1. Developmental Biology

An Eya1-Notch axis specifies bipotential epibranchial differentiation in mammalian craniofacial morphogenesis

  1. Haoran Zhang
  2. Li Wang
  3. Elaine Yee Man Wong
  4. Sze Lan Tsang
  5. Pin-Xian Xu
  6. Urban Lendahl
  7. Mai Har Sham  Is a corresponding author
  1. The University of Hong Kong, China
  2. Mount Sinai School of Medicine, United States
  3. Karolinska Institutet, Sweden
https://doi.org/10.7554/eLife.30126
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Cite this article
  1. Haoran Zhang
  2. Li Wang
  3. Elaine Yee Man Wong
  4. Sze Lan Tsang
  5. Pin-Xian Xu
  6. Urban Lendahl
  7. Mai Har Sham
(2017)
An Eya1-Notch axis specifies bipotential epibranchial differentiation in mammalian craniofacial morphogenesis
eLife 6:e30126.
https://doi.org/10.7554/eLife.30126
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8 figures, 3 tables and 1 additional file

Figures

Figure 1 with 1 supplement
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Cranial placode and pharyngeal arch development in wildtype and Eya1-/- mouse embryos.

(A) Schematic summary of the development of cranial placodes and pharyngeal arches (PA) in mouse embryos. The pre-placodal region, marked by expression of Eya and Six families of genes at E8.0, is …

https://doi.org/10.7554/eLife.30126.002
Figure 1—source data 1

Source data relating to Figure 1—figure supplement 1E.

https://doi.org/10.7554/eLife.30126.004
Figure 1—figure supplement 1
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TUNEL assay in WT and Eya1-/- E9.5 embryos.

(A–D) TUNEL assay on coronal sections of WT and Eya1-/- embryos at E9.5, at the level above the pharyngeal clefts (dorsal) and at the level of the proximal PA. More apoptotic cells (arrowheads) were …

https://doi.org/10.7554/eLife.30126.003
Figure 2 with 1 supplement
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Differences in pharyngeal arch phenotypes in Eya1-/- and Six1-/- embryos.

(A–C’) In situ hybridization on whole-mount and coronal sections of Eya1 and Six1 in WT embryos at E8.5 (n = 5) and E9.5 (n = 5). (D–I) Immunostaining for Sox2 and Sox3 on coronal sections of E9.5 WT…

https://doi.org/10.7554/eLife.30126.005
Figure 2—figure supplement 1
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Expression of Eya1, Fgf8, Fgf15, and Fgfr1 in the pharyngeal ectoderm of E9.5 embryos.

(A) Immunostaining for Eya1 on coronal section of WT embryos at E9.5 (n = 5). The region demarcated by white dotted box in (A) is magnified in (A’), showing expression of Eya1 protein in both the …

https://doi.org/10.7554/eLife.30126.006
Figure 3 with 1 supplement
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Bipotential Sox2+ progenitors give rise to both neurogenic and non-neuronal epibranchial placodal cells.

(A–D) Sox2, Fgf3 and Neurog2 expression in whole-mount WT embryos at E8.0, E8.5, E8.75 and E9.5. At E8.0, the pharyngeal surface ectoderm was induced to form the thickened posterior placode, which …

https://doi.org/10.7554/eLife.30126.007
Figure 3—figure supplement 1
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Analysis of bipotential Sox2+ progenitors of the neurogenic and non-neuronal lineages in the epibranchial placodes.

(A–D’) Co-immunostaining for Neurog2/Sox2 on transverse sections of WT at 5ss and of Eya1-/- embryos at 5ss, 8ss, 16ss and 25ss (n = 3/stage). (E) Schematic diagram showing the development of Sox2+ …

https://doi.org/10.7554/eLife.30126.008
Figure 4 with 2 supplements
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Downregulated Notch signaling in the ectoderm-derived pharyngeal epithelium of Eya1-/- embryos.

(A–D and A’–D’) Immunostaining for Notch1 ICD by V1744, which specifically recognizes Notch1 ICD (A–B’); or by C20, which recognizes an internal epitope on Notch1 ICD and thus both cleaved and …

https://doi.org/10.7554/eLife.30126.009
Figure 4—figure supplement 1
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Expression of Notch signaling factors in the ectoderm-derived pharyngeal epithelium of WT and Eya1-/- embryos.

