A Matter of Time

When a vertebrate embryo develops, a group of cells self-organizes into the neural tube, eventually becoming the brain and the spinal cord. This involves specific signals, but how these signals are interpreted by developing cells remains unclear. A team of researchers at the Institute of Science and Technology Austria (ISTA) now has more insights—thanks to miniature 2D organs and rubbery silicone molds.

Imagine a deep forest. Every blade of grass, flower, bush or tree has a different shape and role. Yet they all thrive on a gradient of sunlight that filters through the canopy. The tissues of vertebrate animals, including humans, develop in a similar fashion. The difference: instead of light, the driving factors are signaling molecules called “morphogens”.

An interdisciplinary team of researchers at the Institute of Science and Technology Austria (ISTA) now found new insights into these morphogens in the developing neural tube—the brain-and-spinal-cord-to be. Using lab-produced organoids—miniature tissues that mimic the embryonic spinal cord—the scientists were able to observe and study the morphogen signaling dynamics during spinal cord development. The corresponding protocol and results are published in Star Protocol and Developmental Cell respectively.

Cells self-organize into bigger structures

Morphogens are produced at specific locations in growing organs and spread through tissues, forming gradients of concentration. Depending on the morphogen concentration, cells take on different roles and form self-organized patterns of cell types that underlie the structure of mature organs. Such is the case when stem cells (precursor cells) transform into the neural tube in a developing embryo. 

Here, morphogens such as “BMPs” (Bone morphogenetic proteins) and so called “Wnts” are the driving signals to form new patterns. “How these morphogens form signaling gradients in growing tissues is poorly understood,” explains Anna Kicheva. “Finding out more about them—and how morphogen production is regulated—is a key aspect of understanding how the entire tissue develops.”

Stefanie Lehr, a PhD student in Kicheva’s group, took on this question. “It is difficult to study the neural tube development process in the embryo itself because one needs to manipulate the tissue at specific times in specific locations,” explains Lehr.

Sharing expertise

“At first, we generated precursors of spinal cord cells and treated them with BMP,” Lehr continues. “To our surprise, after adding BMP, the cells turned into all the different cell types of the dorsal spinal cord and beautifully organized themselves into an ordered pattern. This pattern also changed over time in a specific way, much like in the real tissue.” But how come? What is the mechanism behind that? “Once we saw this, we wanted to establish a quantitative way to study this phenomenon,” she adds. To do so, two things were needed: an in vitro (outside the living organism; in a Petri dish) approach that makes the experimental observation easier and reproducible, and the theoretical expertise to analyze such a complex system. The latter was provided by ISTA colleagues Edouard Hannezo and David Brückner, who both study self-organization and development from a theoretical standpoint. For the in vitro approach, Lehr and colleagues collaborated with Jack Merrin, a staff scientist at ISTA’s Nanofabrication Facility (NFF).

Miniature 2D organs in a flexible cage

The scientists opted to produce organoids. Organoids are simplified versions of organs produced in a Petri dish that have the same key features as their counterparts in the organism. To reproducibly grow stem cells into organoids with a similar cell type organization to the actual neural tube, geometric constraints were necessary. “Conventional micro-patterning in the Petri dish was not an option for us, as the cell types we are generating are motile and are rapidly multiplying,” Lehr says.

Together with Jack Merrin, the scientists developed a stencil system. They utilized photolithography—a system that employs light to create a pattern on a substrate—to fabricate a mold. This mold was then used to produce the stencils, rubbery silicon sheets with small holes. Once placed on a Petri dish, precursor cells are cultured within the holes, forming organoids. The stencils are then removed to allow colony growth, cell movement, and neural tube development, closely mimicking the natural conditions in the embryo.

“This method gives us an exciting opportunity to study tissue growth and pattern formation in a quantitative manner using organoids,” Kicheva adds enthusiastically.

Appearing & Disappearing

Using the stencil approach, the scientists continued to closely investigate BMP’s role in self-organization. The scientists saw that there is a BMP morphogen signaling gradient in the organoid from the onset. Interestingly, this gradient emerges quickly, then fades away, only to reappear again. Similar dynamics are likely to occur in the embryo as well.

Hannezo and Brückner formulated a mathematical model to help unravel the underlying mechanism. The theoretical analysis, combined with experimental validation, showed that this behavior is driven by interlinked negative and positive feedback loops operating on different time scales.

Initially, cells, particularly at the periphery, are highly sensitive to BMP and a signaling gradient is established. This early gradient, on the one hand, induces inhibitors that rapidly shut it down (negative feedback). On the other hand, the early gradient also activates Lmx1a—a crucial protein in the development of the neural tube. Lmx1a then slowly activates BMP and causes the BMP gradient to reappear (positive feedback). This late gradient of BMP signaling then continues to drive the further specialization of cell types. This mechanism not only allows the pathway to be reused over time but also helps to time subsequent developmental events.

Development is temporarily organized

Previously, it was considered that the concentration of morphogens determines a cell’s fate during development. This study now adds a growing body of evidence that the temporal changes, rather than the absolute concentrations, are critical for understanding how cell fates are organized in tissues. Put simply, it is not about the amount of a trigger, but rather how it evolves over time.

“We found that a simple trigger (here: BMP) induces a complex response in which a gradient is self-generated in the tissue and undergoes complex temporal dynamics—this is essentially the type of response that underlies the capacity of embryos and organoids to self-organize,” Kicheva says.

Understanding how cells are organize themselves into functional organs is a fundamental question in developmental and stem cell biology. Furthermore, it is medically relevant and crucial for advancing regenerative medicine and tissue engineering—two fields of applied science focusing on replacing damaged or diseased tissue with functional tissue.

Publications:

S. Lehr, D. Brückner, T. Minchington, M. Greunz-Schindler, J. Merrin, E. Hannezo, A. Kicheva. 2024. Self-organized pattern formation in the developing neural tube by a temporal relay of BMP signaling. Dev Cell. DOI: 10.1016/j.devcel.2024.10.024

S. Lehr, J. Merrin, M. Kulig, T. Minchington, A. Kicheva. Fabrication of elastomeric stencils for patterned stem cell differentiation. 2024. Star Protocol. DOI: 10.1016/j.xpro.2024.103187

Funding information:

Work in Kicheva lab was supported by funding from the European Research Council under Horizon Europe: grant 101044579, and the Austrian Science Fund (FWF): Grant DOI 10.55776/F78. S. Lehr was supported by Gesellschaft für Forschungsförderung Niederösterreich m.b.H. fellowship SC19-011. D. Brückner was supported by the NOMIS foundation as a NOMIS Fellow and by an EMBO Postdoctoral Fellowship (ALTF 343-2022).