Simultaneous production of diverse neuronal subtypes during early corticogenesis

Author's response

Elia Magrinelli & Denis Jabaudon shared

Response to Questions Arising:

1. How much molecular diversity is encoded already in neural progenitors versus acquired postmitotically?

• Elia Magrinelli (EM): This is a key point for which we don’t have a definitive answer. Using a limited set of molecular markers, we find that neuron type-specific characteristics are progressively implemented rather than present from their differentiation onset on, but differences involving other genes could be present early on just as well. A more comprehensive molecular analysis of simultaneously-born neurons throughout corticogenesis and at sequential stages of their differentiation would be required to answer this question.

2. What are the molecular mechanisms for the progressive restriction of fate potential during corticogenesis?
3. What is the contribution of epigenetic modifications and post-transcriptional regulation to lineage specification in the cortex?

• EM: Well, one of the questions we are interested in in the lab is whether there is indeed a fate restriction, as opposed to “simply” a fate progression, as not much is yet known on this topic in mammals. In Drosophila, the seven-up gene regulates the progressive switch of specific neuroblasts cell autonomously during CNS development (Kanai et al. 2005), but external factors, including non-obviously genetic ones might be involved. We have recently shown for example that progression in the membrane potential of progenitors regulates their fate progression (Vitali et al., 2018). As for epigenetics and posttranscriptional aspects, this is an intense area of research right now. As techniques of tagging and isolating specific and coordinated subsets of differentiating neurons and progenitors become more efficient and available, epigenetic investigations on the subject could provide interesting outcomes.

Response to additional questions

1. Why did you pursue this study?

• EM: Time dependency in cortical development is a well-known concept, probably one of the first to be encountered while studying cortical development, although it is still relatively poorly understood in mammals. With FlashTag labeling, we were able to study this process with a high temporal resolution and thus decided to better characterize this process. We observed a substantial difference in the span of laminar distributions in early- vs. late-born neurons, which we thought was interesting because neurons in distinct cortical layers have distinct connectivities, functions and evolutionary histories.

2. How is your method (i.e. Flashtag) better than existing approaches?

• EM: FlashTag allows specific labeling of isochronic/isocyclic cohorts of M-phase progenitors and their progeny. It is based on the fact that during cell division, that in neuronal birth, ventricular zone progenitors are transiently in contact with the ventricles, where FlashTag is injected and from where it diffuses inside cells (Telley et al. 2016, Govindan et al. 2018). This strategy thus allows to focus on the progeny of a population derived from a highly homogeneous population of progenitors. Nucleotide substitute pulse-labeling also has a high temporal resolution when in combination (Takahashi et al. 1999), but does not distinguish between distinct types of progenitors, rendering lineage analyses difficult.

3. To what extend can the differences in diverse verse homogenous neuronal production can be explained by the switch from direct to indirect neurogenesis?

• EM: It is actually unclear whether such a switch exists since indirect neurogenesis is present also at early stages of corticogenesis (Vitali et al. 2018, Càrdenas et al. 2018) but generally speaking yes, direct neurogenesis is more prevalent early on. But yes, it is possible that indirect neurogenesis acts to “buffer” stochastic differences in newborn neuron ground states, yielding more homogeneous final populations. Our labeling strategy selects for directly born neurons and depending on how strongly BP contribute to the final population at any given time, we might observe stronger differences between our labelling approach and previous nucleotide substitute pulse-labeling methods.

4. Primates and humans are characterized by expanded upper cortical layers, do you think that upper-layer production can be less homogenous in those species compared to mice?

• EM: The data we have on primate (including human) development suggests that expansion of the superficial layers results from an increase in the diversity of intermediate progenitor subtypes, thought to be reflected by the expansion of the outer subventricular zone. Seminal data from the Rakic lab (1974) using tritiated thymidine showed very sharply laminarly delineated populations of neurons following labeling at late embryonic stages (the high temporal resolution here is allowed by long developmental durations), so it seems like we would be getting the same type of results. Inter-areal differences might play a bigger role in species with large brains, which we haven’t examined here.

5. What is your opinion on intrinsic versus extrinsic mechanisms for the progressive restriction of fate potential in cortical progenitors?

• EM: Again, in the lab we think at this stage it is safer to talk about progression in fate potential rather than a restriction. The scientific literature contains examples of extrinsic and intrinsic mechanisms and both are likely involved, albeit to different extents at different stages of corticogenesis. With the advent of organoid preparations, it could soon be possible to better tease out this complex field.

References:

Kanai MI., et. al. & Hiromi Y. seven-up Controls switching of transcription factors that specify temporal identities of Drosophila neuroblasts.  Dev Cell (2005).

Govindan, S, Oberst, P., and Jabaudon, D. In Vivo Pulse-Labeling of Isochronic Cohorts of Cells in the Central Nervous System Using FlashTag. bioRxiv, 286831 (2018).

Takahashi, T., et al. Sequence of Neuron Origin and Neocortical Laminar Fate: Relation to Cell Cycle of Origin in the Developing Murine Cerebral Wall. J. Neurosci. 19, 10357–71 (1999).

Telley, L., et al. Sequential Transcriptional Waves Direct the Differentiation of Newborn Neurons in the Mouse Neocortex. Science 351, 1443–6 (2016).

Rakic P. Neurons in rhesus monkey visual cortex: Systematic relation between time of origin and eventual disposition. Science (1974)