(A, A’ and A’’) Co-immunostaining of Notch1 (C20) with Eya1 and Sox2 in rostral-proximal pharyngeal ectoderm of WT at E9.5. Notch1 receptor is expressed in both nucleus and cytoplasm in the …

https://doi.org/10.7554/eLife.30126.010
Figure 4—figure supplement 2
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Complete western blots for Figure 4M.
https://doi.org/10.7554/eLife.30126.011
Figure 5 with 1 supplement
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Over-expression of Notch1 ICD restores pharyngeal segmentation and Fgf3 expression in the pharyngeal epithelium of Eya1-/- embryos.

(A–D) Whole mount fluorescence and section immunostaining for EYFP in Pax2-Cre; R26REYFP (A, B) and GFP in Pax2-Cre;RosaN1-IC (C, D) at E9.5, to visualize Cre efficiency and expression of ectopic …

https://doi.org/10.7554/eLife.30126.012
Figure 5—figure supplement 1
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Over-expression of Notch1 ICD by Pax2-Cre, B2-r4-Cre and Sox2-CreER restored pharyngeal segmentation and morphogenesis in Eya1-/- embryos.

(A–F) In situ hybridization showing Fgf15, Hey1 and Spry1 expression in Pax2-Cre; RosaN1-IC and Pax2-Cre; RosaN1-IC; Eya1-/- embryos at E9.5 (n = 3). (G–J) Lateral view of Sox2-CreER; RosaN1-IC, Sox2…

https://doi.org/10.7554/eLife.30126.013
Figure 6 with 2 supplements
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Eya1 stabilizes and dephosphorylates Notch1 ICD.

(A) Co-immunoprecipitation (Co-IP) analysis using 293T cells with (+) or without (-) transfected Myc-Notch1 ICD and Flag-Eya1, as indicated (n = 6). Pull-down assays (IP) using anti-Flag or anti-Myc …

https://doi.org/10.7554/eLife.30126.014
Figure 6—source data 1

Source data relating to Figure 6E.

https://doi.org/10.7554/eLife.30126.017
Figure 6—source data 2

Source data relating to Figure 6F.

https://doi.org/10.7554/eLife.30126.018
Figure 6—source data 3

Source data relating to Figure 6G.

https://doi.org/10.7554/eLife.30126.019
Figure 6—figure supplement 1
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Eya1 does not affect serine or tyrosine phosphorylation level of Notch1 ICD.

(A) Coomassie staining of SDS-PAGE showing purified Flag-Notch1 ICD, Flag-T2122A, Flag-Eya1 from 293T cells. (B–C) Analysis of phosphorylation status of Notch1 ICD. Transfected 293T cells in the …

https://doi.org/10.7554/eLife.30126.015
Figure 6—figure supplement 2
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Complete western blots for Figure 6 and Figure 6—figure supplement 1.
https://doi.org/10.7554/eLife.30126.016
Figure 7 with 2 supplements
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Eya1 targets the T2122 phosphorylation site of Notch1 ICD.

(A) Schematic diagram showing Notch1 ICD protein domains. Alignment of Notch1 ICD peptide sequence (from a.a. 2119) with c-Myc (from a.a. 55), positions of the phospho-dead (T2122A) and …

https://doi.org/10.7554/eLife.30126.020
Figure 7—source data 1

Source data relating to Figure 7B.

https://doi.org/10.7554/eLife.30126.023
Figure 7—source data 2

Source data relating to Figure 7D.

https://doi.org/10.7554/eLife.30126.024
Figure 7—source data 3

Source data relating to Figure 7E.

https://doi.org/10.7554/eLife.30126.025
Figure 7—figure supplement 1
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Eya1 does not affect the T2487 phosphorylation site of Notch1 ICD, related to Figure 7.

(A) Co-immunoprecipitation analysis of 293T cells transfected with indicated cDNAs. Pull-down assays using anti-HA antibody and immunoblot with anti-Flag and anti-HA. Input was 5% of the amount of …

https://doi.org/10.7554/eLife.30126.021
Figure 7—figure supplement 2
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Complete western blots for Figure 7 and Figure 7—figure supplement 1.
https://doi.org/10.7554/eLife.30126.022
Figure 8
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Model illustrating the role of Eya1 and Notch in regulating neurogenic and non-neuronal lineages during epibranchial placode development.

