Cis-regulatory architecture of a brain signaling center predates the origin of chordates

  • An Erratum to this article was published on 27 July 2016

Abstract

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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: A common cis-regulatory signature in SBE1-like enhancers.
Figure 2: In vivo requirement for the SBE1 transcription factor collective.
Figure 3: Identification of SBE5 as a functional SBE1 homolog.
Figure 4: The ancient origin of SBE1 predates the chordate phylum.

Accession codes

Primary accessions

Gene Expression Omnibus

Change history

  • 16 May 2016

    In the version of this article initially published, the received date for the manuscript was incorrectly listed as 28 January 2015. The correct date is 28 January 2016. The error has been corrected in the HTML and PDF versions of the article.

References

  1. 1

    Neph, S. et al. An expansive human regulatory lexicon encoded in transcription factor footprints. Nature 489, 83–90 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. 2

    Rada-Iglesias, A. et al. A unique chromatin signature uncovers early developmental enhancers in humans. Nature 470, 279–283 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. 3

    Visel, A. et al. ChIP-seq accurately predicts tissue-specific activity of enhancers. Nature 457, 854–858 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. 4

    Yue, F. et al. A comparative encyclopedia of DNA elements in the mouse genome. Nature 515, 355–364 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. 5

    Kiecker, C. & Lumsden, A. Hedgehog signaling from the ZLI regulates diencephalic regional identity. Nat. Neurosci. 7, 1242–1249 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. 6

    Vieira, C. & Martinez, S. Sonic hedgehog from the basal plate and the zona limitans intrathalamica exhibits differential activity on diencephalic molecular regionalization and nuclear structure. Neuroscience 143, 129–140 (2006).

    Article  CAS  PubMed  Google Scholar 

  7. 7

    Hébert, J.M. & Fishell, G. The genetics of early telencephalon patterning: some assembly required. Nat. Rev. Neurosci. 9, 678–685 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. 8

    Jessell, T.M. Neuronal specification in the spinal cord: inductive signals and transcriptional codes. Nat. Rev. Genet. 1, 20–29 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. 9

    Scholpp, S. & Lumsden, A. Building a bridal chamber: development of the thalamus. Trends Neurosci. 33, 373–380 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. 10

    Frazer, K.A., Elnitski, L., Church, D.M., Dubchak, I. & Hardison, R.C. Cross-species sequence comparisons: a review of methods and available resources. Genome Res. 13, 1–12 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. 11

    Arnold, C.D. et al. Quantitative genome-wide enhancer activity maps for five Drosophila species show functional enhancer conservation and turnover during cis-regulatory evolution. Nat. Genet. 46, 685–692 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. 12

    Fisher, S., Grice, E.A., Vinton, R.M., Bessling, S.L. & McCallion, A.S. Conservation of RET regulatory function from human to zebrafish without sequence similarity. Science 312, 276–279 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. 13

    Hare, E.E., Peterson, B.K., Iyer, V.N., Meier, R. & Eisen, M.B. Sepsid even-skipped enhancers are functionally conserved in Drosophila despite lack of sequence conservation. PLoS Genet. 4, e1000106 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. 14

    Ludwig, M.Z., Bergman, C., Patel, N.H. & Kreitman, M. Evidence for stabilizing selection in a eukaryotic enhancer element. Nature 403, 564–567 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. 15

    Vierstra, J. et al. Mouse regulatory DNA landscapes reveal global principles of cis-regulatory evolution. Science 346, 1007–1012 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. 16

    Busser, B.W. et al. A machine learning approach for identifying novel cell type–specific transcriptional regulators of myogenesis. PLoS Genet. 8, e1002531 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. 17

    De Val, S. et al. Combinatorial regulation of endothelial gene expression by Ets and Forkhead transcription factors. Cell 135, 1053–1064 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. 18

    Erives, A. & Levine, M. Coordinate enhancers share common organizational features in the Drosophila genome. Proc. Natl. Acad. Sci. USA 101, 3851–3856 (2004).

    Article  CAS  PubMed  Google Scholar 

  19. 19

    Junion, G. et al. A transcription factor collective defines cardiac cell fate and reflects lineage history. Cell 148, 473–486 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. 20

    Kratsios, P., Stolfi, A., Levine, M. & Hobert, O. Coordinated regulation of cholinergic motor neuron traits through a conserved terminal selector gene. Nat. Neurosci. 15, 205–214 (2012).

