Objective:

2-Deoxy-D-glucose (2DG) has previously been shown to influence not only glycolysis but also other pathways like mannose-dependent glycosylations. This project aims at identifying the effect 2DG on the proper development of the vertebrate anterior-posterior (AP) body axis by recapitulating sequential HOX gene expression in vitro. Known consequences of 2DG, ranging from the inhibition of glycolysis to the dysregulation of glycosylations, are explored in more detail in the context of developmental tempo.

Methods:

To analyse the effects on AP axis development, we differentiate human induced pluripotent stem cells into neuromesodermal progenitor (NMP) cells. These cells are then challenged with 2DG to analyse its effect on the sequential expression of HOX genes via specific luciferase reporters. Additional chemical compounds are included to assess distinct effects of 2DG on different cellular processes.

Results:

We show that 2DG massively delays the onset of HOX gene expression. Despite this, inhibition of the glycolytic pathway specifically is followed by small effects on HOX dynamics only. However, targeting global glycosylation patterns of cellular proteins indeed delays the onset of HOX gene expression similarly compared to 2DG. More specifically, N‑glycosylations appear to be of major importance for the accurate control of the HOX timer.

Conclusion:

While metabolic inhibition has previously been shown to affect the segmentation clock, metabolic effects seem to be less important for HOX dynamics in vitro. We find that inhibiting glycosylations in NMPs can mimic the effects of 2DG, while metabolic inhibition fails to. How these findings translate to effects on the segmentation clock and how these processes are linked remains to be explored further.