Adam B. Cadwallader

The Rho kinase Rock2b establishes anteroposterior asymmetry of the ciliated Kupffer's vesicle in zebrafish

The vertebrate body plan features a consistent left-right (LR) asymmetry of internal organs. In several vertebrate embryos, motile cilia generate an asymmetric fluid flow that is necessary for normal LR development. However, the mechanisms involved in orienting LR asymmetric flow with previously established anteroposterior (AP) and dorsoventral (DV) axes remain poorly understood. In zebrafish, asymmetric flow is generated in Kupffers vesicle (KV). The cellular architecture of KV is asymmetric along the AP axis, with more ciliated cells densely packed into the anterior region. Here, we identify a Rho kinase gene, rock2b, which is required for normal AP patterning of KV and subsequent LR development in the embryo. Antisense depletion of rock2b in the whole embryo or specifically in the KV cell lineage perturbed asymmetric gene expression in lateral plate mesoderm and disrupted organ LR asymmetries. Analyses of KV architecture demonstrated that rock2b knockdown altered the AP placement of ciliated cells without affecting cilia number or length. In control embryos, leftward flow across the anterior pole of KV was stronger than rightward flow at the posterior end, correlating with the normal AP asymmetric distribution of ciliated cells. By contrast, rock2b knockdown embryos with AP patterning defects in KV exhibited randomized flow direction and equal flow velocities in the anterior and posterior regions. Live imaging of Tg(dusp6:memGFP)pt19 transgenic embryos that express GFP in KV cells revealed that rock2b regulates KV cell morphology. Our results suggest a link between AP patterning of the ciliated Kupffers vesicle and LR patterning of the zebrafish embryo.

Post-transcriptional regulation of satellite cell quiescence by TTP-mediated mRNA decay

Skeletal muscle satellite cells in their niche are quiescent and upon muscle injury, exit quiescence, proliferate to repair muscle tissue, and self-renew to replenish the satellite cell population. To understand the mechanisms involved in maintaining satellite cell quiescence, we identified gene transcripts that were differentially expressed during satellite cell activation following muscle injury. Transcripts encoding RNA binding proteins were among the most significantly changed and included the mRNA decay factor Tristetraprolin. Tristetraprolin promotes the decay of MyoD mRNA, which encodes a transcriptional regulator of myogenic commitment, via binding to the MyoD mRNA 3′ untranslated region. Upon satellite cell activation, p38α/β MAPK phosphorylates MAPKAP2 and inactivates Tristetraprolin, stabilizing MyoD mRNA. Satellite cell specific knockdown of Tristetraprolin precociously activates satellite cells in vivo, enabling MyoD accumulation, differentiation and cell fusion into myofibers. Regulation of mRNAs by Tristetraprolin appears to function as one of several critical post-transcriptional regulatory mechanisms controlling satellite cell homeostasis. DOI: http://dx.doi.org/10.7554/eLife.03390.001

Combinatorial expression patterns of heparan sulfate sulfotransferases in zebrafish: I. The 3-O-sulfotransferase family

Heparan sulfate (HS) is an unbranched chain of repetitive disaccharides, which specifically binds ligands when attached to the cell surface or secreted extracellularly. HS chains contain sulfated domains termed the HS fine structure, which gives HS specific binding affinities for extracellular ligands. HS 6‐O‐sulfotransferases (6‐OST) catalyze the transfer of sulfate groups to the 6‐O position of glucosamine residues of HS. We report here the characterization and developmental expression analysis of the 6‐OST gene family in the zebrafish. The zebrafish 6‐OST gene family consists of four conserved vertebrate orthologues, including a gene duplication specific to zebrafish. We examined the mRNA expression patterns in several tissues/organs throughout early zebrafish development, including early cleavage stages, eyes, somites, brain, internal organ primordial, and pectoral fin development. Members of the 6‐OST gene family have spatially and temporally distinct restricted expression, suggesting in vivo functional differences exist between members of this family. Developmental Dynamics 235:3432–3437, 2006.