    Article  CAS  Google Scholar 

  21. 21

    Visel, A., Minovitsky, S., Dubchak, I. & Pennacchio, L.A. VISTA Enhancer Browser—a database of tissue-specific human enhancers. Nucleic Acids Res. 35, D88–D92 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. 22

    Epstein, D.J., McMahon, A.P. & Joyner, A.L. Regionalization of Sonic hedgehog transcription along the anteroposterior axis of the mouse central nervous system is regulated by Hnf3-dependent and -independent mechanisms. Development 126, 281–292 (1999).

    CAS  PubMed  Google Scholar 

  23. 23

    Pavesi, G., Mereghetti, P., Mauri, G. & Pesole, G. Weeder Web: discovery of transcription factor binding sites in a set of sequences from co-regulated genes. Nucleic Acids Res. 32, W199–W203 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. 24

    Brown, C.D., Johnson, D.S. & Sidow, A. Functional architecture and evolution of transcriptional elements that drive gene coexpression. Science 317, 1557–1560 (2007).

    Article  CAS  PubMed  Google Scholar 

  25. 25

    Smith, R.P. et al. Massively parallel decoding of mammalian regulatory sequences supports a flexible organizational model. Nat. Genet. 45, 1021–1028 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. 26

    Holland, P.W.H., Booth, H.A.F. & Bruford, E.A. Classification and nomenclature of all human homeobox genes. BMC Biol. 5, 47 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. 27

    Acampora, D., Avantaggiato, V., Tuorto, F. & Simeone, A. Genetic control of brain morphogenesis through Otx gene dosage requirement. Development 124, 3639–3650 (1997).

    CAS  PubMed  Google Scholar 

  28. 28

    Sakurai, Y. et al. Otx2 and Otx1 protect diencephalon and mesencephalon from caudalization into metencephalon during early brain regionalization. Dev. Biol. 347, 392–403 (2010).

    Article  CAS  PubMed  Google Scholar 

  29. 29

    Scholpp, S. et al. Otx1l, Otx2 and Irx1b establish and position the ZLI in the diencephalon. Development 134, 3167–3176 (2007).

    Article  CAS  PubMed  Google Scholar 

  30. 30

    Juraver-Geslin, H.A., Gómez-Skarmeta, J.L. & Durand, B.C. The conserved barH-like homeobox-2 gene barhl2 acts downstream of orthodentricle-2 and together with iroquois-3 in establishment of the caudal forebrain signaling center induced by Sonic Hedgehog. Dev. Biol. 396, 107–120 (2014).

    Article  CAS  PubMed  Google Scholar 

  31. 31

    Fernandez-L, A. et al. YAP1 is amplified and up-regulated in hedgehog-associated medulloblastomas and mediates Sonic hedgehog–driven neural precursor proliferation. Genes Dev. 23, 2729–2741 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. 32

    Rosenbluh, J. et al. β-catenin-driven cancers require a YAP1 transcriptional complex for survival and tumorigenesis. Cell 151, 1457–1473 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. 33

    Jeong, Y. et al. Spatial and temporal requirements for sonic hedgehog in the regulation of thalamic interneuron identity. Development 138, 531–541 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. 34

    Shen, Y. et al. A map of the cis-regulatory sequences in the mouse genome. Nature 488, 116–120 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. 35

    Irimia, M. et al. Conserved developmental expression of Fezf in chordates and Drosophila and the origin of the zona limitans intrathalamica (ZLI) brain organizer. Evodevo 1, 7 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. 36

    Pani, A.M. et al. Ancient deuterostome origins of vertebrate brain signalling centres. Nature 483, 289–294 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. 37

    Irimia, M. et al. Comparative genomics of the Hedgehog loci in chordates and the origins of Shh regulatory novelties. Sci. Rep. 2, 433 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. 38

    Sugahara, F. et al. Involvement of Hedgehog and FGF signalling in the lamprey telencephalon: evolution of regionalization and dorsoventral patterning of the vertebrate forebrain. Development 138, 1217–1226 (2011).

    Article  CAS  Google Scholar 

  39. 39

    Takatori, N., Satou, Y. & Satoh, N. Expression of hedgehog genes in Ciona intestinalis embryos. Mech. Dev. 116, 235–238 (2002).

    Article  CAS  Google Scholar 

  40. 40

    Shubin, N., Tabin, C. & Carroll, S. Deep homology and the origins of evolutionary novelty. Nature 457, 818–823 (2009).

    Article  CAS  PubMed  Google Scholar 

  41. 41

    Liu-Chittenden, Y. et al. Genetic and pharmacological disruption of the TEAD–YAP complex suppresses the oncogenic activity of YAP. Genes Dev. 26, 1300–1305 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. 42

    Zhao, L. et al. Disruption of SoxB1-dependent Sonic hedgehog expression in the hypothalamus causes septo-optic dysplasia. Dev. Cell 22, 585–596 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. 43

    Suda, Y. et al. The same enhancer regulates the earliest Emx2 expression in caudal forebrain primordium, subsequent expression in dorsal telencephalon and later expression in the cortical ventricular zone. Development 137, 2939–2949 (2010).

