Bone Abstracts

Objectives: Hypophosphatasia (HPP) is a rare hereditary disease, leading to deficits in bone and tooth mineralization, muscular as well as neurological abnormalities due to decreased enzymatic activity of the tissue-nonspecific alkaline phosphatase (TNAP, encoded by the alpl gene). In this project, the zebrafish (Danio rerio) will be established as a new and valuable animal model for HPP research. Consequently, endogenous TNAP expression should be analyzed in different zebrafish samples and a set of staining methods should be established for future analysis of TNAP in zebrafish. In addition, first functional experiments should clarify consequences of gain- and loss-of-function variants in the zebrafish model.

Methods: In order to analyze the endogenous and tissue-specific TNAP function in zebrafish, Alkaline Phosphatase (AP) activity-assays (CSPD-assays) were performed in various tissue lysates. In parallel, ELF® 97 endogenous phosphatase staining was established on cryosections of different zebrafish tissues and stages to clarify spatio-temporal distribution. Additional functional experiments were performed by RNA microinjections into one-cell stage zebrafish embryos in order to establish short-time TNAP overexpression as well as TNAP Morpholino knockdown.

Results: CSPD-assays revealed AP activity in all analyzed tissues, with the highest detectable levels in the eyes, skin, and heart. The TNAP-specific inhibitor levamisole was able to diminish the detectable signals dependent on the respective concentration in all analyzed tissues apart from gut and liver, indicating the expression of other AP isoforms in these tissues. Furthermore, ELF® 97 staining showed tissue restricted TNAP activity in cornea, bones, musculature, gills, kidneys, and brain of zebrafish. For functional investigations either RNAs derived from patient specific TNAP isoforms were microinjected into zebrafish embryos to establish short-time overexpression or a TNAP Morpholino knockdown was performed. This set of experiments clearly showed TNAP’s influence on early developmental processes and clarified the functional consequences of different TNAP mutations.

Conclusions: TNAP activity was detected and could be localized in different zebrafish tissues using either CSPD-assay or ELF® 97 staining. Furthermore, short-time overexpression and knockdown of TNAP could be established and illustrate the feasibility of zebrafish for functional TNAP analyses. Taken together, the zebrafish seems to be a promising in vivo model for HPP, but further refinements are required to fully establish it as a new animal model for the investigation of new therapies for HPP.

¹ S.G. and D.L. contributed equally to this work.

² F.J. received honoraria for lectures and advice from Alexion.

Disclosure: Franz Jakob received honoraria for lectures and advice from Alexion.