RESEARCH

Individuals of the same species can differ widely in size, but their organs have reproducible proportions and patterns of cell types. We are interested in understanding how this reproducibility of tissue patterning is achieved in the developing spinal cord. The main model organism used in the lab is mouse, but we also work with chick embryos, as well as mouse ES cell differentiation.

Our focus is on secreted signalling molecules, called morphogens. Morphogens control the specification of an elaborate gene expression pattern, which ultimately determines the spatial arrangement of the different types of neuron precursors along the dorsoventral axis of the developing spinal cord. At the same time, morphogens control the growth of the tissue by influencing the decisions of cells to divide or exit the cell cycle. Our goal is to better understand how morphogen signalling levels are controlled and how cells interpret morphogen signalling to determine their cell fate and cell cycle progression. Understanding the feedbacks between morphogen signaling, cell fate specification and tissue growth will allow us to gain insight into the mechanisms that ensure that spinal cord development proceeds in a reproducible and stereotypical way in every vertebrate embryo.

The questions about size, reproducibility, patterns and dynamics that we ask are inherently quantitative, hence in our work we aim to obtain quantitative and dynamic data. To combine these data with mathematical modelling and theoretical descriptions, we have a mix of experimentalists and theorists in the group and we work in close collaboration with biophysicists.

Integration of morphogen signaling pathways along the DV axis

In the spinal cord, 14 transcriptionally distinct neural progenitor types are arrayed along the dorsoventral axis. This pattern is controlled by Shh, BMP and Wnt signaling. Ligands that activate these signaling pathways are produced at the opposite poles of the tissue (Shh ventrally, BMPs and Wnts dorsally). We recently showed that the response of neural progenitors to BMP and Shh signaling is determined by the dynamics of a downstream transcriptional network and that this mechanism of morphogen interpretation can account for the observed precision of tissue patterning (Zagorski et al, 2017). The questions that we are currently investigating are: what is the role of Wnt signaling in pattern formation in the spinal cord? Is there cross-talk between Shh and BMP signaling upstream of the target genes?

opposing gradients 2

A) A section through a mouse neural tube, expressing a Shh signalling reporter (green) and stained for pSmad1/5/8, a readout of BMP signalling (red). B) A set of signalling profiles that have been quantified from such images. C) Decoding map of the response to BMP and Shh in neural progenitors. The positional identities from ventral (blue) to dorsal (red) that cells adopt when exposed to different levels of BMP and Shh signaling are color coded.

Morphogen control of tissue growth

In the neural tube, the morphogens Shh and Wnt promote proliferation. However, the rate of proliferation is not proportional to the level of morphogen signalling, instead it is uniform across the tissue. Our goal is to find out how cells interpret Shh and Wnt signalling to tune their proliferation rate. We will use and develop an vitro system based on chick neural tube explants, where we can monitor with live imaging the response of individual cells to defined levels of morphogens. We will further delve into the molecular mechanism behind the dynamics that we observe.

live explants

Snapshots from a time-lapse of chick neural tube explant, which is exposed to 4nM Shh at time 0h. The explant expresses a Shh reporter (GBS-H2B-Venus) and the fluorescence intensity shown is normalized to a control plasmid. Even though the concentration of Shh in the medium is constant throughout the experiment, the downstream signaling activity decreases over time – a phenomenon known as temporal adaptation.

Morphogen gradient formation

The formation of morphogen concentration profiles with specific shape and temporal dynamics is key for tissue patterning and size control. Cells are not passive substrates over which morphogen gradients form – they actively produce, degrade and transport morphogen, while at the same time they divide and dilute the morphogen concentration. We will study how these different properties contribute to the formation of the Shh morphogen gradient in the neural tube. Shh is produced and secreted by the notochord and floor plate, which are located ventrally, and makes a ventral-to-dorsal gradient of concentration. The shape of the gradient changes dynamically over time. One particular question that we will address is what is the relationship between the growing size of the Shh source (notochord and floor plate) during development and the shape of the gradient. We will use mouse imaging and genetics, ES cell differentiation and mathematical modeling in this project.

shhgradsshhquannt

Figures from Cohen et al, Nature Comm. (2015): 6, 6709. In this study, we showed how the Shh gradient shape changes during neural tube development.