During PA development, the Eya1+/Sox2+ placodal progenitors formed at E8.0 are separated into two distinct cell populations by E9.0: the Neurog2+ neurogenic cells and the Sox2+/Fgf3+ non-neuronal …

https://doi.org/10.7554/eLife.30126.026

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
antibodyGoat polyclonal anti-Sox2Neuromics (Minnesota, USA)GT15098-100, RRID: AB_21958001/500, IHC
antibodyGoat polyclonal anti-Sox3R and D systems (Minnesota, USA)AF2569, RRID: AB_22399331/500, IHC
antibodyRat monoclonal anti-Jagged1DSHB (Iowa, USA)Ts1.15h, RRID: AB_5283171/300, IHC
antibodyRabbit monoclonal anti-E-cadherin (24E10)Cell Signaling Technology (Massachusetts, USA)3195, RRID: AB_22914711/1000, IHC
antibodyMouse polyclonal anti-Eya1(A01)Abnova (Taiwan)H00002138-A01, RRID: AB_5632411/300, IHC, WB
antibodyGoat polyclonal anti-Notch1 (C20)Santa Cruz Biotechnology (Texas, USA)sc-6014; RRID: AB_6503361/400, IHC, WB, IP
antibodyRabbit polyclonal anti-Cleaved Notch1Abcam (Hong Kong)ab52301, RRID: AB_8817261/500, IHC, WB
antibodyRabbit polyclonal anti- Cleaved Notch1 (Val1744)Cell Signaling Technology2421S,RRID: AB_23142041/500, IHC, WB
antibodyMouse monoclonal anti-Notch3BioLegend (California, USA)130502, RRID: AB_12277351/1000, IHC
antibodyMouse monoclonal anti- Phospho-Threonine-ProlineCell Signaling Technology9391S, RRID: AB_3318011/3000, WB
antibodyRabbit monoclonal anti-Phospho-Serine-Proline-Proline motif [pSPP]Cell Signaling TechnologyCat#143901/1000, WB
antibodyMouse monoclonal anti-p-Tyrosine (PY99)Santa Cruz Biotechnologysc-7020, RRID:AB_6281231/1000, WB
antibodyMouse monoclonal anti-Neurog2R and D systemsMAB3314, RRID:AB_21495201/1000, IHC
antibodyMouse polyclonal anti-Islet1DSHBPCRP-ISL1-1A9, RRID:AB_26187751/400, IHC
antibodyRabbit polyclonal anti-GFPAbcamab6556, RRID:AB_3055641/1000, IHC
antibodyRabbit polyclonal anti-Hes1Abcamab71559, RRID:AB_12095701/500, WB
antibodyRabbit polyclonal anti-Hey1AbcamAB22614, RRID:AB_4471951/500, WB
antibodyMouse monoclonal anti-GAPDH [6C5]AbcamAb8245, RRID:AB_21074481/10000, WB
antibodyMouse monoclonal anti-FLAG(R) M2Sigma-Aldrich (Missouri, USA)F1804, RRID:AB_2620441/500 for IP, 1/4000 for WB
antibodyRabbit monoclonal anti-FlagSigma-Aldrich (Missouri, USA)F7425, RRID:AB_4396871/4000, WB
antibodyMouse monoclonal anti-c-Myc (9E10)Santa Cruz Biotechnologysc-40, RRID:AB_6272681/1000, WB
antibodyMouse monoclonal anti-TubulinDSHBCat#AA4.3-s; RRID: AB_5797931/5000, WB
antibodyRabbit polyclonal anti-HA-probeSanta Cruz Biotechnologysc-805, RRID:AB_6316181/1000, WB
antibodyDonkey anti-Goat IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488Thermo Fisher Scientific (Hong Kong)A-11055, RRID:AB_25341021/500, IHC
antibodyDonkey anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488Thermo Fisher ScientificA21206, RRID:AB_25357921/500, IHC
antibodyDonkey anti-Rat IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488Thermo Fisher ScientificA-21208, RRID:AB_25357941/500, IHC
antibodyDonkey anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 555Thermo Fisher ScientificA-31570, RRID:AB_25361801/500, IHC
antibodyDonkey anti-Goat IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 594Thermo Fisher ScientificA-11058, RRID:AB_25341051/500, IHC
antibodyDonkey anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, HRPThermo Fisher ScientificA16017, RRID:AB_25346911/5000, WB
antibodyDonkey anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, HRPThermo Fisher ScientificA16035, RRID:AB_25347091/5000, WB
antibodyDonkey anti-goat HRP ConjugateSanta Cruz Biotechnologysc-2020, RRID:AB_6317281/5000, WB
antibodyAnti-Digoxigenin-AP, Fab fragmentsRoche (Germany)Cat#110932749101/2000, ISH
peptide, recombinant protein3XFLAG peptideSigma-Aldrich (Missouri, USA)Cat# F4799
chemical compound, drugX-tremeGENE 9 DNA Transfection ReagentRocheCat#6365779001
chemical compound, drugProteinase KSigma-Aldrich (Missouri, USA)Cat#P6556
chemical compound, drugCycloheximideSigma-Aldrich (Missouri, USA)Cat#C7698
chemical compound, drugTamoxifenSigma-Aldrich (Wisconsin, USA)Cat#06734
chemical compound, drugZ-Leu-Leu-Leu-alSigma-Aldrich (Wisconsin, USA)Cat#2211
chemical compound, drugγ-Secretase inhibitor IXCalbiochem (California, USA)Cat#565770
chemical compound, drugPhosStopRocheCat#04906837001
chemical compound, drugCOmplete protease inhibitor cocktailRocheCat#04693132001
chemical compound, drugrProtein G AgaroseInvitrogen (Hong Kong)Cat#15920–010
chemical compound, drugDAPISigma-Aldrich (Missouri, USA)Cat# D9542
