Searching Specific Markers for Early Pancreas Development in Mouse Embryo
Pancreatic development
To study early pancreas development, it has primary importance to determine specific factors governing formation and differentiation of pancreatic buds contributed by surrounding tissues. Figure below summurizes early development stages of pancreas and surrounding tissues. At the 15 somite stage the notochord touch the neural tube and the gut (A) . When there are 20 somites you can see dorsal aorta is between the gut and the notochord (B). Finally, when 28 somite stage mesenchyme surrounds the whole gut and the dorsal bud forms[1]. In addition, we know that BMPs and BMP receptors are present during early pancreatic development. Therefore, we not only make and test new probes for early pancreas development but also for Bmp signaling. The ultimate goal is to find essential factors in this process and making and characterizing the phenotype of a conditional or KO mice to further understand the early pancreas development and especially pancreatic bud formation in mice.
Figure: Early development of the dorsal pancreas[1]
Strategy and Methodology:
1. Probe preparation
2. In situ hybridization on whole embryos
3. In situ hybridization on sections
I have used several procedures during my work such as probe preparation, mouse handling and dissection of embryos, sectioning of embryos embedded in paraplast, in situ hybridizations on whole embryos and sections. Firstly, we started with preparation of a couple of probes that is considered as possible markers for pancreas bud formation such as Id1, Id2, Osr1, Osr2, BmpR1b, mActRIIA, and mActRIIB. Then, we dissected whole embryos when they are at E10.5 and prepared for in situ hybridization and tested new probes on these whole embryos. We looked at under the microscope for staining of pancreatic region and then we sectioned stained embryos to look at closer. From those, we think of Osr1 is specific for pancreas. We specifically repeated in situ hybridization after sectioning embryos for Osr1 to get a better signal and staining to overcome issues related with penetration. From our observation, when you do in situ after sectioning you get better staining and you get rid of penetration issue. We used Pdx1 stained sections provided by Ally as a pancreatic marker to find out whether our probes stained pancreas at E10.5 or not.
Summary of probes:
| Probes | Information |
Id1 | Important for homeostasis in glandular and protective epithelia, reported to be involved in cell behavior in epidermis, digestive tract, pancreas, liver and so on. [2] Id proteins are key BMP target genes dictating cell fate. [3]
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Id2 | Similar to Id1, Id2 has been described in cell growth activation and inhibition of differentiation in pancreas. [2]
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Osr1 | Osr1 gene is expressed in the intermediate mesoderm.[4] Later, it is been showed that it marked dorsal pancreas at stage E11.5. [5]
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Osr2 | Osr2 is known to be expressed at sites of epithelial-mesenchymal interactions during development such as tooth and kidney.[6]
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BmpR1b | BmpR1b is one of subunit of the Bone morphogenetic protein (BMP) receptor. It is expressed in sclerotome, gut mesenchyme and mesenchymal cells at the core of the limb bud at E10.5.[7]
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ActRIIA | ActRIIA and ActRIIB knockout mutants homozygous for both receptors did not form mesoderm. [8]
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ActRIIB | Allendorph and colleagues showed that BMP-3 binds Activin Receptor type II b (ActRIIb)[9]
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References:
1. Slack, J.M., Developmental biology of the pancreas. Development, 1995. 121(6): p. 1569-80.
2. Coppe, J.P., A.P. Smith, and P.Y. Desprez, Id proteins in epithelial cells. Exp Cell Res, 2003. 285(1): p. 131-45.
3. ten Dijke, P., et al., Controlling cell fate by bone morphogenetic protein receptors. Mol Cell Endocrinol, 2003. 211(1-2): p. 105-13.
4. So, P.L. and P.S. Danielian, Cloning and expression analysis of a mouse gene related to Drosophila odd-skipped. Mech Dev, 1999. 84(1-2): p. 157-60.
5. Grieshammer, U., P. Agarwal, and G.R. Martin, A Cre transgene active in developing endodermal organs, heart, limb, and extra-ocular muscle. Genesis, 2008. 46(2): p. 69-73.
6. Lan, Y., et al., Osr2, a new mouse gene related to Drosophila odd-skipped, exhibits dynamic expression patterns during craniofacial, limb, and kidney development. Mech Dev, 2001. 107(1-2): p. 175-9.
7. Yi, S.E., et al., The type I BMP receptor BMPRIB is required for chondrogenesis in the mouse limb. Development, 2000. 127(3): p. 621-30.
8. Song, J., et al., The type II activin receptors are essential for egg cylinder growth, gastrulation, and rostral head development in mice. Dev Biol, 1999. 213(1): p. 157-69.
9. Allendorph, G.P., et al., BMP-3 and BMP-6 structures illuminate the nature of binding specificity with receptors. Biochemistry, 2007. 46(43): p. 12238-47.