Beta cells Progenitors and its

Differentiation in Zebrafish

By,

Dorisa Jenifer .W

A brief overview

• Diabetes is a disease of high blood sugar. In India, more than 62 million

individuals are currently diagnosed with diabetes. In 2013 it was estimated that

over 382 million people throughout the world had diabetes.

•Researchers demonstrate that 70 per cent of protein-coding human genes are

related to genes found in the zebrafish and that 84 per cent of genes known to be

associated with human disease have a zebrafish counterpart.

•Thus, studying zebrafish behaviour in response to a drug or compound can

give us a better understanding of what the drug effects might be on humans as

well

A Drug Discovery Model

Alternatives for Insulin Production

• Beta (β) cells are unique cells in the pancreas that produce, store

and release the hormone insulin located in the area of the pancreas known

as the Islets of Langerhans.

• The appropriate function of insulin-producing pancreatic beta-cells is

crucial for the regulation of glucose homeostasis, and its impairment leads

to diabetes mellitus, the most common metabolic disorder in man.

• Even though insulin is a life saver, it does not cure the disease. Insulin

injections themselves are not without risk. Improvements in the

treatment of diabetes will come from a better understanding of how

insulin is made in the pancreas and released into the bloodstream, and

how it promotes uptake of circulating glucose by tissues including

muscle and fat.

Organs of Zebrafish

• The pancreas serves two major functions

(i) production of digestive enzymes by exocrine cells

(ii) Regulation of blood sugar by endocrine cells

• The mature exocrine part of pancreas consist of acinar cells connected to the intestine via a highly branched ductal tree.

• The endocrine part of pancreas is comprised of islets of Langerhans that are primarily scattered through the central regions of the organ. Several cells comprise the islet:

1. β cells (50-80%) and tend to segregate the islet core,

2. Glucagon producing alpha cells are the next most common type.

3. δ cells that produce somatostatin,

4. PP cells that produce pancreatic polypeptides, and

5. ϵ cells that produce ghrelin

• The zebrafish main pancreas is located on the right side of the adult fish, attached to the lateral aspect of the duodenum by the pancreatic duct. Typically, one large islet and 3–6 smaller islets occupy the main pancreas of the adult zebrafish. The tail of the pancreas is embedded with single β-cells.

Pancreatic Transcription Factors

• Transcription factor is a protein molecule which specifically binds to DNA

sequences, and hence controls the rate of transcription of genetic data’s from DNA

to messenger RNA.

• During pancreatic developmental stage, transcription factors require certain

sequential regulated expression to produce regular organogenesis.

• Some specific transcription factors are activated during pancreatic development at

early stage and β-cell differentiation, which may be switched off as the β-cell

matures slowly. Some transcription factors like, Pdx-1, Pax 4, Ptf1a are involved in

both early β-cell differentiation and mature β-cell function.

• In this research, the focus is on Transcription factors Pdx1, and Ptf1a.

Cell fate choices

β-cells expressing Pdx1

Pancreatic lineages

Mechanism of Insulin Secretion

Routes of β cells

Strategies to generate new β-cells

β cells in 18 somite stage

CONDITION NUMBER OF BETA CELLS ( 7 dpf)

Ganciclovir (GCV) treated 40±1

Untreated 30±2

RATE OF BETA CELL REPLIATION:

While performing beta cell nuclei counting through confocal analysis, E. Moro et all

has found out a linear growth in number of beta cell nuclei, with an average of 15%

per day in larval stages of zebrafish. (2009)

Collectrin/ TMEM27

• In the pancreas, TMEM27 is produced in beta cells specifically.

• Studies has shown a potential positive role of TMEM27 in glucose-stimulated

insulin exocytosis, another study linked TMEM27 production to beta cell

proliferation.

• The latter study also hypothesised that TMEM27, which is cleaved and shed into

the extracellular space, might be used as a beta cell mass biomarker.

• It plays the role of controlling insulin exocytosis by regulating formation

of the SNARE (soluble N-ethylmaleimide-sensitive-factor attachment protein

receptor) complex in pancreatic beta cells.

• Streptozotocin (STZ), a nitrosourea causes DNA damage after entering β-cells

through the Glut2 receptor. This is used to induce diabetes and can be used as a

control.

Developmental Stages

Ensembl data of Pdx1

Genome sequence of Pdx1

•ACGCTCAGACTGCAGGTAGAGCAGAGGTCCTGATCAGGGGCAGGG

CGCTGGCTCATGTGCTCGTGTACGGCACGGTTTCCCCGGTCTATGG

CAATCATGAATCGGGAAGAGCATTACTATCCGCCTAACCACCTGTAC

AAGGACTCTTGTGCCTTCCAGAGACACCCCAACGAAGACTACAGCC

AAAACCCTCCACCGTGTCTTTATATGAGACAGGCACATTCAGTATAC

GCCTCACCATTGGGCGCACAGGACCAGCCAAATCTTACCGACATTAC

TTCTTATAACATGTCGAGCCGGTATGATCTGGCAGGGCCTCATCTTC

ACCTTCCCCAAACTTCACAGACATCTCTACAGTCGCTCGGGGGTTAC

GGAGACTCTCTGGACCTCTGCGGGGATCGGAACAGATACCATCTCC

CATTTCCGTGGATGAAGTCAACCAAATCTCACACGCACGCATGGAAA

GGACAGTGGACAG

•These are the exon sequences of pdx1 gene obtained from ensembl.

Imaging β cells