Michael P. Sarras

Inhibition of zebrafish fin regeneration using in vivo electroporation of morpholinos against fgfr1 and msxb

Increased interest in using zebrafish as a model organism has led to a resurgence of fin regeneration studies. This has allowed for the identification of a large number of gene families, including signaling molecules and transcription factors, which are expressed during regeneration. However, in cases where no specific inhibitor is available for the gene product of interest, determination of a functional role for these genes has been difficult. Here we demonstrate that in vivo electroporation of morpholino oligonucleotides is a feasible approach for protein knock‐down during fin regeneration. Morpholino oligonucleotides against fgfr1 and msxb were utilized and knock‐down of both proteins resulted in reduced fin outgrowth. Importantly, Fgfr1 knock‐down phenocopied outgrowth inhibition obtained with an Fgfr1 inhibitor. Furthermore, this method provided direct evidence for a functional role for msxb in caudal fin regeneration. Finally, knock‐down of Fgfr1, but not Msxb, affected the blastemal expression of msxc, suggesting this technique can be used to determine epistasis in genetic pathways affecting regeneration. Thus, this convenient reverse genetic approach allows researchers to quickly (1) assess the function of genes known to be expressed during fin regeneration, (2) screen genes for functional relevance during fin regeneration, and (3) assign genes to the molecular pathways underlying fin regeneration. Developmental Dynamics 235:336–346, 2006.

Cre‐mediated site‐specific recombination in zebrafish embryos

Cre‐mediated site‐specific recombination has become an invaluable tool for manipulation of the murine genome. The ability to conditionally activate gene expression or to generate chromosomal alterations with this same tool would greatly enhance zebrafish genetics. This study demonstrates that the HSP70 promoter can be used to inducibly control expression of an enhanced green fluorescent protein (EGFP) –Cre fusion protein. The EGFP–Cre fusion protein is capable of promoting recombination between lox sites in injected plasmids or in stably inherited transgenes as early as 2 hr post–heat shock induction. Finally, the levels of Cre expression achieved in a transgenic fish line carrying the HSP70‐EGFP–cre transgene are compatible with viability and both male and female transgenic fish are fertile subsequent to induction of EGFP–Cre expression. Hence, our data suggests that Cre‐mediated recombination is a viable means of manipulating gene expression in zebrafish. Developmental Dynamics 233:1366–1377, 2005.

The expression of tissue inhibitor of metalloproteinase 2 (TIMP-2) is required for normal development of zebrafish embryos

MMP activities are controlled by a combination of proteolytic pro-enzyme activation steps and inhibition by endogenous inhibitors like α2-macroglobulin and the tissue inhibitors of metalloproteinases (TIMPs). TIMPs are the key inhibitors in tissue. The expression of both MMPs and TIMPs is controlled during tissue remodeling to maintain a balance in the turnover of extracellular matrix. Disruption of this balance may result in a broad spectrum of diseases. Additionally, TIMP-2 has been reported to have growth factor activities. To further study the function of TIMP-2 in development, we utilized zebrafish as an experimental model system. We have successfully isolated a TIMP-2 homologue from zebrafish (zTIMP-2). This zebrafish TIMP-2 showed high similarity to human TIMP-2 with all critical features conserved. Whole-mount in situ analysis showed that zTIMP-2 was expressed as early as the one-cell stage indicating a maternal origin. This expression continued through later stages of development. RT-PCR analysis confirmed the early expression pattern from the 16-cell stage through blastula, gastrula and 24-h stages. In addition, at the protein level, immunoreactive zTIMP-2 was detected using antibody against recombinant human TIMP-2. RFP-reporter analysis indicated that TIMP-2 can be secreted into the extracellular space where ECM is forming. Functional studies showed that the balance of TIMP-2 expression is important to normal development as reflected by the fact that both blockage of TIMP-2 translation using antisense morpholino oligonculeotides or increased translation of TIMP-2 using a mRNA microinjection approach resulted in abnormal zebrafish development. This is in contrast to murine knockout studies that indicate that TIMP-2 does not have a major role in mouse embryogenesis.

