Overexpression screen of chromosome 21 genes reveals modulators of Sonic hedgehog signaling relevant to Down syndrome.

Trisomy 21 and mutations in the Sonic hedgehog (SHH) signaling pathway cause overlapping and pleiotropic phenotypes including cerebellar hypoplasia, craniofacial abnormalities, congenital heart defects and Hirschsprung disease. Trisomic cells derived from individuals with Down syndrome possess deficits in SHH signaling, suggesting that overexpression of human chromosome 21 genes may contribute to SHH-associated phenotypes by disrupting normal SHH signaling during development. However, chromosome 21 does not encode any known components of the canonical SHH pathway. Here, we sought to identify chromosome 21 genes that modulate SHH signaling by overexpressing 163 chromosome 21 cDNAs in a series of SHH-responsive mouse cell lines. We confirmed overexpression of trisomic candidate genes using RNA sequencing in the cerebella of Ts65Dn and TcMAC21 mice, model systems for Down syndrome. Our findings indicate that some human chromosome 21 genes, including DYRK1A, upregulate SHH signaling, whereas others, such as HMGN1, inhibit SHH signaling. Individual overexpression of four genes (B3GALT5, ETS2, HMGN1 and MIS18A) inhibits the SHH-dependent proliferation of primary granule cell precursors. Our study prioritizes dosage-sensitive chromosome 21 genes for future mechanistic studies. Identification of the genes that modulate SHH signaling may suggest new therapeutic avenues for ameliorating Down syndrome phenotypes.

Survey of Human Chromosome 21 Gene Expression Effects on Early Development in Danio rerio.

Trisomy for human chromosome 21 (Hsa21) results in Down syndrome (DS), one of the most genetically complex conditions compatible with human survival. Assessment of the physiological consequences of dosage-driven overexpression of individual Hsa21 genes during early embryogenesis and the resulting contributions to DS pathology in mammals are not tractable in a systematic way. A recent study looked at loss-of-function of a subset of Caenorhabditis elegans orthologs of Hsa21 genes and identified ten candidates with behavioral phenotypes, but the equivalent over-expression experiment has not been done. We turned to zebrafish as a developmental model and, using a number of surrogate phenotypes, we screened Hsa21 genes for effects on early embyrogenesis. We prepared a library of 164 cDNAs of conserved protein coding genes, injected mRNA into early embryos and evaluated up to 5 days post-fertilization (dpf). Twenty-four genes produced a gross morphological phenotype, 11 of which could be reproduced reliably. Seven of these gave a phenotype consistent with down regulation of the sonic hedgehog (Shh) pathway; two showed defects indicative of defective neural crest migration; one resulted consistently in pericardial edema; and one was embryonic lethal. Combinatorial injections of multiple Hsa21 genes revealed both additive and compensatory effects, supporting the notion that complex genetic relationships underlie end phenotypes of trisomy that produce DS. Together, our data suggest that this system is useful in the genetic dissection of dosage-sensitive gene effects on early development and can inform the contribution of both individual loci and their combinatorial effects to phenotypes relevant to the etiopathology of DS.

Defective cerebellar response to mitogenic Hedgehog signaling in Down [corrected] syndrome mice.

Trisomy 21 is the cause of Down [corrected] syndrome (DS) which is characterized by a number of phenotypes, including a brain which is small and hypocellular compared to that of euploid individuals. The cerebellum is disproportionately reduced. Ts65Dn mice are trisomic for orthologs of about half of the genes on human chromosome 21 and provide a genetic model for DS. These mice display a number of developmental anomalies analogous to those in DS, including a small cerebellum with a significantly decreased number of both granule and Purkinje cell neurons. Here we trace the origin of the granule cell deficit to precursors in early postnatal development, which show a substantially reduced mitogenic response to Hedgehog protein signaling. Purified cultures of trisomic granule cell precursors show a reduced but dose-dependent response to the Sonic hedgehog protein signal in vitro, demonstrating that this is a cell-autonomous deficit. Systemic treatment of newborn trisomic mice with a small molecule agonist of Hedgehog pathway activity increases mitosis and restores granule cell precursor populations in vivo. These results demonstrate a basis for and a potential therapeutic approach to a fundamental aspect of CNS pathology in DS.