Dynamics of morphogen signalling and cell fate decisions in the dorsal neural tube

Pattern formation in developing organs is controlled by morphogens. These signalling molecules form concentration gradients across tissues, thereby providing positional information that instructs the pattern of cell differentiation. Morphogen gradients are highly dynamic in space and time. Many factors such as morphogen production, spreading, degradation, cellular rearrangements and others could contribute to changes in the gradient shape, yet how the spatiotemporal signalling dynamics arise in many systems is still unclear. We studied the dynamics of morphogen signalling and tissue patterning in the developing vertebrate neural tube. In this system, neural crest, roof plate and distinct dorsal progenitor subtypes are specified in a spatially and temporally ordered manner in response to dorsal-toventral gradients of BMP and WNT signalling activity. How the BMP and WNT gradients are established and interpreted to ensure ordered cell specification is poorly understood. To address this question, we developed a 2D embryonic stem cell differentiation system that captures key features of dorsal neural tube development. In this system, differentiated colonies display remarkable self-organised pattern formation in response to uniformly applied BMP ligand. We established a method of differentiating the colonies using microfabricated stencils, which allowed us to control the initial size and shape of colonies without confining cell migration and colony growth. This led to highly reproducible pattern formation that facilitates quantification. Using this approach, we observed striking two-phase temporal dynamics of BMP signalling in our colonies: a BMP gradient rapidly forms from the periphery to the centre of colonies, subsequently disappears and is re-established again in the second phase. By combining our quantitative data with a data-driven theoretical model, we uncovered a temporal relay mechanism that underlies this biphasic BMP signalling dynamics. The first signalling phase is controlled by fast tissue-autonomous negative feedback that restricts the duration of the initial response to BMP. The early BMP activity gradient moreover controls the spatial organisation of the cell type pattern: the absence of a first phase results in disordered cell type pattern. The second phase is controlled by slow positive regulation of BMP signalling by the transcription factor LMX1A, a key regulator of roof plate identity. WNT promotes the second phase of BMP signalling via positive feedback on LMX1A. Altogether, the mechanism that we uncovered ensures the coupling of sequential developmental events, making pattern formation spatially and temporally organised. Furthermore, this mechanism allows the BMP signalling pathway to be reused in different contexts – first for the establishment of the neural plate border, and subsequently for dorsal neural progenitor patterning. Our study supports a general developmental principle in which multiple morphogens interact with transcriptional networks resulting in complex spatiotemporal signalling dynamics that ultimately drive organised pattern formation.