Photoreceptors generate neuronal diversity in their target field through a Hedgehog morphogen gradient in Drosophila
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Evaluation Summary:
In this paper, Bostock and Fernandes show that photoreceptor axons provide a graded Hedgehog signal to their target region, the lamina, that is highest in the distal and lowest in the proximal lamina. High levels of Hh favor specification of the distally located lamina neurons L2 and L3, while low levels favor specification of the proximal L5 neurons. This graded response raises interesting parallels with the patterning of the vertebrate spinal cord. The paper will be of special interest to those who study optic lobe development, but will also be of more general interest to developmental neurobiology.
This manuscript was co-submitted with: https://www.biorxiv.org/content/10.1101/2021.12.13.472383v2
(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
Defining the origin of neuronal diversity is a major challenge in developmental neurobiology. The Drosophila visual system is an excellent paradigm to study how cellular diversity is generated. Photoreceptors from the eye disc grow their axons into the optic lobe and secrete Hedgehog (Hh) to induce the lamina, such that for every unit eye there is a corresponding lamina unit made up of post-mitotic precursors stacked into columns. Each differentiated column contains five lamina neuron types (L1-L5), making it the simplest neuropil in the optic lobe, yet how this diversity is generated was unknown. Here, we found that Hh pathway activity is graded along the distal-proximal axis of lamina columns, and further determined that this gradient in pathway activity arises from a gradient of Hh ligand. We manipulated Hh pathway activity cell autonomously in lamina precursors and non-cell autonomously by inactivating the Hh ligand and by knocking it down in photoreceptors. These manipulations showed that different thresholds of activity specify unique cell identities, with more proximal cell types specified in response to progressively lower Hh levels. Thus, our data establish that Hh acts as a morphogen to pattern the lamina. Although this is the first such report during Drosophila nervous system development, our work uncovers a remarkable similarity with the vertebrate neural tube, which is patterned by Sonic Hh. Altogether, we show that differentiating neurons can regulate the neuronal diversity of their distant target fields through morphogen gradients.
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Evaluation Summary:
In this paper, Bostock and Fernandes show that photoreceptor axons provide a graded Hedgehog signal to their target region, the lamina, that is highest in the distal and lowest in the proximal lamina. High levels of Hh favor specification of the distally located lamina neurons L2 and L3, while low levels favor specification of the proximal L5 neurons. This graded response raises interesting parallels with the patterning of the vertebrate spinal cord. The paper will be of special interest to those who study optic lobe development, but will also be of more general interest to developmental neurobiology.
This manuscript was co-submitted with: https://www.biorxiv.org/content/10.1101/2021.12.13.472383v2
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also …
Evaluation Summary:
In this paper, Bostock and Fernandes show that photoreceptor axons provide a graded Hedgehog signal to their target region, the lamina, that is highest in the distal and lowest in the proximal lamina. High levels of Hh favor specification of the distally located lamina neurons L2 and L3, while low levels favor specification of the proximal L5 neurons. This graded response raises interesting parallels with the patterning of the vertebrate spinal cord. The paper will be of special interest to those who study optic lobe development, but will also be of more general interest to developmental neurobiology.
This manuscript was co-submitted with: https://www.biorxiv.org/content/10.1101/2021.12.13.472383v2
(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):
In this manuscript, the authors uncover a role for Hh in specifying identities of lamina neurons. They find that Hh, secreted by the photoreceptor neurons, forms a gradient along the developing lamina columns. This gradient is reflected in a gradient of Hh activity in the lamina precursors. The authors then use genetic manipulations to titrate the levels of Hh and find that L1-4 identities are specified at high Hh levels and L5 at low. Thus, like in the vertebrate spinal cord, the authors find that Hh acts like a morphogen to specify different neuronal identities at different concentrations.
