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. 2020 Jun 30:6:43.
doi: 10.1038/s41421-020-0173-z. eCollection 2020.

Analysis on gene modular network reveals morphogen-directed development robustness in Drosophila

Shuo Zhang #  1   2 , Juan Zhao #  1   3 , Xiangdong Lv  1   2 , Jialin Fan  1   2 , Yi Lu  1 , Tao Zeng  1   3 , Hailong Wu  1 , Luonan Chen  1   3   4   5 , Yun Zhao  1   4   6
Affiliations

Analysis on gene modular network reveals morphogen-directed development robustness in Drosophila

Shuo Zhang et al. Cell Discov. .

Abstract

Genetic robustness is an important characteristic to tolerate genetic or nongenetic perturbations and ensure phenotypic stability. Morphogens, a type of evolutionarily conserved diffusible molecules, govern tissue patterns in a direction-dependent or concentration-dependent manner by differentially regulating downstream gene expression. However, whether the morphogen-directed gene regulatory network possesses genetic robustness remains elusive. In the present study, we collected 4217 morphogen-responsive genes along A-P axis of Drosophila wing discs from the RNA-seq data, and clustered them into 12 modules. By applying mathematical model to the measured data, we constructed a gene modular network (GMN) to decipher the module regulatory interactions and robustness in morphogen-directed development. The computational analyses on asymptotical dynamics of this GMN demonstrated that this morphogen-directed GMN is robust to tolerate a majority of genetic perturbations, which has been further validated by biological experiments. Furthermore, besides the genetic alterations, we further demonstrated that this morphogen-directed GMN can well tolerate nongenetic perturbations (Hh production changes) via computational analyses and experimental validation. Therefore, these findings clearly indicate that the morphogen-directed GMN is robust in response to perturbations and is important for Drosophila to ensure the proper tissue patterning in wing disc.

Keywords: Bioinformatics; Developmental biology.

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Conflict of interest statement

Conflict of interestThe authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1. In vivo samples acquired reflecting the morphogen gradients.
a The Drosophila wing imaginal disc diagram. The expression patterns of Ci and Hh specify the A (red region) and P (blue region) compartment of wing imaginal disc, respectively. b The diagram of the adult wing. The whole adult wing is derived from the wing pouch region (red and blue colored region) in wing imaginal disc. There are five veins in the wing. Among them, the first three veins (L1–L3) are developed from the A compartment cells and the other two (L4 and L5) are from the P compartment cells. c Diagram of the wing imaginal disc section view in A compartment. The dot-line circled region is the area subjected to laser capture. d–d‴ Immunostaining of a ptcGal4-uas-GFP wing imaginal disc. GFP signals (d and green in d‴), Ptc signals (d′ and red in d‴) and Ci signals (d′ and blue in d‴). Scale bar, 50 μm. e–k′ The wing disc sections were cut along A–P axis (bright filed). The circled areas were obtained by laser capture. The wing disc sections before laser capture (e–k). The wing disc sections after laser capture (e′–k′). l–p Real-time qPCR results showing the expression levels of the indicated genes at different positions along A–P axis in a wing imaginal disc. q Workflow of Geo-RNA-seq to acquire PGE profiles along A–P axis and bioinformatics analysis.
Fig. 2
Fig. 2. Sample-set integration and binary spatial module state construction.
a A brief diagram of the sample data collection. Data A is composed of gene information of different positions in wing disc A. There are 4524 MRGs in Data A. Data B is composed of gene information of different positions in wing disc B. There are 5396 MRGs in Data B. There are 4217 common genes in both data sets. b Expression dynamics of hh, ci and dpp in sample set integrated with sample sets A and B from RNA-seq data. The Fragments Per Kilobase Million (FPKM) value on the left Y-axis represents the expression of ci and dpp, and the FPKM value of the right Y-axis represents the expression of hh. c PCA of the hierarchical clustering results. The arrow indicates the direction of positions from A to P compartment (A1–B7). d Heat map of the expression of 4217 MRGs in integrated sample set. Heat map shows clear position-based modularization of the active genes. e Binary module states according to the heat map (Blue indicates inactive and red indicates active).
Fig. 3
Fig. 3. GMN constructed by Boolean model.
a The GMN based on Boolean model. M3 is the regulatory terminus of the network, which is directly regulated by the M1, M8, and M9. M7, M8, and M9 form a regulatory triangle at the center of the GMN (the triangle with red outline). b–d The identified gene regulatory interactions among M7, M8, and M9 from previous reports.
Fig. 4
Fig. 4. Computational analyses and biological validation of the GMN robustness of Drosophila wing imaginal disc.
a The attractors of GMN. There are three attractors: attractor normal (SN, 90.23%), attractor abnormal 1 (SabN1, 9.33%) and attractor abnormal 2 (SabN2, 0.44%). b The biggest state-transition tree converged to the attractor SN. The major (physiological) trajectory is labeled with blue lines connected with 13 physiological states along A–P axis. c The distribution of the state overlap ratios between all the other possible trajectories and the major trajectory. The red line is for the original network. Green line is for the randomly perturbed network.
Fig. 5
Fig. 5. Attractor analysis in response to Hh production changes.
a Hypothesis model of how Hh production changes affect the induction status of M8 along A–P axis. b In response to Hh production changes, the ON or OFF status of M8 at different positions is changed accordingly. Percentage of the normal attractor (attractor SN) was calculated based on the corresponding M8 changes. c The histogram of the percentage of normal attractor SN in response to Hh production changes. The vertical coordinate indicates the variations of M8 in response to different Hh levels. The asterisk indicates the physiological state of M8. The upper states of M8 representing the Hh level decrease. The lower states of M8 representing the Hh level increase. The X axis showed the percentage of SN. d–f″ Wing discs with the indicated genotypes were immunostained with anti-Ptc (blue) antibody or anti-Ci (red) antibody. Ptc and Ci signals were presented in Drosophila wing discs with WT (d–d″), Hh overexpression (hhGal4-uas-Hh) (e–e″), or Hh knockdown (hhGal4-Hh RNAiV1402) (f–f″) genotypes. The width changes of Ptc-positive regions (between two white arrows) indicate the corresponding Hh gradient alterations (d–f), which is also in accordance with the Ci change (d′, e′ and f′). g–i Morphology of Drosophila adult wings with the indicated genotypes. An adult wing of WT control (g), an adult wing of Drosophila with Hh overexpression that is driven by hhGal4 in third instar larvae stage (h), an adult wing of Drosophila with reduced Hh production that is achieved by hhGal4-Hh RNAiV1402 in third instar larvae stage (i).
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