Sequential addition of neuronal stem cell temporal cohorts generates a feed-forward circuit in the Drosophila larval nerve cord
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Evaluation Summary:
Wang et al., present a thorough analysis of specific neuronal lineages in the early larval ventral nervous system with the objective to relate the birth order to circuit connectivity and function. The stated key findings of the work are (1) the identification of sharp temporal cohort divisions for the lineages under investigation, (2) synapse formation between neurons of different lineages and temporal cohorts, and (3) the observation that output neurons in this instance are born prior to input neurons. The study raises the question of to what extent these findings can be generalized.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Abstract
How circuits self-assemble starting from neuronal stem cells is a fundamental question in developmental neurobiology. Here, we addressed how neurons from different stem cell lineages wire with each other to form a specific circuit motif. In Drosophila larvae, we combined developmental genetics (twin-spot mosaic analysis with a repressible cell marker, multi-color flip out, permanent labeling) with circuit analysis (calcium imaging, connectomics, network science). For many lineages, neuronal progeny are organized into subunits called temporal cohorts. Temporal cohorts are subsets of neurons born within a tight time window that have shared circuit-level function. We find sharp transitions in patterns of input connectivity at temporal cohort boundaries. In addition, we identify a feed-forward circuit that encodes the onset of vibration stimuli. This feed-forward circuit is assembled by preferential connectivity between temporal cohorts from different lineages. Connectivity does not follow the often-cited early-to-early, late-to-late model. Instead, the circuit is formed by sequential addition of temporal cohorts from different lineages, with circuit output neurons born before circuit input neurons. Further, we generate new tools for the fly community. Our data raise the possibility that sequential addition of neurons (with outputs oldest and inputs youngest) could be one fundamental strategy for assembling feed-forward circuits.
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Evaluation Summary:
Wang et al., present a thorough analysis of specific neuronal lineages in the early larval ventral nervous system with the objective to relate the birth order to circuit connectivity and function. The stated key findings of the work are (1) the identification of sharp temporal cohort divisions for the lineages under investigation, (2) synapse formation between neurons of different lineages and temporal cohorts, and (3) the observation that output neurons in this instance are born prior to input neurons. The study raises the question of to what extent these findings can be generalized.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the …
Evaluation Summary:
Wang et al., present a thorough analysis of specific neuronal lineages in the early larval ventral nervous system with the objective to relate the birth order to circuit connectivity and function. The stated key findings of the work are (1) the identification of sharp temporal cohort divisions for the lineages under investigation, (2) synapse formation between neurons of different lineages and temporal cohorts, and (3) the observation that output neurons in this instance are born prior to input neurons. The study raises the question of to what extent these findings can be generalized.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
This is a fine paper linking birth order to connectivity in a specific example in Drosophila. The stated key findings of the work are (1) the identification of sharp temporal cohort divisions for the lineages under investigation, (2) synapse formation between neurons of different lineages and temporal cohorts, and (3) the observation that output neurons in this instance are born prior to input neurons. The strengths of the manuscript lie in a thorough and solid characterization of all three aspects. The experiments and data are of a high level and the results highly informative for the 'feed-forward circuit' under investigation. I see the main weakness in overinterpretations of results that may rather represent statistically expected findings for a specific study, questioning generalizations and the attempt …
Reviewer #1 (Public Review):
This is a fine paper linking birth order to connectivity in a specific example in Drosophila. The stated key findings of the work are (1) the identification of sharp temporal cohort divisions for the lineages under investigation, (2) synapse formation between neurons of different lineages and temporal cohorts, and (3) the observation that output neurons in this instance are born prior to input neurons. The strengths of the manuscript lie in a thorough and solid characterization of all three aspects. The experiments and data are of a high level and the results highly informative for the 'feed-forward circuit' under investigation. I see the main weakness in overinterpretations of results that may rather represent statistically expected findings for a specific study, questioning generalizations and the attempt to formulate more general principles. While the conclusions regarding (1) leave little room for interpretation, the findings regarding (2) and (3) may require some clarifications, representing a weakness of the manuscript.
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Reviewer #2 (Public Review):
This study investigated the lineage-circuit relationships using Drosophila EL neurons born from the NB3-3 lineage in the ventral nerve cord as a model system. This is an important question that is less well-studied. From the already existing studies, it appears that lineage-circuit relationships defer in different contexts. In some cases, neurons from the same lineage preferentially wire together, while in other cases, neurons from different lineages wire together. In this study, the authors tested their hypothesis that Drosophila ventral nerve cord circuits are formed preferentially between different temporal cohorts in different neuroblast lineages. They first demonstrated convincingly that there is a sharp transition in the connectivity pattern at the border of the temporal cohorts. Next they …
Reviewer #2 (Public Review):
This study investigated the lineage-circuit relationships using Drosophila EL neurons born from the NB3-3 lineage in the ventral nerve cord as a model system. This is an important question that is less well-studied. From the already existing studies, it appears that lineage-circuit relationships defer in different contexts. In some cases, neurons from the same lineage preferentially wire together, while in other cases, neurons from different lineages wire together. In this study, the authors tested their hypothesis that Drosophila ventral nerve cord circuits are formed preferentially between different temporal cohorts in different neuroblast lineages. They first demonstrated convincingly that there is a sharp transition in the connectivity pattern at the border of the temporal cohorts. Next they characterized a feed-forward circuit in which early-born EL neurons serve as the output, and showed that this circuit is formed between different temporal cohorts in different neuroblast lineages, and the output neurons are born before the input neurons in this circuit.