commercial assay or kitIn situ Cell Death Detection Kit, FluoresceinRoche Cat#11684795910
cell line (Human)HEK 293T cellsATCC (Virginia, USA)CRL-3216,
RRID: CVCL_0063
strain, strain background (C57BL/6N)Mouse: C57BL/6NLaboratory Animal Unit at the University of Hong KongN/A
strain, strain background (C57BL/6N)Mouse: Sox2-CreERThe Jackson Laboratory (Arnold et al., 2011)RRID:IMSR_JAX:017593
strain, strain background (C57BL/6N)Mouse: Pax2-CreA. Grove (Ohyama and Groves, 2004)RRID:MMRRC_010569-UNC
strain, strain background (C57BL/6N)Mouse: B2-r4-CreK.S.E. Cheah (Szeto et al., 2009)RRID:MGI:3849737
strain, strain background (C57BL/6N)Mouse: Rosa26N1-ICThe Jackson Laboratory (Murtaugh et al., 2003)RRID:IMSR_JAX:008159
strain, strain background (C57BL/6N)Mouse: R26REYFPThe Jackson Laboratory (Srinivas et al., 2001)RRID:IMSR_JAX:006148
strain, strain background (C57BL/6N)Mouse: Eya1-/-P.X. Xu (Xu et al., 1999)RRID:MGI:3054666
strain, strain background (C57BL/6N)Mouse: Six1-/-P.X. Xu (Laclef et al., 2003)RRID:MGI:2655196
sequence-based reagentFull list of primers for cloning in Table 2N/AN/A
sequence-based reagentFull list of primers for genotyping in Table 1N/AN/A
recombinant DNA reagentIn situ hybridization probe : Dlx5(Liu et al., 1997)N/A
recombinant DNA reagentIn situ hybridization probe : Dlx1(Qiu et al., 1995)N/A
recombinant DNA reagentIn situ hybridization probe : Eya1(David et al., 2001)N/A
recombinant DNA reagentIn situ hybridization probe : Six1(Pandur and Moody, 2000)N/A
recombinant DNA reagentIn situ hybridization probe : Sox2(De Robertis et al., 1997)N/A
recombinant DNA reagentIn situ hybridization probe : Fgf8(Crossley and Martin, 1995)N/A
recombinant DNA reagentIn situ hybridization probe : Fgf3(Wilkinson et al., 1988)N/A
recombinant DNA reagentIn situ hybridization probe : Hes1(Zheng et al., 2000)N/A
recombinant DNA reagentIn situ hybridization probe: Fgfr1Addgene (Massachusetts, USA)Cat# 14005
recombinant DNA reagentIn situ hybridization probe: Crabp1(IMAGE 2922473)N/A
recombinant DNA reagentIn situ hybridization probe: Neurog2(IMAGE 468821)N/A
transfected construct (mouse)pcDNA3.1+AddgeneCat# V790-20
transfected construct (mouse)pcDNA3.1-Myc-N1ICDC.C. Hui (Toronto, Canada)N/A
transfected construct (mouse)pCS2+-N1DEF-MycU. Lendahl
(Chapman et al., 2006)		N/A
transfected construct (mouse)pcDNA5-FRT-TO-N1FL-MycU. Lendahl
(Chapman et al., 2006)		N/A
transfected construct (mouse)P3XFLAG-myc-CMV-26Sigma-Aldrich (Missouri, USA)Cat# E6401
transfected construct (mouse)pCMV-3XFlag-N1ICDThis paperN/AGenerated by cloning the N1ICD fragment into thepCMV-3XFlag-Myc-26 vector between BamHI and EcoRI sites
transfected construct (mouse)pCMV-3XFlag-N1ICDT2122AThis paperN/APoint mutation, primers listed in Table 2
transfected construct (mouse)pCMV-3XFlag-N1ICDT2122DThis paperN/APoint mutation, primers listed in Table 2
transfected construct (mouse)pCMV-3XFlag-N1ICDT2487AThis paperN/APoint mutation, primers listed in Table 2
transfected construct (mouse)Flag-Eya1P.X. Xu (Li et al., 2017)N/A
transfected construct (mouse)HA-Eya1P.X. Xu (Li et al., 2017)N/A
transfected construct (mouse)Flag-Eya1D327NP.X. Xu (Li et al., 2017)N/A
transfected construct (mouse)Flag-Eya1CP.X. Xu (Li et al., 2017)N/A
transfected construct (mouse)Flag-Eya1NP.X. Xu (Li et al., 2017)N/A
transfected construct (mouse)Flag-Eya1-DYYThis paperN/APoint mutation, primers listed in Table 2
transfected construct (mouse)Flag-Eya1-Y4This paperN/APoint mutation, primers listed in Table 2
transfected construct (human)pCMV-Myc CDC4 WT*AddgeneCat# 16652
transfected construct (human)HA-UbiquitinAddgeneCat# 18712
software, algorithmImageJhttp://imagej.nih.gov/ij/ImageJ,
RRID:SCR_003070
software, algorithmPrism Version 6http://www.graphpad.comGraphPad Prism, RRID:SCR_002798
Table 1
Sequences of primers for genotyping.
https://doi.org/10.7554/eLife.30126.027
Mouse linePrimer sequenceSource
Six1 knock-outGenoFF: 5’ TCCCACCACTTCTTATCCTAG 3’
Exon1R: 5’ AGTGAGCAGAGCTGGGAGAG 3’
LacZR: 5’ TCTTCGCTATTACGCCAGCTG 3’		Laclef et al. (2003)
Eya1 knock-outGenoF2: 5’ ATGTCTGGTTTTAGTTAGGC 3’
WR2: 5’ AAACTCAGTCTGGGCACCAAG 3’
Neo240F: 5’ CAAGCAAAACCAAATTAAGGG 3’		Xu et al., 1999
Pax2-Cre
Sox2-CreER
B2-R4-Cre		Cre A: 5’ ACGGAAATCCATCGCTCGACCAGTT 3’
Cre S: 5’ GTCCGGGCTGCCACGACCAA 3’		N/A
RosaN1-ICWT F: 5’ TAACCTGGTGTGTGGGCGTTGT 3’
WT R: 5’ AATCTGTGGGAAGTCTTGTCC 3’
Mutant F: 5’ ACCCTGGACTACTGCGCCC 3’
Mutant R: 5’ CGAAGAGTTTGTCCTCAACCG 3’		Murtaugh et al. (2003)
Table 2
Sequences of primers for cDNA cloning.

