Beth L. Roman

Interaction between alk1 and blood flow in the development of arteriovenous malformations.

Arteriovenous malformations (AVMs) are fragile direct connections between arteries and veins that arise during times of active angiogenesis. To understand the etiology of AVMs and the role of blood flow in their development, we analyzed AVM development in zebrafish embryos harboring a mutation in activin receptor-like kinase I (alk1), which encodes a TGFβ family type I receptor implicated in the human vascular disorder hereditary hemorrhagic telangiectasia type 2 (HHT2). Our analyses demonstrate that increases in arterial caliber, which stem in part from increased cell number and in part from decreased cell density, precede AVM development, and that AVMs represent enlargement and stabilization of normally transient arteriovenous connections. Whereas initial increases in endothelial cell number are independent of blood flow, later increases, as well as AVMs, are dependent on flow. Furthermore, we demonstrate that alk1 expression requires blood flow, and despite normal levels of shear stress, some flow-responsive genes are dysregulated in alk1 mutant arterial endothelial cells. Taken together, our results suggest that Alk1 plays a role in transducing hemodynamic forces into a biochemical signal required to limit nascent vessel caliber, and support a novel two-step model for HHT-associated AVM development in which pathological arterial enlargement and consequent altered blood flow precipitate a flow-dependent adaptive response involving retention of normally transient arteriovenous connections, thereby generating AVMs.

Assembly and patterning of the vascular network of the vertebrate hindbrain

The cranial vasculature is essential for the survival and development of the central nervous system and is important in stroke and other brain pathologies. Cranial vessels form in a reproducible and evolutionarily conserved manner, but the process by which these vessels assemble and acquire their stereotypic patterning remains unclear. Here, we examine the stepwise assembly and patterning of the vascular network of the zebrafish hindbrain. The major artery supplying the hindbrain, the basilar artery, runs along the ventral keel of the hindbrain in all vertebrates. We show that this artery forms by a novel process of medial sprouting and migration of endothelial cells from a bilateral pair of primitive veins, the primordial hindbrain channels. Subsequently, a second wave of dorsal sprouting from the primordial hindbrain channels gives rise to angiogenic central arteries that penetrate into and innervate the hindbrain. The chemokine receptor cxcr4a is expressed in migrating endothelial cells of the primordial hindbrain channels, whereas its ligand cxcl12b is expressed in the hindbrain neural keel immediately adjacent to the assembling basilar artery. Knockdown of either cxcl12b or cxcr4a results in defects in basilar artery formation, showing that the assembly and patterning of this crucial artery depends on chemokine signaling.

Context-Dependent Proangiogenic Function of Bone Morphogenetic Protein Signaling Is Mediated by Disabled Homolog 2

Bone morphogenetic proteins (BMPs) have diverse functions during development in vertebrates. We have recently shown that BMP2 signaling promotes venous-specific angiogenesis in zebrafish embryos. However, factors that confer a context-dependent proangiogenic function of BMP2 signaling within endothelial cells need to be identified. Here, we report that Disabled homolog 2 (Dab2), a cargo-specific adaptor protein for Clathrin, is essential to mediate the proangiogenic function of BMP2 signaling. We find that inhibition of Dab2 attenuates internalization of BMP receptors and abrogates the proangiogenic effects of BMP signaling in endothelial cells. Moreover, inhibition of Dab2 decreases phosphorylation of SMAD-1, 5, and 8, indicating that Dab2 plays an essential role in determining the outcome of BMP signaling within endothelial cells and may provide a molecular basis for a context-dependent proangiogenic function of BMP2 signaling.

Circulating Bmp10 acts through endothelial Alk1 to mediate flow-dependent arterial quiescence

