Optogenetic control of the Bicoid morphogen reveals fast and slow modes of gap gene regulation
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Abstract
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This review reflects comments and contributions by Rachel Lau, Claudia Molina, Arthur Molines, Anup Parchure, Daniel Rios, Rajan Thakur and Sagar Varankar.
This is a very interesting and thorough study using a novel optogenetic approach for Bicoid regulation and uncovers a novel repressive function of this protein. The experiments are very well designed and the figures are easy to follow.
General clarification - would it be possible to comment on whether nos-tub bnt + Bcd LEXY/iRFP-bCD LEXY embryos hatch? What is the consequence of perturbing the gene network on the overall fitness of the animal?
Abstract
‘Our results recapitulate known relationships’ - Is it possible to specify what the genes listed in this sentence represent? The genes referred to here are likely developmental regulators. For non-specialist readers, it would be …
This review reflects comments and contributions by Rachel Lau, Claudia Molina, Arthur Molines, Anup Parchure, Daniel Rios, Rajan Thakur and Sagar Varankar.
This is a very interesting and thorough study using a novel optogenetic approach for Bicoid regulation and uncovers a novel repressive function of this protein. The experiments are very well designed and the figures are easy to follow.
General clarification - would it be possible to comment on whether nos-tub bnt + Bcd LEXY/iRFP-bCD LEXY embryos hatch? What is the consequence of perturbing the gene network on the overall fitness of the animal?
Abstract
‘Our results recapitulate known relationships’ - Is it possible to specify what the genes listed in this sentence represent? The genes referred to here are likely developmental regulators. For non-specialist readers, it would be helpful to include the term which encompasses these genes to specify that their 'results recapitulate known relationships between _'.
Introduction
Recommend adding additional references in this section, pointing out to the source for different statements. Citing the original research paper rather than a review can attribute credit to the original authors (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5953266).
Figure 1
‘Studying Bicoid-dependent gap gene responses using a stimulus-response approach in single-input embryos.(a-b)’ - Figure 1a-b is good for readers not familiar/specialists in the topic to understand gene networks and how they are conventionally characterised.
‘Illumination also produced nuclear export of similar magnitude and spatial precision, with a 4-fold change in nuclear concentration between dark and light conditions and a spatial precision of ∼10-12 μm (1-2 cells) (Figure S1b-d)’ - This is an important point, may be worth showing it in the main figure.
Figure 2
Figure 2A ‘Bicoid was fused to various fluorescent proteins at its N terminus and to the LEXY optogenetic system at its C terminus. 450 nm light illumination exposes LEXY’s nuclear export sequence (NES), leading to an expected decrease in Bicoid transcriptional activity.’ - For clarity, it may be better to show Bcd inside or outside the nucleus in each condition?
Figure 2C ‘Larval cuticles for Bcd-LEXY variants in dark and light conditions. Anterior head and posterior tail structures are indicated with the outlined and shaded arrows. Illuminated embryos exhibit loss of anterior structures or duplication of posterior structures, indicating progressive loss of Bicoid activity.’- Suggest adding a line below each genotype "X-Bcd-LEXY, bcd[E1]/bcd[E1]" encompassing both 'dark' and 'light' panels; to make it clearer that for each genotype there is a control and a light-treated embryo.
Figure 2 f-g ‘EGFP-Bcd-LEXY time course during cycle of optogenetic activation and deactivation. Representative images are shown in f; quantification during a nuclear cycle 14 (NC14) time course of light and dark exposure shown in g. (h) Quantification of import and export kinetics for five LEXY constructs is shown in h; similar kinetics of translocation are observed for fluorescent Bicoid variants and non-Bicoid-containing LEXY constructs.’ - We understand that both panels report EGFP-Bcd-LEXY, with the left panel being after light stimulation (blue border) and the right panel after the light is removed (grey border). Could this be clarified in the figure legend? Also clarify whether time points are in minutes. Would it be possible to comment on the change observed with light in Figure 2f, is that significant?
‘We also measured expression of the canonical Bcd target gene Hunchback (Hb) in embryos harboring each of the three Bcd-LEXY variants (Figure 2d-e)’ - It may be useful to report the images depicting the staining pattern of Hunchback.
‘Nevertheless, all three variants were expressed at higher levels than wild-type Bcd, suggesting that fusion to LEXY and/or a fluorescent protein also partially interferes with Bcd function’ - mCherry-Bcd-LEXY expression seems to be similar to wild type levels, even though it does not rescue very well the bcd mutant phenotype or the Hb expression levels. mCherry tends to form aggregates, could this explain why this construct shows the weakest rescue? This could be discussed along with the interference by the LEXY domain.