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Reviewer #2 (Public Review):
The authors' demonstration of a gradient of Hh is only partially convincing. Their data clearly show the presence of Hh in the distal but not proximal lamina, but it is not clear from Fig. 2C whether there is a significant difference in the levels experienced by the precursors of L1, L4 and L5. They have also found significant effects of complete loss or full activation of Hh signaling on the most distally and proximally located cell types. However, the effect of intermediate levels of Hh signaling on the medially located cell types is less clear. They obtain these intermediate levels by inactivating a temperature-sensitive allele of hh for different lengths of time, leaving open the possibility that distinct cell fates are determined at different developmental times, and they do not show any conditions that …
Reviewer #2 (Public Review):
The authors' demonstration of a gradient of Hh is only partially convincing. Their data clearly show the presence of Hh in the distal but not proximal lamina, but it is not clear from Fig. 2C whether there is a significant difference in the levels experienced by the precursors of L1, L4 and L5. They have also found significant effects of complete loss or full activation of Hh signaling on the most distally and proximally located cell types. However, the effect of intermediate levels of Hh signaling on the medially located cell types is less clear. They obtain these intermediate levels by inactivating a temperature-sensitive allele of hh for different lengths of time, leaving open the possibility that distinct cell fates are determined at different developmental times, and they do not show any conditions that significantly increase the numbers of L1 or L4 neurons, cell types that are predicted to be specified by intermediate Hh levels. In addition, the authors define cell fates using combinations of only three transcription factor markers, leaving it uncertain whether Hh signaling indeed controls cell identities, or simply the expression of these transcription factors.
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Reviewer #3 (Public Review):
Bostock and Fernandes present a novel role for the conserved Hedgehog (Hh) signaling pathway in specifying neuronal fate in the developing lamina of the Drosophila visual system. The authors apply straightforward, yet elegant genetics to demonstrate that high levels of Hh signaling (loss of the inhibitory ptc receptor) lead to distal lamina neuronal fate, with more L2/3 neurons and fewer L5 neurons. In contrast, lower levels of Hh signaling (either through reduced Hh itself or the downstream transcription factor Ci) lead to more proximal neuronal fates with reduced numbers of lamina neurons in total. By analyzing the expression pattern of an endogenously-expressed Hh:GFP fusion protein, the authors suggest that the Hh signal itself comes from the photoreceptors, and is faintly expressed in the axons as they …
Reviewer #3 (Public Review):
Bostock and Fernandes present a novel role for the conserved Hedgehog (Hh) signaling pathway in specifying neuronal fate in the developing lamina of the Drosophila visual system. The authors apply straightforward, yet elegant genetics to demonstrate that high levels of Hh signaling (loss of the inhibitory ptc receptor) lead to distal lamina neuronal fate, with more L2/3 neurons and fewer L5 neurons. In contrast, lower levels of Hh signaling (either through reduced Hh itself or the downstream transcription factor Ci) lead to more proximal neuronal fates with reduced numbers of lamina neurons in total. By analyzing the expression pattern of an endogenously-expressed Hh:GFP fusion protein, the authors suggest that the Hh signal itself comes from the photoreceptors, and is faintly expressed in the axons as they reach the lamina in a distal to proximal gradient. A similar gradient signaling mechanism is a well-known role of the Hh ortholog, Sonic hedgehog (Shh), for controlling neuronal development in the vertebrate neural tube; however, this is the first time such a mechanism has been shown to play a role in Drosophila. Overall, this paper was well-written and the data generally support the authors' key claims, though there are a few minor places where the claims could be strengthened.
The authors use a combination of genetic approaches to show that the Hh signaling pathway is involved in patterning the lamina. These data appear to be strong and convincing, though it could be even stronger with a quantification of the effects of ptc and smo mutant clones shown. However, one of the points that lacks quite as much support is about the origin of Hh, and the gradient of distribution within the lamina. The Hh::GFP fusion protein does seem to indicate that Hh is expressed in the photoreceptors, but as the authors even pointed out, it is unclear whether the localized pattern is intracellular Hh, secreted Hh, or both. Secreted proteins are much harder to localize and can be more easily lost in staining processes compared to intracellular protein, so the actual distribution of Hh could be different in vivo. Furthermore, while ptc activity is low in L5 neurons based on ptc-LacZ expression, ptc activity seems to be strong just below the lamina near the L5 neurons, which could indicate a potential alternative source of Hh from below, or at least a higher Hh activity in this region. This could mean that it's the amount of ptc rather than the distribution of Hh itself that leads to differential specification.
Additionally, while the Hh titration experiment is intriguing and clever, It's not just that the levels of Hh have been changed in that case, but also the timing of when the Hh levels are reduced. Given that Hh is known to disrupt early lamina development, this could play a potentially confounding role.
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