Redefining the Gastrula Organiser in Early Chick Embryo Development
The vertebrate organiser has, since its discovery in 1924, long been considered responsible for patterning the primary body axes and inducing the formation of the embryonic nervous system. Hensen’s node was identified as the equivalent structure in birds due to its ability to induce a complete secondary axis with a regionalised neural tube when grafted to an ectopic location. The node also demonstrates extraordinary regenerative capacity: following surgical ablation, it can completely regenerate, and normal development follows. Previous observations suggest that a developmental delay occurs following organiser ablation, in both chick and frog embryos. We performed single cell RNA sequencing on node-ablated and control chick embryos during the first 5h post-ablation, to investigate any potential associated changes. Regeneration occurred rapidly, but even prior to this, the only differentially expressed genes were associated with the endoderm, reflecting the removal of the node as the source of endoderm cells. Many cells expressing organiser genes remained after ablation, confirmed with HCR staining and RT-qPCR, showing that organiser genes are expressed as a gradient from anterior to posterior along the primitive streak (PS). Grafting sections of the PS from different anteroposterior (AP) levels into host embryos showed that neural induction ability is not confined to the node, but is also distributed as an AP gradient along the PS. An ‘organisation field’ therefore exists in the chick embryo, rather than it being a unique property associated with one morphological structure. Node regeneration occurs through cells moving into the centre of the embryo and becoming respecified to acquire node identity. I asked how the rest of the embryo responds if the node is prevented from regenerating, by attempting to block these movements with a piece of area opaca tissue. This either led to the graft acquiring node identity, or regeneration occurring around the graft. As the node is believed to be induced by the middle of the PS, I next challenged this by bisecting the embryo at HH2-3 to exclude the PS. I found that as long as the lateral epiblast is left intact, node regeneration occurs frequently at HH2. Pushing the lateral parts together to encourage healing, combined with tight confinement of the culture, resulted in an increase in node regeneration at HH3. I propose two hypotheses to explain this: A) the node-inducing region is larger than previously thought, and a threshold of these factors is surpassed when the two lateral regions are brought together, or B) the confinement of the tissue promotes the formation of a contractile centre, triggering the formation of a new primitive streak. Future work will focus on identifying this mechanism. Despite the last 100 years of research, there are still fundamental questions regarding the node’s requirement for neural induction, versus its sufficiency in a graft experiment. The timing of the requirement of signals from the node is unclear, since neural development begins before node development, and neural tissue is often referred to as the ‘default’ fate for epiblast cells, suggesting that neural induction is a passive process. Furthermore, explants of the neural plate at late gastrula stage appear to have already undergone AP patterning, yet the node-derived axial mesoderm retains the ability to induce distinct types of neural tissue found along the AP axis. In other model organisms, the absence of axial mesoderm disrupts forebrain development, suggesting that it plays an important patterning role. Single cell RNA-seq analysis showed that early neural gene expression was unchanged in the first 5h after node ablation, suggesting that the node is not required for their induction or maintenance. HCR-ISH staining showed that the timing of Sox2/Sox1 expression onset, reflecting committed neural tissue, was also unchanged after node ablation. In addition, I cultured the neural plate in the absence of the PS and posterior regions (‘anterior segment cultures’). Their expression of Sox2 revealed that neural fate is stably acquired at HH3+-HH4, concomitantly with node formation. After HH4, neural tissues progress independently of the node, marked by Sox1 expression in anterior segments within the same timeframe as intact embryos. For the first time in intact neural plate tissue, I show that AP patterning is sequential and occurs through planar signalling from the node; fore-/midbrain fates require node-derived signals up to HH4, yet hindbrain fates require posteriorising signals from both the node and non-node regions for a prolonged period to fully stabilise and express Krox-20 in defined rhombomeres. Additionally, the expression of Hox genes after isolation of anterior segments at HH4 suggest that (part of) the spinal cord is specified before HH4, and that it develops via planar signals and an ‘Activation-Transformation’ mechanism. Despite forebrain patterning occurring during gastrula stages, long-term culture of anterior segments reveal that forebrain fates cannot be maintained without underlying node-derived axial mesendoderm, suggesting that signals from this tissue continue to play an important role in anterior fate specification. Despite not observing any developmental delay in the first 5 hours after node-ablation, I show that ablated embryos tend to have shorter body axes, shorter notochords, fewer somites and are less likely to have two hindbrain Krox-20 stripes than controls, suggesting that node-ablated embryos slow or stall their development. I hypothesise that this results from the reallocation of paraxial mesoderm progenitors to become node tissue, delaying somitogenesis onset and extension of the closely associated axial tissues.
brain, chick embryo, embryo development, hensen's node, neural development, organiser, regeneration
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