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

Abstract

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.

Keywords: Hsa21; over expression; zebrafish.

Figure 1

Flowchart showing steps of making the Hsa21 Gene Expression Clone-set and the screen in zebrafish.

Figure 2

Examples of phenotypes observed in the screen. Control embryos are on the left panel and injected embryos are on the right panel. Somites: RWDD2B 100pg injected embryos at 24 hpf with dashed lines to highlight somitic boundaries. Cyclopia: C21ORF84 100pg injected embryos at 5 dpf. Pigment cell migration: CCT8 100pg injected embryos at 4 dpf, arrows indicating melanocytes. Heart: JAM2 100pg injected embryos at 48 hpf.

Figure 3

Candidate genes (SOD1, RWDD2B, CCT8, and JAM2) coinjected with translational blocking morpholino. Hs-RWDD2B, Hs-CCT8 and Hs-SOD1 MOs were targeted against the human mRNA, while jam2 MOs were targeted against the zebrafish ortholog, DR-jam2. 100pg RNA was injected alone, 2 ng MO alone, or both were coinjected. * P < 0.05, ** P < 0.01. JAM2 data adapted from (Li et al. 2016).

Figure 4

Pairwise combinatorial injections of Shh candidate genes. C21ORF84 was coinjected with 6 other genes to look for synthetic effects. C21ORF84 was injected individually at 100pg RNA, the second gene was injected individually at 100pg RNA and then the two were injected together, 100pg each, for a total of 200pg RNA.