Phosphoproteomics-Mediated Identification of Fer Kinase as a Target of Mutant Shp2 in Noonan and LEOPARD Syndrome

Abstract

Noonan syndrome (NS) and LEOPARD syndrome (LS) cause congenital afflictions such as short stature, hypertelorism and heart defects. More than 50% of NS and almost all of LS cases are caused by activating and inactivating mutations of the phosphatase Shp2, respectively. How these biochemically opposing mutations lead to similar clinical outcomes is not clear. Using zebrafish models of NS and LS and mass spectrometry-based phosphotyrosine proteomics, we identified a down-regulated peptide of Fer kinase in both NS and LS. Further investigation showed a role for Fer during development, where morpholino-based knockdown caused craniofacial defects, heart edema and short stature. During gastrulation, loss of Fer caused convergence and extension defects without affecting cell fate. Moreover, Fer knockdown cooperated with NS and LS, but not wild type Shp2 to induce developmental defects, suggesting a role for Fer in the pathogenesis of both NS and LS.

Dual Roles of Fer Kinase Are Required for Proper Hematopoiesis and Vascular Endothelium Organization during Zebrafish Development

“…We generated gene specific probes to fer cDNA for in situ hybridization, to determine the spatio-temporal expression during development. Similar to what was reported by Paardekooper Overman et al [ 20 ], we found fer to be expressed ubiquitously until approximately ten somites, when it became more specifically concentrated toward the head region. However, we also observed fer expression in the primordial region of the dorsal aorta (DA)/cardinal vein (CV), when compared to a fer antisense probe negative control (In Figure 1 B,C, respectively—24 hpf is shown).…”

“…Improper splicing with the fer MO was confirmed using PCR to amplify the region spanning exon 9, using a forward primer in exon 8 (5′-AGTCCACCACAGAGGAGCTG-3′) and a reverse primer in exon 10 (5′-AGTCTGTCCTTGGCTCTTCG-3′) (Figure 2I). Although not shown here, experiments repeated with MOs and PCR primers, as reported by Paardekooper Overman, et al (P-O MO), confirmed specificities for the Fer gene were consistent with data obtained using our fer -MO1 (Fer protein knockdown with both MOs is shown in Supplementary Figure S1 as fer -MO1 and P-O MO) [ 20 ]. Morpholino knockdown was confirmed using a Western blot analysis, using an anti-Fer monoclonal antibody (Cell Signaling).…”

“…In this study, we characterized a novel expression pattern and functional component to Fer during zebrafish embryonic development, which has not previously reported. We report here that not only is Fer required for proper axis determination and formation of head structures during embryogenesis (as reported by [ 20 ], but also for proper organization of the vascular endothelium and hematopoietic proliferation in the trunk region. The absence of Fer results in disorganized trunk vasculature within the region of the cardinal vein (CV), dorsal aorta (DA) and intersegmental vessels (ISV), resulting in subsequent loss of erythrocyte circulation.…”

“…Interestingly, there is greater variability among invertebrates regarding different versions of Fer, such that while fruit flies have been shown to express both versions [ 18 ]; nematodes only express the truncated, but not the full length, version of Fer [ 16 ]. Recently, the full length version of Fer has been shown to be expressed during zebrafish development in a ubiquitous manner early, while later concentrating to the head region and fin buds [ 20 ]. In addition, the same study reported developmental defects ranging from convergent extension to craniofacial defects when knocking down Fer using morpholino technology [ 20 ].…”

“…Recently, the full length version of Fer has been shown to be expressed during zebrafish development in a ubiquitous manner early, while later concentrating to the head region and fin buds [ 20 ]. In addition, the same study reported developmental defects ranging from convergent extension to craniofacial defects when knocking down Fer using morpholino technology [ 20 ].…”