Loss of function of parathyroid hormone receptor 1 induces Notch-dependent aortic defects during zebrafish vascular development.

OBJECTIVE: Coarctation of the aorta is rarely associated with known gene defects. Blomstrand chondrodysplasia, caused by mutations in the parathyroid hormone receptor 1 (PTHR1) is associated with coarctation of the aorta in some cases, although it is unclear whether PTHR1 deficiency causes coarctation of the aorta directly. The zebrafish allows the study of vascular development using approaches not possible in other models. We therefore examined the effect of loss of function of PTHR1 or its ligand parathyroid hormone-related peptide (PTHrP) on aortic formation in zebrafish. APPROACH AND RESULTS: Morpholino antisense oligonucleotide knockdown of either PTHR1 or PTHrP led to a localized occlusion of the mid-aorta in developing zebrafish. Confocal imaging of transgenic embryos showed that these defects were caused by loss of endothelium, rather than failure to lumenize. Using a Notch reporter transgenic ([CSL:Venus]qmc61), we found both PTHR1 and PTHrP knockdown-induced defective Notch signaling in the hypochord at the site of the aortic defect before onset of circulation, and the aortic occlusion was rescued by inducible Notch upregulation. CONCLUSIONS: Loss of function of either PTHR1 or PTHrP leads to a localized aortic defect that is Notch dependent. These findings may underlie the aortic defect seen in Blomstrand chondrodysplasia, and reveal a link between parathyroid hormone and Notch signaling during aortic development.

Diagnosing Smith-Magenis syndrome and duplication 17p11.2 syndrome by RAI1 gene copy number variation using quantitative real-time PCR.

Smith-Magenis syndrome (SMS) and duplication 17p11.2 (dup17p11.2) syndrome are multiple congenital anomalies/mental retardation disorders resulting from either a deletion or duplication of the 17p11.2 region, respectively. The retinoic acid induced 1 (RAI1) gene is the causative gene for SMS and is included in the 17p11.2 region of dup17p11.2 syndrome. Currently SMS and dup17p11.2 syndrome are diagnosed using a combination of clinically recognized phenotypes and molecular cytogenetic analyses such as fluorescent in situ hybridization (FISH). However, these methods have proven to be highly expensive, time consuming, and dependent upon the low resolving capabilities of the assay. To address the need for improved diagnostic methods for SMS and dup17p11.2 syndrome, we designed a quantitative real-time PCR (Q-PCR) assay that measures RAI1 copy number using the comparative C(t) method, DeltaDeltaC(t). We tested our assay with samples blinded to their previous SMS or dup17p11.2 syndrome status. In all cases, we were able to determine RAI1 copy number status and render a correct diagnosis accordingly. We validated these results by both FISH and multiplex ligation-dependent probe amplification (MLPA). We conclude that Q-PCR is an accurate, reproducible, low-cost, and reliable assay that can be employed for routine use in SMS and dup17p11.2 diagnosis.

Zebrafish tissue injury causes upregulation of interleukin-1 and caspase-dependent amplification of the inflammatory response.

Interleukin-1 (IL-1), the 'gatekeeper' of inflammation, is the apical cytokine in a signalling cascade that drives the early response to injury or infection. Expression, processing and secretion of IL-1 are tightly controlled, and dysregulated IL-1 signalling has been implicated in a number of pathologies ranging from atherosclerosis to complications of infection. Our understanding of these processes comes from in vitro monocytic cell culture models as lines or primary isolates, in which a range and spectra of IL-1 secretion mechanisms have been described. We therefore investigated whether zebrafish embryos provide a suitable in vivo model for studying IL-1-mediated inflammation. Structurally, zebrafish IL-1ß shares a ß-sheet-rich trefoil structure with its human counterpart. Functionally, leukocyte expression of IL-1ß was detectable only following injury, which activated leukocytes throughout zebrafish embryos. Migration of macrophages and neutrophils was attenuated by inhibitors of either caspase-1 or P2X7, which similarly inhibited the activation of NF-κB at the site of injury. Zebrafish offer a new and versatile model to study the IL-1ß pathway in inflammatory disease and should offer unique insights into IL-1 biology in vivo.

Tom1l2 hypomorphic mice exhibit increased incidence of infections and tumors and abnormal immunologic response.

Studies have shown that the TOM1 family of proteins, including TOM1 and TOM1L1, are actively involved in endosomal trafficking and function in the immune response. However, much less is known about the function of TOM1L2. To understand the biological importance of TOM1L2 and the potential significance of its cellular role, we created and evaluated Tom1l2 gene-trapped mice with reduced Tom1l2 expression. Mice hypomorphic for Tom1l2 exhibited numerous infections and tumors compared to wild-type littermates. Associated with this increased risk for infection and tumor formation, apparently healthy Tom1l2 hypomorphs also had splenomegaly, elevated B- and T-cell counts, and an impaired humoral response, although at a reduced penetrance. Furthermore, cellular localization studies showed that a Tom1l2-GFP fusion protein colocalizes with Golgi compartments, supporting the role of Tom1l2 in cellular trafficking, while molecular modeling and bioinformatic analysis of Tom1l2 illustrated a structural basis for a functional role in trafficking. These results indicate a role for Tom1l2 in the immune response and possibly in tumor suppression.