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 β-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 micro-samples 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 post-prandial 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.

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, this method makes it difficult to know how much reagent is absorbed or taken up per fish. Some experimental questions, particularly those related to metabolic studies, may be better addressed by delivering a defined quantity to each fish, based on weight. Here we present a method for intraperitoneal (IP) injection into adult zebrafish. Injection is into the abdominal cavity, posterior to the pelvic girdle. This procedure is adapted from veterinary methods used for larger fish. It is safe, as we have observed zero mortality. Additionally, we have seen bleeding at the injection site in only 5 out of 127 injections, and in each of those cases the bleeding was brief, lasting several seconds, and the quantity of blood lost was small. Success with this procedure requires gentle handling of the fish through several steps including fasting, weighing, anesthetizing, injection, and recovery. Precautions are required to minimize stress throughout the procedure. Our precautions include using a small injection volume and a 35G needle. We use Cortland salt solution as the vehicle, which is osmotically balanced for freshwater fish. Aeration of the gills is maintained during the injection procedure by first bringing the fish into a surgical plane of anesthesia, which allows slow operculum movements, and second, by holding the fish in a trough within a water-saturated sponge during the injection itself. We demonstrate the utility of IP injection by injecting glucose and monitoring the rise in blood glucose level and its subsequent return to normal. As stress is known to increase blood glucose in teleost fish, we compare blood glucose levels in vehicle-injected and non-injected adults and show that the procedure does not cause a significant rise in blood glucose.

Permanent neonatal diabetes mellitus is a rare form of insulin-requiring diabetes presenting within the first few weeks or months of life. Mutations in the insulin gene are the second most common cause of this form of diabetes. These mutations are located in critical regions of preproinsulin and are likely to prevent normal processing or folding of the preproinsulin/proinsulin molecule. To characterize these mutations, we transiently expressed proinsulin-GFP fusion proteins in MIN6 mouse insulinoma cells. Our study revealed three groups of mutant proteins: 1) mutations that result in retention of proinsulin in the endoplasmic reticulum (ER) and attenuation of secretion of cotransfected wild-type insulin: C43G, F48C, and C96Y; 2) mutations with partial ER retention, partial recruitment to granules, and attenuation of secretion of wild-type insulin: G32R, G32S, G47V, G90C, and Y108C; and 3) similar to ( 2) but with no significant attenuation of wild-type insulin secretion: A24D and R89C. The mutant insulin proteins do not prevent targeting of wild-type insulin to secretory granules, but most appear to lead to decreased secretion of wild-type insulin. Each of the mutants triggers the expression of the proapoptotic gene Chop, indicating the presence of ER stress.

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 ␤-cell number, directing midline convergence of ␤-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 ␤-cells are located well posterior to the normal pancreatic domain.

To identify regions of the mouse GnRH (mGnRH) promoter that mediate tissue-specific gene expression, transgenic mice have been generated with fragments of mGnRH promoter fused to the luciferase reporter gene. In this manuscript, we examine transgenic mice, generated with -356/+28 bp and -249/+28 bp of the mGnRH gene. Both fragments of mGnRH promoter target ovarian expression of the luciferase transgene, but neuronal luciferase activity is detected only in the mice bearing the -356-bp fragment, suggesting that the DNA sequences essential for directing neuron-specific expression of the GnRH gene are located between -356 and -249 bp. Two consensus binding sites for Otx2 were identified in this promoter region and were confirmed to be functional. EMSAs demonstrated specific binding of Otx2 to the mGnRH promoter, and overexpression of Otx2 increased transcriptional activity of the mGnRH promoter in transient transfection studies. When both Otx2 binding sites were eliminated, overexpression of Otx2 had no effect. GnRH mRNA expression in immortalized GnRH-secreting cell lines was also found to correlate with Otx2 expression. In addition, transgenic mice, bearing the 356 fragment of the mGnRH gene in which the Otx2 binding sites were eliminated, have significantly lower luciferase activity in the neonatal brain compared with mice generated with intact Otx2 binding sites. Luciferase activity was, however, still present in the ovary. Our findings provide evidence that Otx2 may have a critical role in directing tissue-specific expression of the mGnRH gene to the neuron, but not the ovary.