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In JoVE (1)
Other Publications (8)
- Journal of Cellular Biochemistry
- The Anatomical Record. Part A, Discoveries in Molecular, Cellular, and Evolutionary Biology
- Development (Cambridge, England)
- Development (Cambridge, England)
- BioEssays : News and Reviews in Molecular, Cellular and Developmental Biology
- Proceedings of the National Academy of Sciences of the United States of America
- F1000 Biology Reports
- Zebrafish
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Articles by Mary D. Kinkel in JoVE
JoVE General
Intraperitoneale injectie in de Adult zebravis
1Department of Organismal Biology and Anatomy, The University of Chicago, 2Committee on Molecular Metabolism and Nutrition, The University of Chicago, 3Department of Medicine, The University of Chicago
Tonen we intraperitoneale injectie in volwassen zebravissen. We maken gebruik van een 10 ul NanoFil injectiespuit bestuurd door een Micro4 controller en UltraMicroPump III. Deze demonstratie omvat het gebruik van koud water als een verdoving.
Other articles by Mary D. Kinkel on PubMed
Coordinate Down-regulation of Cartilage Matrix Gene Expression in Bcl-2 Deficient Chondrocytes is Associated with Decreased SOX9 Expression and Decreased MRNA Stability
The anti-apoptotic protein Bcl-2 has been shown to function in roles unrelated to apoptosis in a variety of cell types. We have previously reported that loss of Bcl-2 expression alters chondrocyte morphology and modulates aggrecan expression via an apoptosis-independent pathway. Here we show that Bcl-2 is required for chondrocytes to maintain expression of a variety of cartilage-specific matrix proteins. Using quantitative, real-time PCR, we demonstrate that Bcl-2-deficient chondrocytes coordinately down-regulate genes coding for hyaline cartilage matrix proteins including collagen II, collagen IX, aggrecan, and link protein. The decrease in steady-state level of these mRNA transcripts results, in part, from decreased mRNA stability in Bcl-2-deficient chondrocytes. Transcriptional regulation is also likely involved because chondrocytes with decreased Bcl-2 levels show decreased expression of SOX9, a transcription factor necessary for expressing the major cartilage matrix proteins. In contrast, chondrocytes constitutively expressing Bcl-2 have a stable phenotype when subjected to loss of serum factor signaling. These cells maintain high levels of SOX9, as well as the SOX9 targets collagen II and aggrecan. These results suggest that Bcl-2 is involved in a pathway important for maintaining a stable chondrocyte phenotype.
Age-related Expression Patterns of Bag-1 and Bcl-2 in Growth Plate and Articular Chondrocytes
Aging cartilage displays increased chondrocyte apoptosis and decreased responsiveness of chondrocytes to growth factors. The molecular mechanisms responsible for these changes have not been identified. Bag-1 is a Bcl-2-binding protein that promotes cell survival, interacts with a diverse group of cellular proteins, and may integrate multiple pathways involved in controlling cell survival, growth, and phenotype. Bcl-2 is important for maintaining chondrocyte phenotype and delaying terminal differentiation and apoptosis of chondrocytes. Comparatively little is known about the role of Bag-1 in cartilage. Here we show that both growth plate and articular chondrocytes in the mouse express the Bag-1 protein. In the growth plate, Bag-1 expression is prominent in the late proliferative and prehypertrophic chondrocytes, displaying a pattern similar to what has been reported for Bcl-2. Further, the expression of both Bcl-2 and Bag-1 declines with age in the articular cartilage. Growth assays demonstrate that knocking down Bag-1 expression causes a decrease in growth rate. These results suggest that Bag-1 is involved in the regulation of chondrocyte phenotype and cartilage aging.
Retinoids Signal Directly to Zebrafish Endoderm to Specify Insulin-expressing Beta-cells
During vertebrate development, the endodermal germ layer becomes regionalized along its anteroposterior axis to give rise to a variety of organs, including the pancreas. Genetic studies in zebrafish and mice have established that the signaling molecule retinoic acid (RA) plays a crucial role in endoderm patterning and promotes pancreas development. To identify how RA signals to pancreatic progenitors in the endoderm, we have developed a novel cell transplantation technique, using the ability of the SOX32 transcription factor to confer endodermal identity, to selectively target reagents to (or exclude them from) the endodermal germ layer of the zebrafish. We show that RA synthesized in the anterior paraxial mesoderm adjacent to the foregut is necessary for the development of insulin-expressing beta-cells. Conversely, RA receptor function is required in the foregut endoderm for insulin expression, but not in mesoderm or ectoderm. We further show that activation of RA signal transduction in endoderm alone is sufficient to induce insulin expression. Our results reveal that RA is an instructive signal from the mesoderm that directly induces precursors of the endocrine pancreas. These findings suggest that RA will have important applications in the quest to induce islets from stem cells for therapeutic uses.
