human wt MED29 in low concentrations of 5 ng/μl (middle bar) and higher concentrations of 10 ng/μl (bottom bar), showing significantly increased fraction of larva demonstrating positive touch response following rescue with a high concentration but not a low concentration. G Statistical analysis of the proportion of larvae demonstrating a “strong” cerebellar expression (indicated by pvalb7 staining as shown in D among the MED29 morphants (top bar) and MED29 morphants following rescue with plasmids containing human wt MED29 in low concentrations of 5 ng/μl (middle bar) and higher concentrations of 10 ng/μl (bottom bar), showing significantly increased fraction of larva with strong cerebellar expression following rescue with a high concentration but not a low concentration.

Increased and decreased expression levels of MED29 affect neuronal development in culture; reduced expression levels of MED29 affects neuronal migration in vivo.

A Representative confocal images of mouse primary hippocampal neurons transfected with empty vector control (left panel), MED29^WT (middle panel) or MED29^Leu139Pro (right panel). B Analysis of the total neurite length (left panel) and arborization (right panel) normalized to control. Overexpression of MED29WT resulted in a trend towards increased neurite length, and significant increase in arborization, compared to empty vector. Overexpression of MED29Leu139Pro showed similar neurite length and arborization as the empty vector control, and was significantly different compared to MED29WT in the number of branches. C Representative confocal images of mouse primary hippocampal neurons transfected with control shRNA (left panel) or Med29 shRNA (right panel). D Analysis of the total neurite length (left panel) and arborization (right panel) following MED29 shRNA normalized to control shRNA. Knockdown of Med29 impaired neuronal development, resulting in significantly shorter dendrites and reduced arborization, compared to control shRNA. Data are presented as mean ± SEM. The number of neurons and number of batches used for analysis for each condition are indicated in the methods section. **p < 0.01. ***p < 0.001. E Representative images from mouse brains at postnatal day 1, showing the transfected cells (tdTomato+) from the subventricular zone (SVZ) to the cortical plate (CP) for the empty vector control (left panel), MED29^WT (middle panel), and MED29^Leu139Pro (right panel). F Left, cumulative distribution of the transfected neurons at P1 from the cortical plate (CP) to the intermediate zone (IZ). Right, percentage of neurons reaching the superficial layers of the cortex, showing a small but significant reduction in migration following electroporation with the MED29^Leu139Pro vector. G Representative images from mouse brains at postnatal day 1 electroporated with control shRNA or Med29 shRNA, showing the transfected cells (mRFP+) from the SVZ to the cortical plate (CP). H Left, cumulative distribution of the transfected neurons at P1 from the cortical plate (CP) to the intermediate zone (IZ). Right, percentage of neurons reaching the superficial layers of the cortex, demonstrating a significant reduction in migration following electroporation with Med29ShRNA. Data are presented as mean ± SEM. Number of images analyzed for each condition is indicated in the methods section. *p < 0.05, ***p < 0.001.