This study represents a significant further step in the study of lineage-circuit relationship, and the data are solid and well-controlled to support their conclusion within this context being studied. Major achievements and strengths include: 1) Using a combination of genetic experiments, connectome mining and network science approaches, they precisely mapped the birth-order and the morphology and connectivity of all EL neurons, and provided solid and quantitative evidence to a sharp transition in morphology and connectivity that correlated with the border of temporal cohorts. 2) Further, they characterized extensively the four classes of synaptic inputs to early EL neurons, and showed that early EL neurons are embedded within a feed -forward circuit that encodes vibrational onset. 3) They mapped the origin and birth order of the input neurons, and provided evidence to their hypothesis that Drosophila ventral nerve cord circuits are formed preferentially between different temporal cohorts in different neuroblast lineages. 4) The new permanent labeling construct for ts-MARCM will be a very useful tool for the fly community. 5) They showed that within a feedforward circuit, the output neurons are born before the input neurons. This showed that the strict early to early/ late-to-late hypothesis does not apply to all circuits. One weakness is that it is not known whether the birth order for this particular circuit can be generalized to other feedforward circuits.
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Reviewer #3 (Public Review):
Wang. et al explore the relationship between birth order and connectivity of neurons that wire together in a specific circuit. Using a refined single-cell clonal technique, the authors generate embryonic clones to map the birth order of neurons that derive from distinct stem cell lineages yet contribute to the same circuit in the Drosophila ventral nerve cord. Wang et. al map neurons of this circuit to discrete developmental windows, or "temporal cohorts," and show that neurons belonging to early vs. late temporal cohorts have stereotyped morphologies, wiring patterns, and birth orders. They convincingly show that distinct stem cell lineages contribute to the output vs input neurons of the circuit and that the output neurons are born before the input neurons. As a result, the authors provide novel insights …
Reviewer #3 (Public Review):
Wang. et al explore the relationship between birth order and connectivity of neurons that wire together in a specific circuit. Using a refined single-cell clonal technique, the authors generate embryonic clones to map the birth order of neurons that derive from distinct stem cell lineages yet contribute to the same circuit in the Drosophila ventral nerve cord. Wang et. al map neurons of this circuit to discrete developmental windows, or "temporal cohorts," and show that neurons belonging to early vs. late temporal cohorts have stereotyped morphologies, wiring patterns, and birth orders. They convincingly show that distinct stem cell lineages contribute to the output vs input neurons of the circuit and that the output neurons are born before the input neurons. As a result, the authors provide novel insights into the relationship between neurogenesis and circuit assembly.
Strengths
The relationship between birth order and connectivity at a single-cell resolution is a valuable step forward in understanding how cells are wired together during development. By dissecting the circuit into its individual subtypes and working backwards to birthdate the neurons, Wang et al take an unbiased and effective approach to understand how cells involved in sensing vibrational stimuli assemble within the ventral nerve cord.The temporal mapping of neurons within and between lineages is challenging and laborious work and the data presented is clear and convincing. The authors modified the existing ts-MARCM system with the addition of another recombinase to facilitate the visualization of clones at earlier stages of development; this technique will be of use to members of the fly community.
The authors effectively demonstrate how analysis of the connectome data can be used to infer multiple aspects of neuronal development, including whether neurons share a common neuroblast parent (clustering of cell bodies) and their relative birth order (comparative cortex-neurite length).
Weaknesses
A major conclusion of the paper is that the output neurons in a circuit are made before the input neurons. However, the strength of this conclusion is weakened by the fact that ~34% of the interneuron inputs to the EL-early neurons remain unmapped. This includes six neurons that synapse onto the EL-early neurons over 10 times each. It is therefore likely that other lineages contribute neurons that synapse onto the EL-early neurons. Without knowing the relative birthdates of these neurons to the early-EL neurons, the output first-input second conclusion should be tempered.More consideration/discussion should be given to the tTF windows that these cohorts are derived from. For example, it would be intriguing if the early-EL, Ladder and Basin neuronal cohorts are all derived from the same tTF window. This would suggest that wiring specificity within a circuit is driven by the tTFs.
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