Cdx4 is Required in the Endoderm to Localize the Pancreas and Limit Beta-cell Number
Cdx transcription factors have crucial roles in anteroposterior patterning of the nervous system and mesoderm. Here we focus on the role of cdx4 in patterning the endoderm in zebrafish. We show that cdx4 has roles in determining pancreatic beta-cell number, directing midline convergence of beta-cells during early pancreatic islet formation, and specifying the anteroposterior location of foregut organs. Embryos deficient in cdx4 have a posteriorly shifted pancreas, liver and small intestine. The phenotype is more severe with knockdown of an additional Cdx factor, cdx1a. We show that cdx4 functions within the endoderm to localize the pancreas. Morpholino knockdown of cdx4 specifically in the endoderm recapitulates the posteriorly shifted pancreas observed in cdx4 mutants. Conversely, overexpression of cdx4 specifically in the endoderm is sufficient to shift the pancreas anteriorly. Together, these results suggest a model in which cdx4 confers posterior identity to the endoderm. Cdx4 might function to block pancreatic identity by preventing retinoic acid (RA) signal transduction in posterior endoderm. In support of this, we demonstrate that in cdx4-deficient embryos treated with RA, ectopic beta-cells are located well posterior to the normal pancreatic domain.
On the Diabetic Menu: Zebrafish As a Model for Pancreas Development and Function
Development of the vertebrate pancreas is a complex stepwise process comprising regionalization, cell differentiation, and morphogenesis. Studies in zebrafish are contributing to an emerging picture of pancreas development in which extrinsic signaling molecules influence intrinsic transcriptional programs to allow ultimate differentiation of specific pancreatic cell types. Zebrafish experiments have revealed roles for several signaling molecules in aspects of this process; for example our own work has shown that retinoic acid signals specify the pre-pancreatic endoderm. Time-lapse imaging of live zebrafish embryos has started to provide detailed information about early pancreas morphogenesis. In addition to modeling embryonic development, the zebrafish has recently been used as a model for pancreas regeneration studies. Here, we review the significant progress in these areas and consider the future potential of zebrafish as a diabetes research model.
Cyp26 Enzymes Function in Endoderm to Regulate Pancreatic Field Size
The control of organ size and position relies, at least in part, upon appropriate regulation of the signals that specify organ progenitor fields. Pancreatic cell fates are specified by retinoic acid (RA), and proper size and localization of the pancreatic field are dependent on tight control of RA signaling. Here we show that the RA-degrading Cyp26 enzymes play a critical role in defining the normal anterior limit of the pancreatic field. Disruption of Cyp26 function causes a dramatic expansion of pancreatic cell types toward the anterior of the embryo. The cyp26a1 gene is expressed in the anterior trunk endoderm at developmental stages when RA is signaling to specify pancreas, and analysis of cyp26a1/giraffe (gir) mutant zebrafish embryos confirms that cyp26a1 plays the primary role in setting the anterior limit of the pancreas. Analysis of the gir mutants further reveals that cyp26b1 and cyp26c1 function redundantly to partially compensate for loss of Cyp26a1 function. We used cell transplantation to determine that Cyp26a1 functions directly in endoderm to modulate RA signaling and limit the pancreatic field. Taken together with our finding that endodermal expression of cyp26 genes is subject to positive regulation by RA, our data reveal a feedback loop within the endoderm. Such feedback can maintain consistent levels of RA signaling, despite environmental fluctuations in RA concentration, thus ensuring a consistent size and location of the pancreatic field.
Recent Advances in Pancreas Development: from Embryonic Pathways to Programming Renewable Sources of Beta Cells
In recent years, there has been significant progress in understanding the detailed mechanisms of pancreas development. These studies have in turn influenced research aimed at producing pancreatic islet cells from stem cells. Here, we review recent progress in both of these areas.
Blood Sugar Measurement in Zebrafish Reveals Dynamics of Glucose Homeostasis
The adult zebrafish has the potential to become an important model for diabetes-related research. To realize this potential, small-scale methods for analyzing pancreas function are required. The measurement of blood glucose level is a commonly used method for assessing beta-cell function, but the small size of the zebrafish presents challenges both for collecting blood samples and for measuring glucose. We have developed methods for collecting microsamples of whole blood and plasma for the measurement of hematocrit and blood glucose. We demonstrate that two hand-held glucose meters designed for use by human diabetics return valid results with zebrafish blood. Additionally, we present methods for fasting and for performing postprandial glucose and intraperitoneal glucose tolerance tests. We find that the dynamics of zebrafish blood glucose homeostasis are consistent with patterns reported for other omnivorous teleost fish.
