Doctor of Philosophy

dissertationHeparan sulfate proteoglycans (HSPGs) are biologically relevant molecules composed of core proteins and glycosaminoglycan (GAG) chains. The location of HSPGs, on the cell surface or in the extracellular matrix, and their structural heterogeneity place them at a unique advantage to influence signaling pathways and cellcell or cell-matrix interactions. Most of the genes that code for the core proteins and the enzymes that build and modify the GAG chains have been identified. One type of modification, 3-O-sulfation, is catalyzed by a family of 3-O-sulfotransferases (3-OSTs) in zebrafish. Gene expression studies suggest they could modulate different steps of zebrafish development, but the specific roles of each 3-OST have not been elucidated. My dissertation focused on the functions a particular 3-OST, 3-OST-7, perform in zebrafish heart development. To elucidate the functions of 3-OST-7 in zebrafish heart development, I knocked down 3-OST-7 using morpholinos and found that 3-OST-7 controls ventricular contraction. Analysis of the noncontracting ventricle phenotype in 3-OST-7 morphants demonstrated that tropomyosin4 is required to mediate 3-OST-7 regulation of ventricular contraction, placing 3-OST-7 upstream of a novel pathway that controls coordinated sarcomere assembly. Further analysis of the 3-OST-7 knockdown model illustrated that 3-OST-7 functions in two distinct pathways that regulate ventricular maturation. First, 3-OST-7 is necessary for transforming the isometric ventricular cardiomyocytes into their elongated form. Second, 3-OST-7 shares a feedback loop with bmp4 signaling to regulate contraction. These findings demonstrated the specificity of 3-OST-7 action towards influencing cardiac development. Moreover, these studies highlighted the strength of the 3-OST-7 knockdown model in teasing apart the relationship between structure and function. I anticipate that further investigation of this model could advance our understanding of the interrelationships between HSPGs and the signaling pathways that orchestrate cardiac formation

Nascent adhesions shorten the period of lamellipodium protrusion through the Brownian ratchet mechanism

Directional cell migration is driven by the conversion of oscillating edge motion into lasting periods of leading edge protrusion. Actin polymerization against the membrane and adhesions control edge motion, but the exact mechanisms that determine protrusion period remain elusive. We addressed this by developing a computational model in which polymerization of actin filaments against a deformable membrane and variable adhesion dynamics support edge motion. Consistent with previous reports, our model showed that actin polymerization and adhesion lifetime power protrusion velocity. However, increasing adhesion lifetime decreased the protrusion period. Measurements of adhesion lifetime and edge motion in migrating cells confirmed that adhesion lifetime is associated with and promotes protrusion velocity, but decreased duration. Our model showed that adhesions\u27 control of protrusion persistence originates from the Brownian ratchet mechanism for actin filament polymerization. With longer adhesion lifetime or increased adhesion density, the proportion of actin filaments tethered to the substrate increased, maintaining filaments against the cell membrane. The reduced filament-membrane distance generated pushing force for high edge velocity, but limited further polymerization needed for protrusion duration. We propose a mechanism for cell edge protrusion in which adhesion strength regulates actin filament polymerization to control the periods of leading edge protrusion. [Media: see text]

3-OST-7 regulates BMP-dependent cardiac contraction.

The 3-O-sulfotransferase (3-OST) family catalyzes rare modifications of glycosaminoglycan chains on heparan sulfate proteoglycans, yet their biological functions are largely unknown. Knockdown of 3-OST-7 in zebrafish uncouples cardiac ventricular contraction from normal calcium cycling and electrophysiology by reducing tropomyosin4 (tpm4) expression. Normal 3-OST-7 activity prevents the expansion of BMP signaling into ventricular myocytes, and ectopic activation of BMP mimics the ventricular noncontraction phenotype seen in 3-OST-7 depleted embryos. In 3-OST-7 morphants, ventricular contraction can be rescued by overexpression of tropomyosin tpm4 but not by troponin tnnt2, indicating that tpm4 serves as a lynchpin for ventricular sarcomere organization downstream of 3-OST-7. Contraction can be rescued by expression of 3-OST-7 in endocardium, or by genetic loss of bmp4. Strikingly, BMP misregulation seen in 3-OST-7 morphants also occurs in multiple cardiac noncontraction models, including potassium voltage-gated channel gene, kcnh2, affected in Romano-Ward syndrome and long-QT syndrome, and cardiac troponin T gene, tnnt2, affected in human cardiomyopathies. Together these results reveal 3-OST-7 as a key component of a novel pathway that constrains BMP signaling from ventricular myocytes, coordinates sarcomere assembly, and promotes cardiac contractile function

