ZeRuth Lab Research

Research Overview

Our lab is broadly interested in understanding how transcription factor networks control development and physiology and characterizing how mutations in transcription factors give rise to disease. We are currently investigating transcription factor networks that mediate the development of the endocrine pancreas and its proper function in the mature organism. Using the zebrafish as a model organism, we hope to 1) understand how endocrine lineages are specified in the pancreas, 2) to characterize the signals that regulate β cell mass both neonatally and in response to nutrient excess, and 3) to delineate the molecular events that result in β cell dysfunction preceding type 2 diabetes. Additionally, we are using zebrafish along with in vitro techniques to 4) determine the role a tiny, hair-like organelle called the primary cilium plays in regulating blood glucose homeostasis. We have particular interest in the Krüppel-like transcription factor, Glis3, which has variants that have been implicated in the development of a number of diseases including neonatal, type 1, and type 2 diabetes and may be important in each of the above processes. We hope this research will lead to the identification of therapeutic targets for the treatment of diabetes and other metabolic diseases and ultimately allow for the generation of insulin-producing β cells in the lab that can be used for transplantation therapy.

Zebrafish Pancreas Development

The zebrafish (Danio rerio) has emerged as a powerful model for the study of pancreas development for several reasons including short generation time, rapid development, and amenability to genetic techniques. Indeed, a single female can give rise to 200-300 eggs at a time and the transparent embryos develop externally allowing for efficient visualization of developmental processes. Importantly, the zebrafish pancreas is similar to the mammalian pancreas both morphologically and functionally and the developmental programs that promote pancreatic development appear to be highly evolutionarily conserved. The zebrafish additionally has several unique features lacking in rodent models such as compartmentalized endocrine cell formation in the dorsal bud that allows for discerning factors required for endocrine versus exocrine differentiation and the ability to regenerate β-cells following destruction. These factors in combination with its ability to be used in high-throughput genetic and small molecule screens make the zebrafish a powerful model for the study of pancreas development and diabetes.

The vertebrate pancreas is comprised of three major cell types: acini, ductal, and endocrine. The role of the acinar cells is to secrete digestive enzymes which are then carried by the ducts into the duodenum. The endocrine cells make up a very small proportion of the pancreas (5-10%) and are arranged in clusters called islets of Langerhans. The major role of the pancreatic islets is to maintain glucose homeostasis by secreting hormones directly into the bloodstream through neighboring capillaries

The endocrine pancreas is made up of 5 major cell types: glucagon producing alpha cells, insulin producing beta cells, somatostatin producing delta cells, ghrelin producing epsilon cells, and pancreatic polypeptide producing PP cells. Beta cells are the most numerous endocrine cells and are typically located toward the center of the islet and surrounded by the other cell types. Insulin is released in response to high blood glucose levels to signal the uptake of glucose by peripheral tissues such as muscle and fat to be used as energy. Conversely, during periods of low blood glucose, glucagon is secreted by the alpha cells to signal the breakdown of glucose stored in the liver so that it can be used as energy. The remaining three endocrine cell types help fine tune the regulation of insulin and glucagon.

In both zebrafish and mammals, the pancreas develops from two buds arising from an evagination of the foregut to form the dorsal and ventral anlagen that then form a single organ after subsequent rotation and fusion. All acinar, ductal, and endocrine cells of the pancreas arise from early multipotent progenitor cells expressing specific transcription factors such as Pdx1, Sox9, Pax6, and Nkx2.2. The presence or absence of specific transcription factors guides the differentiation of the pancreatic progenitors into their terminal cell types.

Pancreas development in zebrafish occurs similarly to that observed in mammals. By 24 hpf the dorsal bud arises from the foregut endoderm while the ventral bud emerges later by 32 hpf. In contrast to mammals, the dorsal anlage in zebrafish gives rise exclusively to endocrine cells while the ventral anlage gives rise to ductal and exocrine cells as well as to additional endocrine cells. The endocrine cells arising from the dorsal anlage give rise to what is known as the principal islet by 24 hpf. Additional endocrine cells within the ventral anlage migrate toward and join the principal islet after the two buds fuse at 44 hpf. The secondary islets form several days later from a population of endocrine progenitor cells located along the intrapancreatic duct. As in mammals, the pancreas in zebrafish emerges from a pool of pancreatic progenitor cells expressing transcription factors including nkx2.2 and pdx1, which is detected by 14 hpf.

Following the specification and development of the pancreas, the islet mass must be regulated in order to meet the demands of the organism. The processes by which β cell mass is regulated postnatally differs from those employed during development. During the patterning of the pancreas, the endocrine cells arise predominately through the directed differentiation of precursor cells to an endocrine fate. Postnatally, however most new β cells arise through proliferation of existing β cells. In the neonatal period, β cell replication leads to an increase in overall islet mass. This period of proliferation is transient and islet mass remains stable in both human and zebrafish adults. A compensatory mechanism has however been reported in obese individuals whereby the β cell mass increases in order to deal with the increased demands presented. Currently, very little is known about the signals that initiate the expansion of β cells. Given that decreased β cell mass is characteristic of both type 1 and type 2 diabetes, it is important that a better understanding of the pathways that regulate β cell mass be gained. Our lab is interested in identifying and characterizing transcription factors involved in the specification of endocrine cells in the pancreas during development and their subsequent maturation and expansion.

Gli-similar 3

Gli-similar proteins (Glis1-3) form a sub-family of Kruppel-like zinc finger transcription factors that are closely related to the Gli family of proteins. As Kruppel-like proteins, the Glis family members contain five tandem Cys₂His₂ zinc fingers constituting a DNA binding domain that recognizes specific elements in the regulatory regions of target genes. The human GLIS3 gene is located on chromosome 9p24.2 and encodes an approximately 95 kDa protein containing in addition to the centrally located zinc finger motif, a C-terminal transactivation domain and a relatively large N-terminus. Glis3 is most highly expressed in the kidney and has been additionally detected in the thymus, lung, uterus, ovary, testis, brain, thyroid, and in the endocrine and ductal pancreas.

In humans, GLIS3 deficiency is associated with a rare syndrome characterized by neonatal diabetes mellitus and hypothyroidism. Depending on the nature of the mutation, some patients also develop hepatic fibrosis, glaucoma, osteopenia, polycystic kidney disease, bilateral sensorineural deafness, facial dysmorphism, and mild mental retardation. A number of genome-wide association studies (GWAS) have also implicated GLIS3 as a risk locus for the development of type I and type II diabetes as well as for Alzheimer's disease. Similar to what has been observed in humans, Glis3 knockout in mice gave rise to pups with severe polycystic kidney disease and neonatal diabetes characterized by hyperglycemia and hypoinsulinemia that survived just days after birth. Finally, Glis3 has been observed in mature beta cells where it acts as a positive regulator of insulin transcription and plays critical roles in maintenance and normal physiological function. We are working to characterize Glis3 function, identify Glis3 target genes, and better understand how Glis3 dysfunction leads to diseases such as diabetes.