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

Journal name:
Nature Genetics
Volume:
48,
Pages:
575–580
Year published:
DOI:
doi:10.1038/ng.3542
Received
Accepted
Published online
Corrected online

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.

At a glance

Figures

  1. A common cis-regulatory signature in SBE1-like enhancers.
    Figure 1: A common cis-regulatory signature in SBE1-like enhancers.

    (a) Heads of transgenic embryos (E11.5) showing X-gal staining from reporter constructs for mouse SBE1 and selected human SBE1-like enhancers from the VISTA Enhancer Browser (VM, ventral midbrain; VPD, ventroposterior diencephalon). Chromosome positions are indicated (NCBI37/mm9 (mouse) and GRCh37/hg19 (human)). (b) Shared motifs identified by Weeder (motifs 1–5) and by JASPAR and UniPROBE (motif 6) that are significantly enriched in SBE1-like enhancers. Transcription factors matching a particular motif that are also expressed in the SBE1 domain appear in blue. (c) DNA sequence alignment (MAFFT version 7) of a core region of SBE1 from representative vertebrate species showing the positions of shared motifs. Deeply conserved SBE1 nucleotides are shaded in gray. (dg) Luciferase reporter assays performed in COS-1 cells transfected with reporter constructs encoding mouse homologs of SBE1-like enhancers (x axes) and expression vectors encoding Otx2 (d), Barhl2 (e), Otx2 and Barhl2 (f), and Yap1 and Tead2 (g). Comparison of luciferase activity is to the activity observed in cells transfected with empty vector (dashed lines). Bar graphs are color-coded to match transcription factors with their binding sites (motifs). Black bars represent mutant SBE1 reporter constructs in which a particular motif was deleted. Lower concentrations of the Otx2 and Barhl2 expression vectors were used in f than in d and e to demonstrate the synergy between these transcription factors. Each bar represents the average of at least three experiments performed in triplicate. (hj) ChIP–qPCR performed with chromatin isolated from E10.5 embryonic mouse brain (B) and limb bud (L) for Otx2 (h) and from transfected COS-1 cells for Barhl2 (i) and Tead2 (j). qPCR results represent an average of at least three biological replicates. The negative-control (NC) and positive-control (PC) primers in h amplify sequence upstream of SBE1 and within an Emx2 forebrain enhancer, respectively. Error bars in all graphs represent standard deviation of the mean (s.d.m.): *P < 0.05, **P < 0.01, ***P < 0.001, Student's t test.

  2. In vivo requirement for the SBE1 transcription factor collective.
    Figure 2: In vivo requirement for the SBE1 transcription factor collective.

    (ad) X-gal staining of transgenic embryos expressing wild-type SBE1-lacZ (a) or the mutant versions SBE1Δmotif1-lacZ (b), SBE1Δmotif6-lacZ (c) and SBE1Δmotif2/2.1-lacZ (d) at E10.5. The extent of zli staining is indicated by the length of each red bracket. The number of stained embryos out of the total number of embryos carrying a given transgene is indicated. Scale bars, 1 mm. (e) Schematic of an E10.5 embryo demonstrating the approach for measuring the spatial distribution of X-gal staining or Shh expression in the zli with respect to head size. (fi) Whole-mount in situ hybridization for Shh in control embryos (f), Barhl2−/− embryos (n = 4) (g), and embryos with conditional disruption of one (h) or both (i) alleles of Yap1 (n = 4) at E10.5. Scale bars, 1 mm. (j) Quantification of the spatial distribution of X-gal staining (blue bars) or Shh expression (purple bars) in the zli normalized to head size. WT, wild type. Error bars represent s.d.m.: *P < 0.01, **P < 0.001, ***P < 0.0001, Student's t test.

  3. Identification of SBE5 as a functional SBE1 homolog.
    Figure 3: Identification of SBE5 as a functional SBE1 homolog.

    (a) UCSC Genome Browser view (NCBI37/mm9) of a 1-Mb interval upstream of Shh showing ChIP-seq signal enrichment for chromatin marks (H3K27ac and H3K4me1) associated with enhancer activity in whole brain (E14.5)34. The locations of SBE1- and SBE5-related peaks (outlined in blue) and other Shh CNS-enhancer-related peaks (outlined in gray) are shown as well as the shuffled arrangements of the motifs (colored boxes). (b) Activation of an SBE5-luciferase reporter construct by members of the SBE1 transcription factor collective. (ce) ChIP–qPCR analysis shows SBE5 enrichment in Otx2-bound chromatin from E10.5 mouse brain (B) but not limb bud (L) (c), as well as in Barhl2-bound (d) and Tead2-bound (e) chromatin from COS-1 cells. The negative-control and positive-control primers in c amplify sequences upstream of SBE1 and within an Emx2 forebrain enhancer, respectively. Error bars represent s.d.m.: **P < 0.01, ***P < 0.001, Student's t test. Each bar in be represents the average of at least three experiments performed in triplicate. (f) X-gal staining of a transgenic embryo (E10.5) expressing SBE5-lacZ in a similar pattern to SBE1-lacZ (compare with Fig. 2a). (gj) Whole-mount in situ hybridization for Shh on a control embryo (g), an embryo with SBE1 deletion (h), an embryo with SBE5 deletion (i) and an embryo with deletion of both SBE1 and SBE5 (j) (E10.5). ShhΔSBE1/ΔSBE1 embryos show reduced Shh expression in the ventral midbrain and caudal diencephalon (red arrow). ShhΔSBE5/ΔSBE5 embryos display a partial truncation in Shh zli expression (red bracket). ShhΔSBE1ΔSBE5/ΔSBE1ΔSBE5 embryos are devoid of Shh in the entire SBE1 domain. Scale bars, 0.5 mm.

