2025/09/15 by Wilhelmus A. Brouwer, Luca Braccioli · 2 voices
Decision Sciences · Engineering · #Academic Publishing and Open Access #Biomedical and Engineering Education
paper · pdf · doi:10.1242/dev.205199
A major breakthrough in the field of mammalian embryology is the development of stem-cell based embryo models. Derived from pluripotent stem cells, such as embryonic stem cells or induced pluripotent stem cells, they can recapitulate various stages of embryogenesis, ranging from pre-implantation development to organogenesis (Arias et al., 2022; Shahbazi and Pasque, 2024). One specific stem cell-based model that is gaining popularity to study post-implantation embryogenesis is the gastruloid model. Gastruloids are made by aggregating a few hundred cells in a low-adhesion environment for 2 days. By subsequently providing them with a pulse of the Wnt-agonist Chiron for 24 h, gastruloids elongate and differentiate into the three embryonic germ layers. Gastruloids recapitulate anterior–posterior axis formation and the start of organogenesis (Beccari et al., 2018; van den Brink et al., 2014, 2020). When embedded in an extracellular matrix, gastruloids undergo striking morphogenesis, resulting in the formation of a neural tube and somite-like structures (Veenvliet et al., 2020). Gastruloids are increasingly proving to be useful tools to study self-organization, morphogenesis, metabolism and gene regulation during development (Bennabi et al., 2025; McNamara et al., 2024; Merle et al., 2024; Rekaik et al., 2023; Stapornwongkul et al., 2025; Suppinger et al., 2023). However, a major limitation of gastruloids is their lack of anterior embryonic structures, such as the anterior neural tube, which has hampered their use in studying early brain development.Balaskas and colleagues recently overcame this limitation by inducing the formation of anterior neural structures in gastruloids (Balaskas et al., 2025 preprint). They built on their previous work, which showed that the Chiron pulse during gastruloid formation can be substituted by growing gastruloids in hypoxic conditions (López-Anguita et al., 2022). They found that hypoxia activates Wnt signalling through HIF1α. Variations in both oxygen levels and Wnt activity modulate the differentiation trajectories in gastruloids. Interestingly, growing gastruloids in hypoxia without Chiron promotes the formation of mid- and forebrain, which are absent in standard gastruloids. However, their previous findings underscored a central challenge: no single culture condition is currently sufficient to support the formation of a full neural tube in gastruloids.If we cannot yet capture a complete neural tube in gastruloids using one culture condition, why not build them piece by piece? This is the logic behind the assembloid strategy employed by Balaskas and colleagues: engineering tissues separately under the right conditions and combining them in a single model. This approach has been proven highly effective in generating brain organoids (Andersen et al., 2020; Birey et al., 2017). For Balaskas and colleagues, the assembloid strategy yielded gastruloids with anterior neural structures, which they termed ‘hypoxia and an anterior–posterior assembly strategy gastruloids’ or ‘HAP-gastruloids’. Their approach involves culturing three separate aggregates under different oxygen and Wnt signalling conditions, before combining them and embedding them in extracellular matrix. This allowed them to mimic the optimal differentiation environments for each aggregate. The protocol proved highly efficient, yielding high proportions of HAP-gastruloids expressing somite, endoderm, spinal cord and, for the first time, brain markers.HAP-gastruloids can now model a complete neural tube, displaying neural patterning mimicking the full anterior–posterior axis of the embryo. Comparison with mouse embryos revealed that HAP-gastruloids recapitulate anterior–posterior tissue organization at equivalent embryonic stages. To test whether hypoxia also regulates neural patterning in the embryo, they assessed the effect of modulating oxygen levels in ex utero mouse embryos. Neural patterning in ex utero embryos developed with 30% higher efficiency in hypoxia compared to normoxia, confirming the role of hypoxia in mouse embryos. Having established that hypoxia promotes neural development in both HAP-gastruloids and embryos, the authors next investigated the underlying mechanism. They focused on HIF1α, a key regulator of hypoxia responses (Iyer et al., 1998), by generating HIF1α-knockout HAP-gastruloids. Strikingly, HIF1α-knockout HAP-gastruloids showed compromised development of anterior brain structures. These findings suggest that hypoxia may regulate pathways involved in neural patterning, such as TGFβ, BMP and Wnt (Brafman and Willert, 2017).During brain development, the inhibition of TGFβ, BMPs and Wnt signalling pathways is essential to prevent anterior cells from adopting a posterior fate (Brafman and Willert, 2017). The authors hypothesized that loss of HIF1α may disrupt this crucial regulation. To test this, they perturbed TGFβ, BMP and Wnt pathways in HAP-gastruloids. TGFβ signalling induction reduced development of the mid- and forebrain, while TGFβ inhibition drastically increased the percentage of forebrain cells. These results indicate that TGFβ acts as an inhibitor during neural patterning. For BMP signalling, they focused on two different BMP proteins, BMP4 and BMP7. BMP7 selectively affected forebrain development, while BMP4 influenced overall HAP-gastruloid morphology. This suggests that BMP7 also functions as an inhibitor of forebrain neural patterning in HAP-gastruloid formation. Wnt inhibition affected both the anterior and posterior neural tube. Not only did it improve forebrain development, but it also induced the expression of posterior ventral spinal cord markers. Moreover, Wnt inhibition resulted in impaired posterior elongation and reduced expression of somitic and presomitic mesodermal markers. Although Wnt inhibition was restricted to the anterior aggregate, its effects were also evident in the posterior domain. This non-local response points to signalling exchange between aggregates, suggesting inter-tissue communication. In conclusion, Wnt inhibition in the anterior improves neural patterning, but it negatively affects posterior development. If hypoxia is the sole regulator of TGFβ and Wnt in the anterior, then perturbing the levels of TGFβ and Wnt in the anterior aggregate should mimic the effect of hypoxia under normoxic conditions. To test this hypothesis, the authors perturbed TGFβ and Wnt in ‘normoxia and anterior–posterior assembly strategy gastruloids’ or ‘NAP-gastruloids’. Neural patterning in NAP-gastruloids treated with both inhibitors combined resulted in the formation of midbrain and posterior forebrain. However, NAP-gastruloids do not develop any anterior forebrain structures. Together, these experiments highlight that hypoxia cannot be simply substituted by Wnt and BMP inhibition to induce anterior neural fates.A major limitation of gastruloids has been their inability to form anterior neural tissues. With HAP-gastruloids, this limitation is finally overcome. Neural patterning can finally be studied in a stem cell-based embryo model that spans the full anterior–posterior axis.The crucial advance in achieving improved neural patterning lies in the assembloid strategy. While earlier efforts attempted to create local signalling hubs within a single aggregate (Xu et al., 2021; Yamada et al., 2024), Balaskas and colleagues instead exposed multiple aggregates to distinct signalling environments before assembling them (Balaskas et al., 2025 preprint). This strategy addresses the inherent limitations of inducing organizer-like hubs in isolation. At the same time, it reveals a key principle: the specialization of multiple tissues in HAP-gastruloids requires a degree of segregation before these tissues can be successfully combined.The successful approach taken by Balaskas and colleagues opens a new frontier but also raises many questions: how does hypoxia drive differentiation towards anterior neural fates? How is the hypoxic response tuned along the anterior–posterior axis in the embryo? Are the interactions between the different aggregates mirroring interactions occurring in the embryo? And if so, how are such compartmentalized signalling environments established in vivo? Balaskas and colleagues have established HAP-gastruloids as a versatile platform to explore how hypoxia and spatially compartmentalized signals converge to guide tissue organization in early neural tube formation.