Blood flow plays crucial roles in vascular development, remodeling and homeostasis, but the molecular pathways required for transducing flow signals are not well understood. In zebrafish embryos, arterial expression of activin receptor-like kinase 1 (alk1), which encodes a TGFβ family type I receptor, is dependent on blood flow, and loss of alk1 mimics lack of blood flow in terms of dysregulation of a subset of flow-responsive arterial genes and increased arterial endothelial cell number. These data suggest that blood flow activates Alk1 signaling to promote a flow-responsive gene expression program that limits nascent arterial caliber. Here, we demonstrate that restoration of endothelial alk1 expression to flow-deprived arteries fails to rescue Alk1 activity or normalize arterial endothelial cell gene expression or number, implying that blood flow may play an additional role in Alk1 signaling independent of alk1 induction. To this end, we define cardiac-derived Bmp10 as the crucial ligand for endothelial Alk1 in embryonic vascular development, and provide evidence that circulating Bmp10 acts through endothelial Alk1 to limit endothelial cell number in and thereby stabilize the caliber of nascent arteries. Thus, blood flow promotes Alk1 activity by concomitantly inducing alk1 expression and distributing Bmp10, thereby reinforcing this signaling pathway, which functions to limit arterial caliber at the onset of flow. Because mutations in ALK1 cause arteriovenous malformations (AVMs), our findings suggest that an impaired flow response initiates AVM development.

Intravenous Microinjections of Zebrafish Larvae to Study Acute Kidney Injury

In this video article we describe a zebrafish model of AKI using gentamicin as the nephrotoxicant. The technique consists of intravenous microinjections on 2 dpf zebrafish. This technique represents an efficient and rapid method to deliver soluble substances into the bloodstream of zebrafish larvae, allowing for the injection of 15-20 fish per hour. In addition to AKI studies, this microinjection technique can also be used for other types of experimental studies such as angiography. We provide a detailed protocol of the technique from equipment required to visual measures of decreased kidney function. In addition, we also demonstrate the process of fixation, whole mount immunohistochemistry with a kidney tubule marker, plastic embedding and sectioning of the larval zebrafish. We demonstrate that zebrafish larvae injected with gentamicin show morphological features consistent with AKI: edema, loss of cell polarity in proximal tubular epithelial cells, and morphological disruption of the tubule.

Loss of unc45a precipitates arteriovenous shunting in the aortic arches

Aortic arch malformations are common congenital disorders that are frequently of unknown etiology. To gain insight into the factors that guide branchial aortic arch development, we examined the process by which these vessels assemble in wild type zebrafish embryos and in kurzschluss(tr12) (kus(tr12)) mutants. In wild type embryos, each branchial aortic arch first appears as an island of angioblasts in the lateral pharyngeal mesoderm, then elaborates by angiogenesis to connect to the lateral dorsal aorta and ventral aorta. In kus(tr12) mutants, angioblast formation and initial sprouting are normal, but aortic arches 5 and 6 fail to form a lumenized connection to the lateral dorsal aorta. Blood enters these blind-ending vessels from the ventral aorta, distending the arteries and precipitating fusion with an adjacent vein. This arteriovenous malformation (AVM), which shunts nearly all blood directly back to the heart, is not exclusively genetically programmed, as its formation correlates with blood flow and aortic arch enlargement. By positional cloning, we have identified a nonsense mutation in unc45a in kus(tr12) mutants. Our results are the first to ascribe a role for Unc45a, a putative myosin chaperone, in vertebrate development, and identify a novel mechanism by which an AVM can form.

Dynamic analysis of BMP-responsive smad activity in live zebrafish embryos

Bone morphogenetic proteins (BMPs) are critical players in development and disease, regulating such diverse processes as dorsoventral patterning, palate formation, and ossification. These ligands are classically considered to signal via BMP receptor‐specific Smad proteins 1, 5, and 8. To determine the spatiotemporal pattern of Smad1/5/8 activity and thus canonical BMP signaling in the developing zebrafish embryo, we generated a transgenic line expressing EGFP under the control of a BMP‐responsive element. EGFP is expressed in many established BMP signaling domains and is responsive to alterations in BMP type I receptor activity and smad1 and smad5 expression. This transgenic Smad1/5/8 reporter line will be useful for determining ligand and receptor requirements for specific domains of BMP activity, as well as for genetic and pharmacological screens aimed at identifying enhancers or suppressors of canonical BMP signaling. Developmental Dynamics 240:682–694, 2011.

