Genomic approaches have predicted hundreds of thousands of tissue-specific cis-regulatory sequences, but the determinants critical to their function and evolutionary history are mostly unknown1,2,3,4. Here we systematically decode a set of brain enhancers active in the zona limitans intrathalamica (zli), a signaling center essential for vertebrate forebrain development via the secreted morphogen Sonic hedgehog (Shh)5,6. We apply a de novo motif analysis tool to identify six position-independent sequence motifs together with their cognate transcription factors that are essential for zli enhancer activity and Shh expression in the mouse embryo. Using knowledge of this regulatory lexicon, we discover new Shh zli enhancers in mice and a functionally equivalent element in hemichordates, indicating an ancient origin of the Shh zli regulatory network that predates the chordate phylum. These findings support a strategy for delineating functionally conserved enhancers in the absence of overt sequence homologies and over extensive evolutionary distances.
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We thank J. Richa and his staff at the Transgenic and Chimeric Mouse Facility (Perelman School of Medicine, University of Pennsylvania) for assistance with transgenic mouse generation. We also thank S. Liebhaber, K. Kaestner, C. Brown, K. Zaret and members of the Epstein laboratory for helpful discussions and comments on the manuscript. This work was funded by grants from the National Institutes of Health, R01 NS039421 (D.J.E.) and R21 EY023104 (L.G.), and the National Science Foundation, 1258169 (C.J.L.), and by a predoctoral fellowship from the Louis-Jeantet Foundation (O.S.).
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 A 116-bp core region of SBE1 is necessary and sufficient to regulate enhancer activity in transgenic embryos.
(a) Schematic of the Shh gene showing the position of SBE1 (blue oval) in the second intron. Multiz alignment from the UCSC Genome Browser of representative vertebrate species shows a high degree of conservation in the core region (CR). (b) Whole-mount view of Shh expression at E10.5. (c,d) X-gal staining of E10.5 embryos expressing 531-bp full-length SBE1 or 116-bp CR(3×) reporter construct in the ventral midbrain (vm), ventroposterior diencephalon (vpd) and zli. Scale bars, 0.5 mm. (e) Results of the in vivo transgenic reporter assay to determine the role of the CR (purple) in mediating SBE1 activity.
X-gal staining of transgenic embryos (E11.5) expressing lacZ reporter constructs under the transcriptional control of the indicated human (hs) or mouse (mm) regulatory sequences. Each of the embryos shows some degree of staining for SBE1-like elements in the ventral midbrain, ventroposterior diencephalon and zli. The X-gal staining in the ventral hindbrain and spinal cord of most embryos results from Hsp68 promoter activity. Embryos with their IDs in red were selected for shared motif analysis as described in Figure 1.
(a–g) UCSC Genome Browser snapshots showing the mouse chromosomal position (mm9) for each of the seven SBE1-like enhancers (red) with respect to the nearest gene expressed in the SBE1 domain. The RNA-seq track from the SBE1 region (E10.5) is displayed in blue. Sequence reads are indicated along the y axis. Scale bars are shown for each genomic interval.
(a) An E10.5 embryo from the 429M20eGFP BAC transgenic line showing GFP fluorescence in the SBE1 domain (dotted line), encompassing the ventral midbrain, ventroposterior diencephalon and zli. Reporter activity is also detected in the floor plate of the hindbrain and spinal cord. RNA was isolated from cells outlined by the dashed line for RNA-seq analysis. (b) A heat map of gene expression in the SBE1 domain showing FPKM values for transcriptional regulators of SBE1. (c–f) Whole-mount in situ hybridization for candidate transcription factors expressed in the SBE1 domain at E10.5. Scale bars, 1 mm.
(a) Sequence logo based on the position weight matrix (PWM) of motif 1 (Otx) from the seven most specific SBE1-like enhancers (top). Comparison of the PWMs for a 10-nt sequence encompassing the consensus Otx binding site, GATTA, derived from 53 SBE1-like enhancers (middle) and 200 random genomic sequences matched for GC content and length (bottom). Adenine nucleotides preferentially flank the GATTA core sequence of the Otx binding site in SBE1-like enhancers as compared to random genomic sequence (**P < 0.01, ***P < 0.001, Fisher’s exact test). (b) Results of luciferase reporter assays in COS-1 cells cotransfected with increasing doses of Otx2 and wild-type (WT) or mutant versions of SBE1-luciferase reporter constructs. The mutations include deletion of motif 1 (blue), point mutations within the GATTA consensus sequence (magenta) and point mutations immediately adjacent to the GATTA binding site in motif 1 (green). (c) ChIP-qPCR performed with chromatin isolated from COS-1 cells that were cotransfected with FLAG-Otx2 and wild-type or mutant versions of SBE1-luciferase constructs as described in b. qPCR results represent an average of three biological replicates (*P < 0.05, **P < 0.01, two-sided Student’s t test). Error bars in all graphs represent s.d.m.
