Gene Regulatory Networks of Epidermal and Neural Fate Choice in a Chordate

Abstract

Neurons are a highly specialized cell type only found in metazoans. They can be scattered throughout the body or grouped together, forming ganglia or nerve cords. During embryogenesis, centralized nervous systems develop from the ectoderm, which also forms the epidermis. How pluripotent ectodermal cells are directed toward neural or epidermal fates, and to which extent this process is shared among different animal lineages, are still open questions. Here, by using micromere explants, we were able to define in silico the putative gene regulatory networks (GRNs) underlying the first steps of the epidermis and the central nervous system formation in the cephalochordate amphioxus. We propose that although the signal triggering neural induction in amphioxus (i.e., Nodal) is different from vertebrates, the main transcription factors implicated in this process are conserved. Moreover, our data reveal that transcription factors of the neural program seem to not only activate neural genes but also to potentially have direct inputs into the epidermal GRN, suggesting that the Nodal signal might also contribute to neural fate commitment by repressing the epidermal program. Our functional data on whole embryos support this result and highlight the complex interactions among the transcription factors activated by the signaling pathways that drive ectodermal cell fate choice in chordates.

Keywords: EvoDevo; amphioxus; cell fate; gene regulatory network; neural induction.

Fig. 1.

Pipeline used for parallel analysis of ATAC-seq and RNA-seq data and for the putative GRN construction. The different steps presented correspond to the analysis and putative GRN construction at the G5 stage. The numbers in the circles correspond to differentially expressed genes (RNA-seq, blue for gene overexpressed in activin-treated explants, orange for genes overexpressed in untreated explants) or to differentially accessible chromatin regions (ATAC-seq, blue for regions more accessible in activin-treated explants, orange for regions more accessible in untreated explants). The detailed pipeline is presented on the left for the “all neural (G5)” genes (genes overexpressed in activin-treated explants and showing more accessible peaks in activin-treated explants).

Fig. 2.

GO term enrichment and putative GRN for “all neural (G5)” and “all epidermal (G5)” genes. (a) GO term enrichment visualization for the “all neural (G5)” gene list. (b) GO term enrichment visualization for the “all epidermal (G5)” gene list. For (a) and (b), GO terms that were not connected to others are not represented. (c) Putative GRN constructed using the “all neural (G5)” gene RNA-seq and ATAC-seq data. (d) Putative GRN constructed using the “all epidermal (G5)” gene RNA-seq and ATAC-seq data. In (c) and (d), the SMAD transcription factors are in purple, the transcription factors that belong to the “all neural (G5)” genes are in blue, the transcription factors that are only overexpressed in activin-treated explants are in light blue, the transcription factors whose expression is not different between untreated and activin-treated explants are in gray, the transcription factors that belong to the “all epidermal (G5)” genes are in orange, the transcription factors that are only overexpressed in untreated explants are in light orange. Edges between SMAD, SoxB1, Dlx, and the other nodes are highlighted in color, the others are in gray. Transcription factors that have the same combination of edges are grouped into unique nodes.

Fig. 3.

GO term enrichment and putative GRN for “all neural (N2)” and “all epidermal (N2)” genes. (a) GO term enrichment visualization for the “all neural (N2)” gene list. (b) GO term enrichment visualization for the “all epidermal (N2)” gene list. For (a) and (b), GO terms that were not connected to others are not presented. (c) Putative GRN constructed using the “all neural (N2)” gene RNA-seq and ATAC-seq data. (d) Putative GRN constructed using the “all epidermal (N2)” gene RNA-seq and ATAC-seq data. In (c) and (d), edges between Smad2/3, SoxB1a, Neurogenin, Dlx, and the other nodes are highlighted in color, the others are in gray. Color code is as in fig. 2.

Fig. 4.

Analysis of “all neural (G5)” genes expression in whole embryos. Mfuzz clustering graphs are presented on the left. The color code reflects “Membership” values calculated by Mfuzz (magenta for high values and green for low values of Membership score). Corresponding box plots (center line, mean; box limits, upper and lower quartiles; points, outliers; box in magenta = box with the highest mean expression), enrichment in TFBS of the ATAC-seq peaks with differential accessibility corresponding to the core genes of each cluster, and GO term enrichment of these core genes are presented.

Fig. 5.

Expression patterns of genes of the epidermal and neural putative gastrula stage GRNs analyzed by in situ hybridization. The expression of epidermal genes (Rfx1/2/3, Dlx, Ovol, Grhl, Klf1/2/4, FoxJ1, Fhl2, Tfap2) and neural genes (Pou3fl, SoxB1a, Neurogenin, Snail, Zeb, Tcf15, Stox, Insm, Pou3f) were analyzed at eight-cell, morula, blastula, G0, G1, G2, G4, G5, N0, N2, N3, and N4 stages. Side views are presented with anterior to the left and dorsal to the top for gastrula and neurula stages. In the drawings, the presumptive ectoderm is highlighted in light blue, the epidermis territory in turquoise, and the forming CNS in dark blue. At N4 stage, Rfx1/2/3 is expressed in the cerebral vesicle (arrowhead) and Klf1/2/4 in the anterior endoderm (arrowhead). Nr6a is expressed during neurulation in the cerebral vesicle region (arrowheads). Stox is expressed at N3 and N4 stages in the posterior endoderm (arrowheads) and in the anterior endoderm at N4 stage (double arrowhead). Insm is expressed in the neural plate/tube as well as in the epidermal sensory neurons at neurula stages (arrowheads). Scale bar = 50 µm.

Fig. 6.

Functional analysis of putative GRN nodes. In situ hybridization for Elav, Neurogenin, SoxB1a, and K1 were undertaken on control embryos and on embryos injected with SoxB1a-EN, Dlx, or Klf1/2/4 mRNA and fixed at the N2 stage (19 hpf at 19°C). Side views with anterior to the left and dorsal to the top. Scale bar = 50 µm. The number of embryos showing the same expression pattern is indicated in each picture.