    Article  CAS  PubMed  Google Scholar 

  44. 44

    Symmons, O. et al. Functional and topological characteristics of mammalian regulatory domains. Genome Res. 24, 390–400 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. 45

    Ruf, S. et al. Large-scale analysis of the regulatory architecture of the mouse genome with a transposon-associated sensor. Nat. Genet. 43, 379–386 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. 46

    Ding, Q. et al. BARHL2 differentially regulates the development of retinal amacrine and ganglion neurons. J. Neurosci. 29, 3992–4003 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. 47

    Mahoney, J.E., Mori, M., Szymaniak, A.D., Varelas, X. & Cardoso, W.V. The hippo pathway effector Yap controls patterning and differentiation of airway epithelial progenitors. Dev. Cell 30, 137–150 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. 48

    Riccomagno, M.M., Martinu, L., Mulheisen, M., Wu, D.K. & Epstein, D.J. Specification of the mammalian cochlea is dependent on Sonic hedgehog. Genes Dev. 16, 2365–2378 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. 49

    Ran, F.A. et al. Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 8, 2281–2308 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. 50

    Jeong, Y., El-Jaick, K., Roessler, E., Muenke, M. & Epstein, D.J. A functional screen for sonic hedgehog regulatory elements across a 1 Mb interval identifies long-range ventral forebrain enhancers. Development 133, 761–772 (2006).

    Article  CAS  PubMed  Google Scholar 

  51. 51

    Grant, G.R. et al. Comparative analysis of RNA-Seq alignment algorithms and the RNA-Seq unified mapper (RUM). Bioinformatics 27, 2518–2528 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. 52

    Pavesi, G. & Pesole, G. Using Weeder for the discovery of conserved transcription factor binding sites. Curr. Protoc. Bioinformatics Chapter 2, Unit 2.11 (2006).

  53. 53

    Newburger, D.E. & Bulyk, M.L. UniPROBE: an online database of protein binding microarray data on protein–DNA interactions. Nucleic Acids Res. 37, D77–D82 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. 54

    Sandelin, A., Alkema, W., Engström, P., Wasserman, W.W. & Lenhard, B. JASPAR: an open-access database for eukaryotic transcription factor binding profiles. Nucleic Acids Res. 32, D91–D94 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. 55

    Lowe, C.J. et al. Dorsoventral patterning in hemichordates: insights into early chordate evolution. PLoS Biol. 4, e291 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

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.).

Author information

Affiliations

Authors

Contributions

Y.Y. and D.J.E. conceived the project, designed the experiments and wrote the manuscript. Y.Y. performed the cotransfection, transgenic mouse, gene expression and ChIP assays. P.J.M. performed the transgenic hemichordate reporter assays. Y.J. performed the transgenic mouse reporter assays with core region constructs. Y.-T.Z. performed the statistical analysis. Y.Y. and A.N.K. performed the motif analysis. A.M.P. and C.J.L. provided reagents and advice on the hemichordate experiments. Y.Y., L.G., O.S., W.V.C. and F.S. generated mutant mouse lines and provided embryos.

Corresponding author

Correspondence to Douglas J Epstein.

Ethics declarations

Competing interests

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.

Supplementary Figure 2 SBE1-like enhancers from the VISTA Enhancer Browser.

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.

Supplementary Figure 3 Genomic location of SBE1-like enhancers.

(ag) 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.

Supplementary Figure 4 RNA-seq profile of the SBE1 domain.

(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. (cf) Whole-mount in situ hybridization for candidate transcription factors expressed in the SBE1 domain at E10.5. Scale bars, 1 mm.

Supplementary Figure 5 An extended Otx binding site (motif 1) is required for SBE1 activity.

(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.

Supplementary Figure 6 SBE1–transcription factor interactions.

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).

Supplementary Figure 7 In vivo requirement of motifs 4 and 5 for SBE1 activity.

(ac) 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).

Supplementary Figure 8 Sequence alignment of SBE1 with SBE5 or skSBE1.

(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.

Supplementary Figure 9 Shh expression in the zli is codependent on the SBE1 and SBE5 enhancers.

(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. (bf) 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.

Supplementary Figure 10 Gene regulatory network underlying Shh expression in the zli.

(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 information

Supplementary Text and Figures

Supplementary Figures 1–10 (PDF 5758 kb)

Supplementary Table 1

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)

Supplementary Table 2

This table contains a list of the primers used in this study. (XLSX 32 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

Further reading