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A Hox regulatory network of hindbrain segmentation is conserved to the base of vertebrates

Abstract

A defining feature governing head patterning of jawed vertebrates is a highly conserved gene regulatory network that integrates hindbrain segmentation with segmentally restricted domains of Hox gene expression. Although non-vertebrate chordates display nested domains of axial Hox expression, they lack hindbrain segmentation. The sea lamprey, a jawless fish, can provide unique insights into vertebrate origins owing to its phylogenetic position at the base of the vertebrate tree1,2,3. It has been suggested that lamprey may represent an intermediate state where nested Hox expression has not been coupled to the process of hindbrain segmentation4,5,6. However, little is known about the regulatory network underlying Hox expression in lamprey or its relationship to hindbrain segmentation. Here, using a novel tool that allows cross-species comparisons of regulatory elements between jawed and jawless vertebrates, we report deep conservation of both upstream regulators and segmental activity of enhancer elements across these distant species. Regulatory regions from diverse gnathostomes drive segmental reporter expression in the lamprey hindbrain and require the same transcriptional inputs (for example, Kreisler (also known as Mafba), Krox20 (also known as Egr2a)) in both lamprey and zebrafish. We find that lamprey hox genes display dynamic segmentally restricted domains of expression; we also isolated a conserved exonic hox2 enhancer from lamprey that drives segmental expression in rhombomeres 2 and 4. Our results show that coupling of Hox gene expression to segmentation of the hindbrain is an ancient trait with origin at the base of vertebrates that probably led to the formation of rhombomeric compartments with an underlying Hox code.

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Figure 1: Conserved segmental activity of jawed vertebrate enhancers in zebrafish and lamprey.
Figure 2: Expression of segmental regulators and hox genes in the lamprey hindbrain.
Figure 3: Identification of enhancers from the lamprey hox2 locus.
Figure 4: Comparison of enhancer activity and segmental gene expression in lamprey supports an origin of the hindbrain GRN at the base of vertebrates.

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Accession codes

Primary accessions

GenBank/EMBL/DDBJ

Data deposits

The sequences for the lamprey hox1w and kreisler transcripts have been deposited in GenBank under accession numbers KM087087 (hox1w) and KM087088 (kreisler). All original source data have been deposited in the Stowers Institute Original Data Repository and are available online at http://odr.stowers.org/websimr/.

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Acknowledgements

We thank S. Green for sharing expertise, methods and managing the lamprey facility; T. Sauka-Spengler for in situ hybridization advice; M. Simoes-Costa and B. Uy for help with reporter constructs; J. McEllin, C. Nolte, C. Scott and L. Wiedemann for discussions and assistance with lamprey hox genes; M. Distel and R. Koster for the r3/r5-mCherry zebrafish line; M. Miller for graphic design; A. Ikmi for manuscript comments and the Stowers Institute aquatics facility for zebrafish care. H.J.P. and R.K. were supported by the Stowers Institute (RK grant #2013-1001). M.E.B. was supported by grants R01NS086907 and R01DE017911.

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Authors and Affiliations

Authors

Contributions

H.J.P., R.K. and M.E.B. conceived this research program. H.J.P. conducted the experiments. H.J.P., R.K. and M.E.B. jointly analysed the data, discussed the ideas and interpretations and wrote the manuscript.

Corresponding author

Correspondence to Robb Krumlauf.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Gnathostome enhancer elements selected for reporter analysis.

Schematic diagrams depicting the gnathostome enhancer elements assayed for activity in zebrafish and lamprey embryos in this study. The endogenous genomic positions of the enhancer elements (green boxes) are shown relative to the genes that they regulate. Known trans-acting factors are listed above the elements, while the corresponding regulatory modules and their combined activity domains are detailed below the elements. For each element, the species from which it was cloned are listed on the right. Figure adapted with permission from figure 4.2 in ref. 9.

Extended Data Figure 2 Segmental activity of additional jawed vertebrate enhancers in zebrafish and lamprey.

GFP reporter expression mediated by gnathostome enhancer elements in zebrafish and lamprey embryos. Dorsal views are shown, with anterior to the top. For zebrafish, two images of the same embryo are shown, presenting GFP plus brightfield (top) and GFP plus endogenous r3r5-mCherry (middle) signals. The zebrafish otic vesicle is circled. m, mouse; zf, zebrafish.

Extended Data Figure 3 Segmental patterns of GFP reporter expression in transgenic zebrafish lines.

Lateral (top) and dorsal (middle) views of 30 hpf transgenic (F1) zebrafish embryos show combined brightfield illumination and segmental GFP reporter expression in the hindbrain mediated by five different gnathostome enhancer elements. The corresponding transient transgenic GFP expression patterns mediated by these elements are shown in Fig. 1b and Extended Data Fig. 2. When available, GFP lines were crossed with the endogenous r3r5-mCherry reporter line as a reference (bottom). The otic vesicle is circled. m, mouse; zf, zebrafish.

Extended Data Figure 4 Developmental time course of GFP reporter expression mediated by lamprey and gnathostome regulatory elements in lamprey embryos.

Developmental stages st18–26 are shown. All embryos are positioned such that the hindbrain is viewed dorsally, with anterior to the top, except for mouse Hoxb4 at st22, which is viewed laterally with anterior to the left. For hoxb2 a weaker r6 stripe begins to appear at st23. Black boxes indicate no GFP expression mediated by that element at that developmental stage. In both fish and lamprey, expression driven by the gnathostome Hoxb1 enhancers appears to be temporally dynamic, starting broad and refining with time, which is probably caused by autoregulation within this element. However, we cannot rule out the possibility that the enhancers used may be missing some repressor elements that are required for fine-tuning. m, mouse; zf, zebrafish.

Extended Data Table 1 Zebrafish reporter assay statistics
Extended Data Table 2 Lamprey reporter assay statistics

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Parker, H., Bronner, M. & Krumlauf, R. A Hox regulatory network of hindbrain segmentation is conserved to the base of vertebrates. Nature 514, 490–493 (2014). https://doi.org/10.1038/nature13723

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