Syk and Zap-70 function redundantly to promote angioblast migration

AbstractSpleen tyrosine kinase (Syk) plays critical roles in B-cell and T-cell development, the maintenance of vascular integrity, and proper partitioning of the blood vascular and lymphatic vascular system. Here, we utilize the zebrafish as an in vivo system to demonstrate novel roles for Syk and the related kinase Zeta associated protein (Zap-70) in promoting angioblast migration. Partial knockdown of either gene results in early angiogenic delay of the intersegmental vessels, dorsal intersegmental vessel patterning defects, and partial loss of the thoracic duct. Higher dose knockdown of both genes results in little to no angiogenic sprouting of the intersegmental vessels, a phenotype which resembles knockdown of vegfa. Di-phosphorylated ERK, an effector of the vegfa pathway, is also downregulated in the aorta of syk:zap double morphants. Over-expression of syk under the control of a blood-specific or vascular-specific promoter rescues sprouting defects after loss of vegfa. Together these results suggest that syk and zap-70 function redundantly in an early progenitor to promote the migration of intersegmental vessel angioblasts and lymphangioblasts that contribute to the thoracic duct, either downstream of, or in parallel to vegfa

Variation in the structure and function of invertebrate-associated bacterial communities

Microorganisms are intricately involved in the ecology of many insects, often contributing to host fitness and forming evolutionarily stable associations. The interactions between hosts and microbes can significantly alter their evolutionary trajectories, enabling them to adapt to novel environmental conditions. In this thesis I have examined how host ecology can shape the interactions of bacteria with insects of agricultural and epidemiological importance. I have described the bacterial communities associated with Bactrocera oleae (the olive fruit fly) and generated draft genome sequences for several members of the gut microbiota, including the symbiotic bacterium “Candidatus Erwinia dacicola”. Comparative genomic analyses indicate that Ca. E. dacicola and a novel facultative bacterium Tatumella TA1 may perform key nutritional functions for the host, including the synthesis of essential amino acids and ammonia assimilation from host nitrogenous waste products. Tatumella TA1 is consistently associated with all life stages of populations collected in Israel and Crete at low relative abundance, and encodes large adhesion proteins that may assist in attachment to the host epithelium or other members of the microbiota in the B. oleae gut. I have also examined the variation in frequency and relative abundance of facultative microbes that infect several Glossina spp. (the tsetse fly): the sole vector of African trypanosomes in Sub-Saharan Africa. In addition to three vertically transmitted endosymbionts (Wigglesworthia, Sodalis, and Wolbachia), tsetse flies are infected with two additional potential reproductive manipulators: Spiroplasma and Rickettsia, and a novel strain of Klebsiella. The draft genomes generated for these taxa over the course of this thesis provide the opportunity for future studies in to their role in host biology and how community interactions can shape the transmission and evolutionary dynamics of host-associated microbes

Lack of co-localization of mural cell and the ectomesenchymal neural crest marker Fli1a.

<p>Confocal images of 4 and 7(acta2:mCherry) and nuclear neural crest marker (fli1a:nEGFP^y7) using ventrally staged embryos. (A) Mural cell and neural crest markers are expressed along the ventral aorta (A’) and aortic arch artery region (A”) of the 4 dpf embryo. (B) Mural cell and neural crest markers are expressed along the ventral aorta (B’) and aortic arch region (B”) at 7 dpf. There appears to be little to no co-localization of fluorescent markers at both 4 and 7 dpf. Scale bar in A represents 100 µm. Insets (A’, A”, B’, B”) are 100 µm in length. VA =  Ventral Aorta, AAA =  Aortic Arch Arteries. Arrowheads depict cells that no do not co-localize.</p

Morphology of vascular and visceral mural cells in acta2:EGFP transgenic fish.

<p>(A) Ventral pharyngeal region of a 4 dpf double transgenic Tg(acta2:EGFP)^ca7; Tg(kdrl:mCherry)^ci5 (mural cells are green and endothelial cells are red) zebrafish shows extensive mural cell coverage of the ventral aorta (VA) and lesser coverage on the smaller aortic arches (AA) or opercular artery (ORA). (B) Wholemount adult ventral aorta and attached afferent branchial arteries shows extensive smooth muscle coverage. (C) Lateral view of the gut (g) and swim bladder (b) of a 14 dpf double transgenic Tg(acta2:EGFP)^ca7; Tg(kdrl:mCherry)^ci5 zebrafish shows radial and circumferential smooth muscle on both gut and swim bladder, but sparse mural cells on the dorsal aorta (DA) and no visible cells on the posterior cardinal vein (PCV). Scale bar in A represents 25 µm. Scale bar in B and C represents 100 µm.</p

Vascular mural cells of the ventral head are very stable over time.