  4. The ancient origin of SBE1 predates the chordate phylum.
    Figure 4: The ancient origin of SBE1 predates the chordate phylum.

    (a) Schematic of the Shh and hh gene structures in mouse and S. kowalevskii (acorn worm), respectively, showing the position of SBE1 (blue oval) in the second intron of both species. The shuffled arrangement of the SBE1-like motifs (colored boxes) within the 1.1-kb skSBE1 sequence is shown. (b) Otx2 and Yap–Tead2 but not Barhl2 were sufficient to activate skSBE1-driven luciferase activity in cotransfection assays performed in COS-1 cells. Error bars represent s.d.m.: ***P < 0.001, Student's t test; NS, not significant. Each bar represents the average of at least three experiments performed in triplicate. (c,d) Transgenic mouse embryos expressing mouse SBE1-lacZ (c) and skSBE1-lacZ (d) reporter constructs at E10.5 show similar patterns of X-gal staining in the ventral midbrain, ventroposterior diencephalon and zli (arrows). Ectopic staining outside of these domains is likely due to the site of transgene integration. Scale bars, 1 mm. (e) hh expression in a hemichordate embryo at 48 hours post-fertilization. Scale bar, 100 μm. (fi) Transgenic S. kowalevskii embryos expressing mNeonGreen in a narrow band of cells at the prospective proboscis–collar boundary from skSBE1 (n = 6/60 injected) (f); mmSBE1 (n = 5/55 injected) (g); mmSBE5 (n = 5/15 injected) (h); and control (gbx promoter only; n = 0/60 injected) (i) reporter constructs. Scale bars, 100 μm. (j) The evolutionary trajectory of Shh expression in the zli and hh expression in zli-like structures (black arrows) correlates with presence of the SBE1 motif cluster.

  5. A 116-bp core region of SBE1 is necessary and sufficient to regulate enhancer activity in transgenic embryos.
    Supplementary Fig. 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.

  6. SBE1-like enhancers from the VISTA Enhancer Browser.
    Supplementary Fig. 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.

  7. Genomic location of SBE1-like enhancers.
    Supplementary Fig. 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.

  8. RNA-seq profile of the SBE1 domain.
    Supplementary Fig. 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.

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

  10. SBE1-transcription factor interactions.
    Supplementary Fig. 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).

  11. In vivo requirement of motifs 4 and 5 for SBE1 activity.
    Supplementary Fig. 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).

  12. Sequence alignment of SBE1 with SBE5 or skSBE1.
    Supplementary Fig. 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.

  13. Shh expression in the zli is codependent on the SBE1 and SBE5 enhancers.
    Supplementary Fig. 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.

  14. Gene regulatory network underlying Shh expression in the zli.
    Supplementary Fig. 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.

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Gene Expression Omnibus

Change history

Corrected online 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.

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Author information

  1. These authors contributed equally to this work.

    • Paul J Minor &
    • Ying-Tao Zhao

Affiliations

  1. Department of Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

    • Yao Yao,
    • Ying-Tao Zhao,
    • Anna N King &
    • Douglas J Epstein
  2. Hopkins Marine Station, Department of Biology, Stanford University, Pacific Grove, California, USA.

    • Paul J Minor,
    • Ariel M Pani &
    • Christopher J Lowe
  3. Department of Genetic Engineering, College of Life Sciences and Graduate School of Biotechnology, Kyung Hee University, Yongin-si, Republic of Korea.

    • Yongsu Jeong
  4. Developmental Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany.

    • Orsolya Symmons &
    • François Spitz
  5. Department of Ophthalmology, University of Rochester Medical Center, Rochester, New York, USA.

    • Lin Gan
  6. Columbia Center for Human Development, Department of Medicine, Pulmonary Allergy Critical Care, Columbia University Medical Center, New York, New York, USA.

    • Wellington V Cardoso

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.

Competing financial interests

The authors declare no competing financial interests.

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Supplementary information

Supplementary Figures

  1. Supplementary Figure 1: A 116-bp core region of SBE1 is necessary and sufficient to regulate enhancer activity in transgenic embryos. (306 KB)

    (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.

  2. Supplementary Figure 2: SBE1-like enhancers from the VISTA Enhancer Browser. (652 KB)

    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.

  3. Supplementary Figure 3: Genomic location of SBE1-like enhancers. (424 KB)

    (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.

  4. Supplementary Figure 4: RNA-seq profile of the SBE1 domain. (234 KB)

    (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.

  5. Supplementary Figure 5: An extended Otx binding site (motif 1) is required for SBE1 activity. (183 KB)

    (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.

  6. Supplementary Figure 6: SBE1–transcription factor interactions. (75 KB)

    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).

  7. Supplementary Figure 7: In vivo requirement of motifs 4 and 5 for SBE1 activity. (132 KB)

    (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).

  8. Supplementary Figure 8: Sequence alignment of SBE1 with SBE5 or skSBE1. (739 KB)

    (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.

  9. Supplementary Figure 9: Shh expression in the zli is codependent on the SBE1 and SBE5 enhancers. (109 KB)

    (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.

  10. Supplementary Figure 10: Gene regulatory network underlying Shh expression in the zli. (128 KB)

    (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.

PDF files

  1. Supplementary Text and Figures (6,254 KB)

    Supplementary Figures 1–10

Excel files

  1. Supplementary Table 1 (53,613 KB)

    This table lists the genomic positions of SBE1 and the 52 SBE1-like enhancers in the human (hg19) and mouse (mm9) genome.

  2. Supplementary Table 2 (33,470 KB)

    This table contains a list of the primers used in this study.

Additional data