‘nos-tub bnt, are devoid of all three A-P patterning cues and produce a posterior-like gene expression state throughout the embryo (Figure S2a).’ - The manuscript reports use of the triple mutant line bnt (mutant for bcd, nos and tsl) plus nos-tub, but in Figure S2a the genotype is stated as a double mutant, for nos and tsl but not for bcd. Is this correct?
Figure 3
‘Spatially-uniform, single-input embryos to enable optogenetic interrogation of specific gap genes.’- suggest adding a wt panel, showing what's on f but in the same way as b-e panels.
‘Mapping approximate embryonic positions represented by dark and light conditions in each genetic background. Bottom: diagram from Ref. 2 quantifying gap gene expression as a function of A-P position, with posterior = 100% EL. Top: the approximate position based on gap gene expression for each optogenetic variant under in illuminated (open circle) and dark conditions (blue circle).’ - Is panel f a schematic rather than a data figure? If so, could this be specified in the figure legend.
‘This pattern was altered dramatically in the presence of uniformly expressed Bcd-LEXY, which drove an anterior-like of gt and hb transcription in the dark, shifting to a mid-embryo-like state of hb and Kr transcription in the light (Figure 3c). Uniform iRFP-Bcd-LEXY embryos transcribed gt, hb, and Kr in the dark, shifting to Kr and kni expression in the light (Figure 3d).’ - From the characterization experiments in Fig1, the Bcd-LEXY is a stronger inducer of anterior-like pattern than iRFP-Bcd-LEXY showing a wider Hb pattern. However in the dark, Hb transcription seems more pronounced in iRFP-Bcd-LEXY than Bcd-LEXY, could this be commented on?
‘2P excitation is ideal because it can be used for simultaneous GFP and mCherry imaging without triggering AsLOV2 excitation (Figure S3b; Video S5-6)’ - This setup is a great approach to combine optogenetic stimulations while still allowing to use other fluorophores like GFP.
Figure 4
Figure 4b ‘Example of light stimulation and two-color imaging of NLS-mCherry-LEXY and MCP foci for a gt MS2 transcriptional reporter. Images show ventral regions of representative embryos in the absence or presence of a 450 nm light input delivered in a stripe in the middle of the embryo.’ - Is it possible to report the length of stimulation?
Figure c-e - The scheme of representation used to depict which regions of a wildtype embryo are recapitulated by each of the uniformly expressed embryos is really helpful.
Figure 4i - Really good way of summarising the data for individual figures, to help readers remember the dynamics of specific genes in response to their regulators.
Figure 5
‘constant-light and constant-dark stimuli were used as controls’- would it be possible to comment on the lack of difference here?
‘Conversely, a light-induced increase in nuclear Bcd triggered a similarly rapid but smaller-amplitude decrease in kni transcription (Figure 6b). Just as in the case of gt, the stability of the high-kni-expressing state may be indicative of positive autoregulation of kni expression by its own protein product. Together, these data suggest that Bcd can act as an apparent repressor of kni expression, an unexpected role for Bcd which is typically considered to perform only transcriptional activation functions. The initiation of kni transcription within 2 min after Bcd nuclear export is only compatible with a direct regulatory link, not Bcd-induced expression of an intermediate repressor.’ - Very interesting data. As a possible follow up, it may be worth testing the tagged Bcd-LEXY constructs since they have different levels of activation and will have a different potency as repressors.
Figure 6
Suggest to include images along with the quantification.
Discussion
The authors have done an amazing job of introducing and efficiently utilising an approach to study the regulation of gene networks in this study. While understanding the morphological outcomes of the embryos may not be a key result in the study, it may be relevant to include some discussion on how combining morphological data along with the gene expression networks and how different the uniformly expressing embryos are from wild type embryos, with respect to comprehending the physiological proximity of their system.
Methods
‘The construct was further recombined to the Sp marker on the same chromosome to mark the transgene’- Probably not an issue but since Sp is an allele of Wg, is it possible to clarify if this may have an influence on the interpretation of the results?
‘We created a custom MATLAB script to analyze and visualize time-lapse MS2 counts.’ - A great asset of the work is the use of MS2 foci for quantifications instead of simple fluorescence intensity or other more indirect assays. This provides a direct numeric approximation for the perturbations.
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