<p>(A–E) Single images taken from a confocal microscopy timelapse video. Images were collected at 102 hpf (A) and every three hours for 12 hours (B–E). Insets (A’–E’) show a higher magnification of the ventral aorta, where mural cells that are present at the beginning of the timelapse are still present at the end of the timelapse with no cytokinesis. Arrowheads depict mural cells throughout the timelapse that appear to have little movement. Scale bar represents 100 µm.</p

Acta2 promoter/enhancer construct design and expression in zebrafish.

<p>(A) A zebrafish (Dr) enhancer/promoter construct was constructed from the proximal promoter and first intron sequence of the zebrafish <i>acta2</i> gene, and contains three highly conserved CArG binding sites also found in the mouse (Mm) <i>acta2</i> proximal promoter and first intron. (B) Comparison of zebrafish CaRG boxes A and B in zebrafish, tilapia and medaka. (C,D) By wholemount in situ hybridization, <i>acta2</i> shows strong expression in the gut (g) at 72 hpf (B), and expressed in the gut, swim bladder (sb), ventral aorta (va), floor plate (fp), aortic arch arteries (aaa), and bulbus arteriosus (ba) at 100 hpf (C). (E,F) Co-localization of wholemount in situ hybridization <i>acta2</i> and anti-GFP staining of the acta2:GFP transgene shows strong expression in the aortic arch arteries (aaa) at 100 hpf. (G,H,I) 4 dpf acta2:EGFP transgenic fish (H) stained with Tagln rabbit polyclonal antibody (G). Merge (I) shows co-localization between acta2:GFP and Tagln. Arrowheads in G–I depict vascular mural cells. Scale bar in G represents 20 µm.</p

Mural cell and endothelial development in the ventral head of larval zebrafish.

<p>Confocal micrographs collected from a ventral point of view show a progressive increase in vessel complexity (red, A, C, E, G) and in density of mural cell coverage of aortic arch vessels (green, B, D, F, H) from 4 dpf (A, B), 7 dpf (C, D), 11 dpf (E, F) through 14 dpf (G, H). Heart expression of acta2:EGFP is maintained. aaa =  aortic arch arteries; va =  ventral aorta; ba =  bulbus arteriosus. Scale bar in A represents 100 µm.</p

Vascular and visceral mural and smooth muscle cells in the trunk.

<p>(A–B) At 4 dpf, acta2:EGFP positive cells (arrows in B) are seen in the ventral portion of the dorsal aorta, but not in other vessels of the trunk. Floor plate (fp) expression of acta2:EGFP is observed in all images. (C–D) At 14 dpf, the distribution of vascular mural cells to the ventral portion of the dorsal aorta only, is still observed. (E) In contrast to the scarce vascular smooth muscle coverage, visceral smooth muscle cells strongly express the acta2:EGFP transgene at 80 hpf. Scale bars represent 100 µm. Green striations are skeletal muscle fibres.</p

Smooth muscle markers are restricted to the developing cardiac outflow tract by 56 hpf.

<p>(A) At 56 hpf, <i>acta2</i> expression is restricted to the developing BA. (B,C) Double transgenic Tg(acta2:EGFP)^ca7; Tg(kdrl:mCherry)^ci5 embryo shows expression of EGFP in both the atrium and ventricle of the heart at 56 hpf, but not in the BA. (D) <i>acta2</i> expression is evident at 78 hpf in the BA in both wholemount and cross section (E) and in transgenic animals (F). (G–I) Expression of <i>acta2</i> continues to be restricted to the BA and ventral aorta (VA) at 100 hpf by in situ hybridization and in transgenic fish. (J–O): Cross sections of the 22 dpf BA show a multilamellar arterial phenotype as visualized by hematoxylin and eosin staining (J), in situ hybridization of <i>acta2</i> (K) and transgenic GFP (nuclei stained blue with DAPI, L). The bulbus vascular wall consists of three layers: an inner intima, middle media, and outer adventitia (Ad, separated by black lines in J). The intima is endothelial (arrowheads point to nuclei of endothelial cells). The media consists of 3–4 cell-thick layers of vascular smooth muscle cells (M, arrows point to nuclei of SMCs). In comparison to the BA, the vascular wall of the VA at 22 dpf is thin (M) but expresses <i>acta2</i> by in situ hybridization (N) and GFP in transgenic animals (O). The endothelium of VA is covered by a thin layer of SMCs (arrowheads point to nuclei of SMCs). (P) In situ hybridization of the wholemount adult heart shows strong staining in the bulbus arteriosus, but not ventricle or atrium, which is localized to the myocardial wall in cross section (Q). (R) Wholemount dissected acta2:EGFP transgenic heart shows stronger expression of GFP in the bulbus arteriosus as compared to ventricle. Staining is also continuous with the ventral aorta. In B,C, F, I, and R, green expression is acta2:EGFP transgene. Scale bar in B, C, F, and I is 100 µm. Scale bar in E, H, and Q is 50 µm. Scale bar in K, L, N, and O is 20 µm.</p