Combinatorial Expression Patterns of Heparan Sulfate Sulfotransferases in Zebrafish: III. 2-O- Sulfotransferase and C5-Epimerases

Heparan sulfate (HS) is an unbranched chain of repetitive disaccharides, which specifically binds ligands when attached to the cell surface or secreted extracellularly. HS chains contain sulfated domains, termed the HS fine structure, which give HS specific binding affinities for extracellular ligands. HS 2‐O‐sulfotransferase (2‐OST) catalyzes the transfer of sulfate groups to the 2‐O position of uronic acid residues of HS. We report here the characterization and developmental expression patterns of 2‐OST in several tissues/organs throughout early zebrafish development, including early cleavage stages, eyes, somites, brain, internal organ primordial, and pectoral fin. The 2‐OST gene has spatially and temporally distinct expression, which is a surprise given the essential role of 2‐OST in HS fine structure formation. Furthermore, although 2‐OST and C5‐epimerase are predicted to be interdependent for protein translocation from the endoplasmic reticulum to the Golgi, their expression is not coordinately regulated during zebrafish development. Developmental Dynamics 236:581–586, 2007.

Differential roles for 3-OSTs in the regulation of cilia length and motility

As cells integrate molecular signals from their environment, cell surface receptors require modified proteoglycans for the robust activation of signaling pathways. Heparan sulfate proteoglycans (HSPGs) have long unbranched chains of repetitive disaccharide units that can be sulfated at specific positions by heparan sulfate O-sulfotransferase (OST) families. Here, we show that two members of the 3-OST family are required in distinct signaling pathways to control left-right (LR) patterning through control of Kupffer’s vesicle (KV) cilia length and motility. 3-OST-5 functions in the fibroblast growth factor pathway to control cilia length via the ciliogenic transcription factors FoxJ1a and Rfx2. By contrast, a second 3-OST family member, 3-OST-6, does not regulate cilia length, but regulates cilia motility via kinesin motor molecule (Kif3b) expression and cilia arm dynein assembly. Thus, two 3-OST family members cell-autonomously control LR patterning through distinct pathways that regulate KV fluid flow. We propose that individual 3-OST isozymes create distinct modified domains or ‘glycocodes’ on cell surface proteoglycans, which in turn regulate the response to diverse cell signaling pathways.

Isolation, Culture, Functional Assays, and Immunofluorescence of Myofiber-Associated Satellite Cells

Adult skeletal muscle stem cells, termed satellite cells, regenerate and repair the functional contractile cells in adult skeletal muscle called myofibers. Satellite cells reside in a niche between the basal lamina and sarcolemma of myofibers. Isolating single myofibers and their associated satellite cells provides a culture system that partially mimics the in vivo environment. We describe methods for isolating and culturing intact individual myofibers and their associated satellite cells from the mouse extensor digitorum longus muscle. Following dissection and isolation of individual myofibers we provide protocols for myofiber transplantation, satellite cell transfection, immune detection of satellite cell antigens, and assays to examine satellite cell self-renewal and proliferation.

Transplantation of Skeletal Muscle Stem Cells

Transplanting adult stem cells provides a stringent test for self-renewal and the assessment of comparative engraftment in competitive transplant assays. Transplantation of satellite cells into mammalian skeletal muscle provided the first critical evidence that satellite cells function as adult muscle stem cells. Transplantation of a single satellite cell confirmed and extended this hypothesis, providing proof that the satellite cell is a bona fide adult skeletal muscle stem cell as reported by Sacco et al. (Nature 456(7221):502-506). Satellite cell transplantation has been further leveraged to identify culture conditions that maintain engraftment and to identify self-renewal deficits in satellite cells from aged mice. Conversion of iPSCs (induced pluripotent stem cells) to a satellite cell-like state, followed by transplantation, demonstrated that these cells possess adult muscle stem cell properties as reported by Darabi et al. (Stem Cell Rev Rep 7(4):948-957) and Mizuno et al. (FASEB J 24(7):2245-2253). Thus, transplantation strategies involving either satellite cells derived from adult muscles or derived from iPSCs may eventually be exploited as a therapy for treating patients with diseased or failing skeletal muscle. Here, we describe methods for isolating dispersed adult mouse satellite cells and satellite cells on intact myofibers for transplantation into recipient mice to study muscle stem cell function and behavior following engraftment .

The Glycocode: Translating Heparan Sulfate Fine Structure into Developmental Function

Heparan sulfate proteoglycans (HSPGs) are an important component of the cell surface and extracellular matrix. HSPGs function in a wide variety of biological processes, including cell adhesion, signaling, migration, and proliferation. HSPGs are an information-dense family consisting of a core protein to which one or more glycosaminoglycan (GAG) chains are attached. The information contained within the GAG chains allows for great complexity and a specificity to bind and regulate binding of growth factors and morphogens. It is therefore no surprise that HSPGs are involved in many developmental processes, such as neural migration, kidney formation, and placentation. Here we explore how the information-rich GAG chains control distinct aspects of development utilizing a “glycocode” model.