Research Article: Novel Reporter Alleles of GSK-3α and GSK-3β

Date Published: November 21, 2012

Publisher: Public Library of Science

Author(s): William B. Barrell, Heather L. Szabo-Rogers, Karen J. Liu, Renping Zhou. http://doi.org/10.1371/journal.pone.0050422

Abstract

Glycogen Synthase Kinase 3 (GSK-3) is a key player in development, physiology and disease. Because of this, GSK-3 inhibitors are increasingly being explored for a variety of applications. In addition most analyses focus on GSK-3β and overlook the closely related protein GSK-3α. Here, we describe novel GSK-3α and GSK-3β mouse alleles that allow us to visualise expression of their respective mRNAs by tracking β-galactosidase activity. We used these new lacZ alleles to compare expression in the palate and cranial sutures and found that there was indeed differential expression. Furthermore, both are loss of function alleles and can be used to generate homozygous mutant mice; in addition, excision of the lacZ cassette from GSK-3α creates a Cre-dependent tissue-specific knockout. As expected, GSK3α mutants were viable, while GSK3β mutants died after birth with a complete cleft palate. We also assessed the GSK-3α mutants for cranial and sternal phenotypes and found that they were essentially normal. Finally, we observed gestational lethality in compound GSK-3β−/−; GSK3α+/− mutants, suggesting that GSK-3 dosage is critical during embryonic development.

Partial Text

Glycogen Synthase Kinase 3 (GSK-3) is a serine/threonine kinase which was first discovered as a regulator of glycogen biosynthesis [1]. Since then, GSK-3 has been shown to phosphorylate many other substrates. One example is β-catenin, an intracellular signalling molecule required in the canonical Wnt signalling pathway. GSK-3 also acts as a key regulator in a number of developmental signalling pathways, including Hedgehog, transforming growth factor-β (TGF-β), nuclear factor of activated T-cells (NF-AT) and insulin/IGF signalling (reviewed in Frame and Cohen, 2001) [2]. This wide-ranging activity has been associated with a broad spectrum of human diseases, such as diabetes, inflammation, neurological disorders and cancer. As a result, GSK-3 inhibitors are being explored for a variety of therapeutic uses (reviewed in Jope, R. et al. 2006) [3]. However, in humans, GSK-3 is expressed from two paralogous genes: GSK-3α and GSK-3β. Although available inhibitors act on both proteins, few studies distinguish between GSK-3α and GSK-3β. In fact, many studies entirely neglect GSK-3α, despite its widespread expression and activity. Here, we describe new genetic tools useful for assessing the similarities and differences in the mammalian GSK-3 genes.

To date, there have been four GSK-3α and nine GSK-3β targeted mutations reported (Table 1); but none of these alleles provide a quick and easy method of assessing expression. While mRNA in situ hybridization is more straightforward, there have been surprisingly few such analyses of GSK-3α and GSK-3β in the literature and frequently, the assumption is that both genes are ubiquitous. However, several groups have recently reported tissue specific expression of GSK-3s [13], [14]. In the mouse, the most thorough analysis has been in the palate [14]. Immunohistochemical analyses are more common; however, many studies focus on specific cell types and do not distinguish between developmental roles of the two genes. Here, we describe lacZ reporter alleles for both GSK-3α and GSK-3β, which are also useful as null alleles. In addition, we describe the skeletal and craniofacial phenotypes of GSK-3α mutant animals, which have not previously been documented.

In mammals, there are two GSK-3 genes encoding GSK-3α, GSK-3β and a less-studied splice isoform GSK-3β2 that appear to have overlapping activities and target specificities [5], [6]. Current evidence linking GSK-3 to a variety of human disorders has led to the exploration of dozens of pharmacological inhibitors of GSK-3. All of these inhibitors bind in the ATP-binding pocket of GSK-3 and cannot differentiate between GSK-3α and GSK-3β [19]. Because GSK-3 inhibition is likely to have broad pleiotropic effects, further studies distinguishing in vivo roles for GSK-3α and GSK-3β will be necessary for better targeting of GSK-3 function.

Source:

http://doi.org/10.1371/journal.pone.0050422