Structure and function of an early divergent form of laminin in hydra: a structurally conserved ECM component that is essential for epithelial morphogenesis

Abstract. As a major component of the extracellular matrix (ECM), laminin has been found in many vertebrate and invertebrate organisms. Its molecular structure is very similar across species lines and its biological function in the ECM has been extensively studied. In an effort to study ECM structure and function in hydra, we have cloned a partial hydra laminin α chain and the full-length hydra laminin β chain using ECM-enriched cDNA libraries. Analysis of deduced amino acid sequences indicated that both polypeptides have high sequence similarity to a number of invertebrate and vertebrate laminin α and β subunits. Rotary shadow analysis of isolated hydra laminin indicates it has a heterotrimeric organization that is characteristic of vertebrate laminins. A putative integrin-class protein was also identified using a cell-binding peptide sequence from the laminin β chain as an affinity probe, indicating that integrins are possible cell surface receptors in hydra. In agreement with previous results for the hydra laminin β chain, in situ hybridization experiments revealed that hydra laminin α chain mRNA is restricted to endodermal cells. As with a number of other hydra ECM components, higher levels of laminin α chain mRNA are localized to regions where cell migration and differentiation are actively undertaken such as the base of tentacles, the peduncle region, buds, regenerating tentacles, and at the head end during regeneration. The role of laminin in morphogenesis was studied using an antisense approach and the results indicated that translation of the laminin α chain is required for head regeneration.

A cnidarian homologue of translationally controlled tumor protein (P23/TCTP).

Abstract A protein homologous to P23, or translationally controlled tumor protein (TCTP), was cloned in Hydra vulgaris, the most ancient type of metazoan from which P23/TCTP has been characterized to date. Hydra P23/TCTP is composed of 184 amino acids and is encoded by a single mRNA of 700 bp. This invertebrate P23/TCTP is well conserved compared to those of other invertebrate and vertebrate species. Expression of Hydra P23/TCTP was confirmed by western blot of Hydra cell lysates using a polyclonal antibody against murine recombinant P23/TCTP. Spatial distribution of P23/TCTP mRNA and protein in Hydra was studied using in situ hybridization and immunostaining, respectively. Hydra P23/TCTP expression along the longitudinal body axis is regulated at both the transcriptional and the translational level. High levels of P23/TCTP mRNA were detected in a subpopulation of cells in the body column. In contrast, no mRNA was evident in the differentiated cells of the head and the foot regions. Coincidentally, P23/TCTP protein also concentrates to the body column, with no detectable protein in the head and foot region. However, despite the existence of P23/TCTP mRNA in both the ectoderm and endoderm in the body column, its protein is localized to the endodermal cells, suggesting a regulatory mechanism at the translational level. Taken together, the expression pattern of P23/TCTP in Hydra correlates with regions in which cell proliferation is actively occurring and its expression is excluded from regions where terminal differentiation has occurred.

Both Hoxc13 orthologs are functionally important for zebrafish tail fin regeneration

Hox genes are re-expressed during regeneration in many species. Given their important role in body plan development, it has been assumed, but not directly shown, that they play a functional role in regeneration. In this paper we show that morpholino-mediated knockdown of either Hoxc13a or Hoxc13b during the process of zebrafish tail fin regeneration results in a significant reduction of regenerative outgrowth. Furthermore, cellular proliferation within the blastema is directly affected in both knockdowns. Hence, similar to the demonstration of unique functions of multiple Hox genes during limb formation, both Hoxc13 orthologs have distinct functions in regeneration.

Structure, expression, and developmental function of early divergent forms of metalloproteinases in hydra.

ABSTRACTMetalloproteinases have a critical role in a broad spectrum of cellular processes ranging from the breakdown of extracellular matrix to the processing of signal transduction-related proteins. These hydrolytic functions underlie a variety of mechanisms related to developmental processes as well as disease states. Structural analysis of metalloproteinases from both invertebrate and vertebrate species indicates that these enzymes are highly conserved and arose early during metazoan evolution. In this regard, studies from various laboratories have reported that a number of classes of metalloproteinases are found in hydra, a member of Cnidaria, the second oldest of existing animal phyla. These studies demonstrate that the hydra genome contains at least three classes of metalloproteinases to include members of the 1) astacin class, 2) matrix metalloproteinase class, and 3) neprilysin class. Functional studies indicate that these metalloproteinases play diverse and important roles in hydra morphogenesis and cell differentiation as well as specialized functions in adult polyps. This article will review the structure, expression, and function of these metalloproteinases in hydra.