Moving Domain Computational Fluid Dynamics to Interface with an Embryonic Model of Cardiac Morphogenesis

Peristaltic contraction of the embryonic heart tube produces time- and spatial-varying wall shear stress (WSS) and pressure gradients (∇P) across the atrioventricular (AV) canal. Zebrafish (Danio rerio) are a genetically tractable system to investigate cardiac morphogenesis. The use of Tg(fli1a:EGFP)y1 transgenic embryos allowed for delineation and two-dimensional reconstruction of the endocardium. This time-varying wall motion was then prescribed in a two-dimensional moving domain computational fluid dynamics (CFD) model, providing new insights into spatial and temporal variations in WSS and ∇P during cardiac development. The CFD simulations were validated with particle image velocimetry (PIV) across the atrioventricular (AV) canal, revealing an increase in both velocities and heart rates, but a decrease in the duration of atrial systole from early to later stages. At 20-30 hours post fertilization (hpf), simulation results revealed bidirectional WSS across the AV canal in the heart tube in response to peristaltic motion of the wall. At 40-50 hpf, the tube structure undergoes cardiac looping, accompanied by a nearly 3-fold increase in WSS magnitude. At 110-120 hpf, distinct AV valve, atrium, ventricle, and bulbus arteriosus form, accompanied by incremental increases in both WSS magnitude and ∇P, but a decrease in bi-directional flow. Laminar flow develops across the AV canal at 20-30 hpf, and persists at 110-120 hpf. Reynolds numbers at the AV canal increase from 0.07±0.03 at 20-30 hpf to 0.23±0.07 at 110-120 hpf (p< 0.05, n=6), whereas Womersley numbers remain relatively unchanged from 0.11 to 0.13. Our moving domain simulations highlights hemodynamic changes in relation to cardiac morphogenesis; thereby, providing a 2-D quantitative approach to complement imaging analysis.

Mechanotransduction in embryonic vascular development

A plethora of biochemical signals provides spatial and temporal cues that carefully orchestrate the complex process of vertebrate embryonic development. The embryonic vasculature develops not only in the context of these biochemical cues, but also in the context of the biomechanical forces imparted by blood flow. In the mature vasculature, different blood flow regimes induce distinct genetic programs, and significant progress has been made toward understanding how these forces are perceived by endothelial cells and transduced into biochemical signals. However, it cannot be assumed that paradigms that govern the mature vasculature are pertinent to the developing embryonic vasculature. The embryonic vasculature can respond to the mechanical forces of blood flow, and these responses are critical in vascular remodeling, certain aspects of sprouting angiogenesis, and maintenance of arterial–venous identity. Here, we review data regarding mechanistic aspects of endothelial cell mechanotransduction, with a focus on the response to shear stress, and elaborate upon the multifarious effects of shear stress on the embryonic vasculature. In addition, we discuss emerging predictive vascular growth models and highlight the prospect of combining signaling pathway information with computational modeling. We assert that correlation of precise measurements of hemodynamic parameters with effects on endothelial cell gene expression and cell behavior is required for fully understanding how blood flow-induced loading governs normal vascular development and shapes congenital cardiovascular abnormalities.

Analysis of early embryonic great-vessel microcirculation in zebrafish using high-speed confocal μPIV.

In the developing cardiovascular system, hemodynamic vascular loading is critical for angiogenesis and cardiovascular adaptation. Normal zebrafish embryos with transgenically-labeled endothelial and red blood cells provide an excellent in vivo model for studying the fluid-flow induced vascular loading. To characterize the developmental hemodynamics of early embryonic great-vessel microcirculation in the zebrafish embryo, two complementary studies (experimental and numerical) are presented. Quantitative comparison of the wall shear stress (WSS) at the first aortic arch (AA1) of wild-type zebrafish embryos during two consecutive developmental stages is presented, using time-resolved confocal micro-particle image velocimetry (μPIV). Analysis showed that there was significant WSS difference between 32 and 48 h post-fertilization (hpf) wild-type embryos, which correlates with normal arch morphogenesis. The vascular distensibility of the arch wall at systole and the acceleration/deceleration rates of time-lapse phase-averaged streamwise blood flow curves were also analyzed. To estimate the influence of a novel intermittent red-blood cell (RBC) loading on the endothelium, a numerical two-phase, volume of fluid (VOF) flow model was further developed with realistic in vivo conditions. These studies showed that near-wall effects and cell clustering increased WSS augmentation at a minimum of 15% when the distance of RBC from arch vessel wall was less than 3 μm or when RBC cell-to-cell distance was less than 3 μm. When compared to a smooth wall, the WSS augmentation increased by a factor of ~1.4 due to the roughness of the wall created by the endothelial cell profile. These results quantitatively highlight the contribution of individual RBC flow patterns on endothelial WSS in great-vessel microcirculation and will benefit the quantitative understanding of mechanotransduction in embryonic great vessel biology, including arteriovenous malformations (AVM).