Reporter assays performed in COS-1 cells cotransfected with SBE1-luciferase and a subthreshold dose of expression construct for Barhl2, Yap1, Tead2 or Otx2 or mixtures of each transcription factor. Only the combination of Otx2 and Barhl2 resulted in synergistic activation of SBE1-luciferase expression (*P < 0.05, two-sided Student’s t test).
(a–c) X-gal staining of transgenic embryos expressing wild-type or mutant versions of the SBE1-lacZ construct at E10.5. The extent of zli staining is indicated by the length of the red bracket. Reduced zli staining was observed upon deletion of motif 4 or 5. The number of stained embryos out of the total carrying a given transgene is indicated. Scale bars, 1 mm. (d) Quantification of the spatial distribution of X-gal staining in the zli normalized to head size (*P < 0.01, **P < 0.001, Student’s t test).
(a) SBE1 has 18.1% sequence identity with SBE5. (b) SBE1 has 23.9% sequence identity with skSBE1. Alignments were performed using the pairwise global alignment tool (Emboss Needle)56 with the default setting.
(a) Genomic map of the Shh gene and a 1-Mb region upstream showing the position of SBE1 and SBE5 (blue ovals), as well as ten other previously described Shh enhancers50,57-59. The 228-kb deletion in the mouse Del(C1-Z) Tracer line (mm9 chr. 5: 29,413,901–29,642,246; referred to herein as ShhΔSBE5) is outlined in red. (b–f) Whole-mount in situ hybridization for Shh on wild-type (WT), ShhΔSBE5/ΔSBE5, ShhΔSBE1ΔSBE5/ΔSBE1ΔSBE5, ShhP1; ShhΔSBE1ΔSBE5/ΔSBE1ΔSBE5 and ShhΔSBE5/SBE5Δ2kb embryos at E10.5. The extent of Shh expression along the length of the zli is highlighted (red bracket). The ShhP1 transgene expresses Shh under the influence of SBE1 and Shh floor plate enhancers 1 and 2 (SFPE1 and SFPE2) and restores Shh expression in the ventral midbrain, ventroposterior diencephalon and zli of SBE1/SBE5 double mutants. ShhP1 embryos also display ectopic Shh expression in the otic vesicle (ov). The loss of Shh expression in the fore and hindlimb buds (fl, hl) of SBE5 mutants is attributed to deletion of the zone of polarizing regulatory sequence (ZRS)60. Because the ZRS is not included on the ShhP1 transgene, Shh limb bud expression is not restored in e. Scale bar, 1 mm. (g) Quantification of the spatial distribution of Shh expression in the zli normalized to head size at E10.5. A smaller (2-kb) deletion of SBE5 generated by CRISPR/Cas9 (SBE5Δ2kb) had the same effect on Shh zli expression as the larger (228-kb) SBE5 deletion allele (ΔSBE5). ***P < 0.0001, n = 7, two-sided Student’s t test.
(a) Schematic of a mouse embryo (E10.5) highlighting the domains of Shh expression under the influence of SBE1 and SBE5 (blue hatched lines), which include the ventral midbrain (mb) and basal plate of prosomeres 1–3 (p1–p3), as well as the zona limitans intrathalamica (zli). Additional sites of Shh expression in the ventral midline of the neural tube are indicated (dark gray). sc, spinal cord; hb, hindbrain; tel, telencephalon. (b) Transcriptional control of Shh zli expression. For Shh to gain expression in the zli, this tissue must first be rendered transcriptionally competent through the cross-repressive interactions of Irx homeoproteins (Irx1b and Irx3) and Fez family zinc-finger proteins (Fez and Fezl), as well as the Wnt-mediated extinction of Gli3 expression5,29,61-63. Once a permissive environment is established in the zli (~E10.0), a transcription factor collective, including Otx1, Otx2, Barhl2, Yap–Tead and possibly unidentified transcription factors (TF-X) is recruited to SBE1 and SBE5 to initiate Shh transcription. An early response to Shh signaling from the zli is repression of Pax6, which, in turn, prevents Shh from being expressed beyond the zli64. A morphogenic gradient of Shh signaling emerges from the zli to promote distinct neuronal identities within the thalamic and prethalamic territories65,66.
Supplementary Figures 1–10 (PDF 5758 kb)
This table lists the genomic positions of SBE1 and the 52 SBE1-like enhancers in the human (hg19) and mouse (mm9) genome. (XLSX 52 kb)
This table contains a list of the primers used in this study. (XLSX 32 kb)
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Yao, Y., Minor, P., Zhao, Y. et al. Cis-regulatory architecture of a brain signaling center predates the origin of chordates. Nat Genet 48, 575–580 (2016). https://doi.org/10.1038/ng.3542
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