The bases in red indicate the mutated sites.

https://doi.org/10.7554/eLife.30126.028
PrimerSequence
N1ICD_T2122A-FCTGCCCTGGGTGGCGCACCCACTCTGTCTCC
N1ICD_T2122A-RGGAGACAGAGTGGGTGCGCCACCCAGGGCAG
N1ICD_T2122D-FCTGCCCTGGGTGGCGATCCCACTCTGTCTCC
N1ICD_T2122D-RGGAGACAGAGTGGGATCGCCACCCAGGGCAG
N1ICD_T2487A-FAGCACCCCTTCCTCGCCCCATCCCCTGAGTC
N1ICD_T2487A-RGACTCAGGGGATGGGGCGAGGAAGGGGTGCT
Eya1DYY-FAGTTCACAGCAGGCCGCTCCGTCTGCTCCCGGCTTTGGCCAG
Eya1DYY-RCTGGCCAAAGCCGGGAGCAGACGGAGCGGCCTGCTGTGAACT
Eya1Y4-FCAGGGTCAGGCCGCACAGGCTGCTAACAGCTCGCCGGCTCCAGCACAC
Eya1Y4-RGTGTGCTGGAGCCGGCGAGCTGTTAGCAGCCTGTGCGGCCTGACCCTG
Hey1-FAGAACTAGTGGATCCATGAAGAGAGCTCAC
Hey1-RCTTGATATCGAATTCTTAGAAAGCTCCGAT
Brn1 (Pou3f3)-FAGAACTAGTGGATCCTCAAACAGCGGCGCA
Brn1 (Pou3f3)-RCTTGATATCGAATTCAGTTCACGGGACCTT
Dll1-FAGAACTAGTGGATCCATCTGTCTGCCAGGG
Dll1-RCTTGATATCGAATTCGCACCGTTAGAACAA
Fgf15-FAGAACTAGTGGATCCCAGTTGCTTTGTGGG
Fgf15-RCTTGATATCGAATTCAGTTCACGGGACCTT

Additional files

Transparent reporting form
https://doi.org/10.7554/eLife.30126.029

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