Evidence for Cell Surface Extracellular Matrix Binding Proteins in Hydra vulgaris

The present study was designed to identify and functionally characterize potential cell surface extracellular matrix binding proteins in Hydra vulgaris. Using [3H]-laminin as a probe, radioreceptor analysis of a dissociated mixed hydra cell preparation indicated that the average number of laminin binding sites per cell was about 10,000 with a dissociation constant of 1.49 nM. These binding sites could be displaced with unlabelled laminin in a dose-dependent manner and with high concentrations (500 nM) of unlabelled fibronectin. No displacement with type-IV collagen and type-I collagen was observed. Immunoscreening studies with a battery of antibodies raised to mammalian extracellular matrix (ECM) binding proteins indicated potential cell surface binding sites for the anti-beta 1 integrin monoclonal antibody, mAb JG22. Cell adhesion studies indicated that mAb JG22 blocked binding of hydra cells to laminin, but did not affect their binding to fibronectin, type-IV collagen, or type-I collagen. Light and electron microscopic immunocytochemical studies indicated that mAb JG22 localized to the basal plasma membrane of ectodermal and endodermal epithelial cells. Immunoprecipitation studies identified tow major bands with masses of about 196 kDa and 150 kDa under reducing conditions, and two bands with masses of > 200 kDa under non-reducing conditions. Functional studies indicated that mAb JG22 could reversibly block morphogenesis of hydra cell aggregates, and could block in vivo interstitial cell migration in hydra grafts. These observations indicate that hydra has cell surface binding sites for ECM components which are functionally important during development of this simple Cnidarian.

Molecular and biological characterization of a zonula occludens-1 homologue in Hydra vulgaris, named HZO-1

Abstract Zonula occludens-1 (ZO-1) is one of the earliest identified molecular components of tight junctions. Sequence analysis has placed ZO-1 into the broader membrane-associated guanylate kinase (MAGUK) protein family that contains such diverse members as postsynaptic density 95 (PSD-95), Drosophila discs large tumor suppressor gene product (dlg-A), p55, and TamA. Studies in both vertebrates and invertebrates have established that the MAGUK family is involved in a wide variety of cellular functions. These functions involve the regulation of such cellular processes as: (1) tight junction formation, (2) cell proliferation, (3) cell differentiation, and (4) neuronal synapse transmission. Extending these studies, we report the presence of a ZO-1 homologue in Hydra vulgaris, a member of the Cnidaria, the second oldest phylum of the animal kingdom. Hydra ZO-1 (HZO-1) is encoded by a single messenger RNA (mRNA) of approximately 6.0 kb that contains an open reading frame of 5,085 bp. The 191 kDa predicted protein consists of a characteristic MAGUK domain structure, including three PSD-95/SAP90, discs-large, ZO-1 (PDZ) domains, a src homology (SH3) domain, and a guanylate kinase (GUK) domain. Western blot analysis using an antibody generated from a synthetic peptide designed from the HZO-1 sequence confirmed the presence of a Hydra protein of the appropriate mass. While whole mount in situ hybridization determined that HZO-1 mRNA was expressed along the entire longitudinal axis of Hydra, cross-sectional analysis established that HZO-1 mRNA expression was restricted to the ectoderm or outer cell layer of the organism’s epithelial bilayer. Consistent with this mRNA expression pattern, immunofluorescence studies localized HZO-1 protein to the apical plasma membrane of ectodermal cells. It is unclear what role HZ0-1 has in the cellular physiology of Hydra; however, immunolocalization studies indicate a conserved plasma membrane-associated function(s), as reported for its counterparts in other invertebrate and vertebrate species. These studies establish that the MAGUK family of proteins with a membrane-associated function arose early during metazoan evolution, even before the divergence of protostomes and deuterostomes.

Regulation of Mouse Pancreatitis-Associated Protein-l Gene Expression during Caerulein-lnduced Acute Pancreatitis

The pancreatitis-associated proteins (PAPs) are major pancreatic secretory proteins during acute pancreatitis. However, mechanisms of regulation of PAP gene expression are poorly understood, and there is a lack of information regarding mouse PAP gene expression. Herein, we employed Northern blotting and RNase protection assays to measure mouse PAP-I mRNA levels in the normal pancreas and intestine, and in the pancreas during caerulein-induced acute pancreatitis. Unexpectedly, we found that mouse PAP-I mRNA levels are constitutively high in the adult pancreas, as well as in the small intestine. Furthermore, mouse pancreatic PAP-I mRNA levels are rapidly and dramatically down-regulated (3 h) after the initiation of caerulein injections, but slowly return to high levels by 72 h. Interestingly, we found that pancreatic PAP-I mRNA levels are also transiently and dramatically down-regulated after L-buthionine-[S,R]-sulfoximine administration. Thus, a correlation between PAP-I mRNA levels and glutathione levels in the mouse pancreas was demonstrated.