3-OST-7 Regulates BMP-Dependent Cardiac Contraction

<div><p>The 3-O-sulfotransferase (3-OST) family catalyzes rare modifications of glycosaminoglycan chains on heparan sulfate proteoglycans, yet their biological functions are largely unknown. Knockdown of 3-OST-7 in zebrafish uncouples cardiac ventricular contraction from normal calcium cycling and electrophysiology by reducing <i>tropomyosin4</i> (<i>tpm4</i>) expression. Normal 3-OST-7 activity prevents the expansion of BMP signaling into ventricular myocytes, and ectopic activation of BMP mimics the ventricular noncontraction phenotype seen in 3-OST-7 depleted embryos. In 3-OST-7 morphants, ventricular contraction can be rescued by overexpression of tropomyosin <i>tpm4</i> but not by troponin <i>tnnt2</i>, indicating that <i>tpm4</i> serves as a lynchpin for ventricular sarcomere organization downstream of 3-OST-7. Contraction can be rescued by expression of 3-OST-7 in endocardium, or by genetic loss of <i>bmp4</i>. Strikingly, BMP misregulation seen in 3-OST-7 morphants also occurs in multiple cardiac noncontraction models, including potassium voltage-gated channel gene, <i>kcnh2</i>, affected in Romano-Ward syndrome and long-QT syndrome, and cardiac troponin T gene, <i>tnnt2</i>, affected in human cardiomyopathies. Together these results reveal 3-OST-7 as a key component of a novel pathway that constrains BMP signaling from ventricular myocytes, coordinates sarcomere assembly, and promotes cardiac contractile function.</p></div

3-OST-7 controls region-specific BMP signaling in differentiating heart.

<p>ISH for <i>tbx2b</i> (A and D), <i>notch1B</i> (B and E) showed normal AV-restricted expression, whereas <i>bmp4</i> expression (C and F) showed ectopic expression in ventricular myocardium of 3-OST-7 morphants at 48 hpf (<i>n</i> = 30 for each group). IHC for P-Smad at 48 hpf showed delocalized expression in nuclei of 3-OST-7 morphant ventricle (H) compared to localized AV canal expression in control (G) (<i>n</i> = 10 for each group). (I) Graph depicting increased P-Smad-positive nuclei in the ventricle several unit distances away from the AV in 3-OST-7 morphants compared to P-Smad-positive nuclei localized in the AV for control (error bars, standard deviation). Imaging of live <i>Tg(BRE:d2GFP)</i> fish (J and L) showed GFP expression localized to the AV junction in control (K) and expanded expression in ventricle in morphant (M). V, ventricle; At, atrium; red arrows point to AV; white dashed lines outline the hearts.</p

3-OST-7 regulates cardiac contraction by constraining BMP signaling.

<p>(A) <i>Tg(hs:bmp2b)</i> heterozygotes were crossed to wild-type zebrafish and embryos were either untreated (no hs) or heat-shocked at 12 hpf. Embryos in each group were scored for ventricular contraction, and then genotyped for presence of heat-shock transgene. Graph depicts percentage contraction of embryos with transgene (green) or without (blue) in each treatment group. Induction of BMP signaling led to ventricular noncontraction. (B) <i>bmp4^st72</i> heterozygotes were crossed and embryos were either uninjected or injected with 3-OST-7 MO. Embryos in each group were scored for ventricular noncontraction, and then genotyped for <i>bmp4</i> mutation (RE, digestion with <i>SpeI</i>). Graph depicts percentage contraction of each genotypic class in uninjected embryos (blue) or embryos injected with 3-OST-7 MO (red). Ventricular noncontraction was rescued in 3-OST-7 morphants by <i>bmp4^st72</i> mutation.</p

Noncontraction is correlated with ectopic <i>bmp4</i> expression.

<p>(A) Graph comparing the percentage of normal contraction with 3-OST-7, <i>kcnh2</i>, and <i>tnnt2</i> MO injections. Error bars, SEM (B) Graph comparing patterns of <i>bmp4</i> expression at 48 hpf among control embryos (injected with 3-OST-5 MO), 3-OST-7 morphants, <i>kchn2</i> morphants, and <i>tnnt2</i> morphants. Loss of contraction correlates with ectopic <i>bmp4</i> expression in the ventricle (AV+V) or throughout the entire heart in 3-OST-7, <i>kcnh2</i> and <i>tnnt2</i> morphants.</p

Model for role of 3-O-sulfation catalyzed by 3-OST-7 in cardiac development.

<p>Under normal conditions, specific 3-OST-7-dependent 3-O-sulfation patterns (pink circles) on endocardial HSPGs constrain <i>bmp4</i> in nonchamber (noncontracting) myocardium (AV junction, red compartment), allowing transcription of <i>tpm4</i> in contracting myocardium (ventricle, green compartment). Tpm4 then stabilizes the sarcomere and ensures proper contraction (Tn, troponin). Knockdown of 3-OST-7 results in loss of 3-O-sulfation, expansion of <i>bmp4</i> and BMP signaling and P-Smad delocalization into ventricular myocardium. High levels of BMP signaling lead to reduced levels of <i>tpm4</i> transcripts and Tpm4 proteins, which then disrupt sarcomere assembly and lead to noncontraction.</p