The TFAP2A-IRF6-GRHL3 genetic pathway is conserved in neurulation

doi:10.1093/hmg/ddz010

. 2019 May 15;28(10):1726-1737.

doi: 10.1093/hmg/ddz010.

The TFAP2A-IRF6-GRHL3 genetic pathway is conserved in neurulation

Youssef A Kousa^{1

2

3}, Huiping Zhu⁴, Walid D Fakhouri⁵, Yunping Lei⁴, Akira Kinoshita⁶, Raeuf R Roushangar¹, Nicole K Patel⁷, A J Agopian⁸, Wei Yang⁹, Elizabeth J Leslie¹⁰, Tamara D Busch¹¹, Tamer A Mansour^{12

13

14}, Xiao Li¹⁵, Arianna L Smith¹², Edward B Li^{16

17}, Dhruv B Sharma¹⁸, Trevor J Williams¹⁹, Yang Chai²⁰, Brad A Amendt¹⁵, Eric C Liao^{16

17}, Laura E Mitchell⁸, Alexander G Bassuk¹¹, Simon Gregory²¹, Allison Ashley-Koch²¹, Gary M Shaw⁹, Richard H Finnell⁴, Brian C Schutte^{1

7

12

22}

Affiliations

¹ Departments of Biochemistry and Molecular Biology.
² Division of Neurology, Childrens National Health System.
³ Center for Neuroscience Research, The Childrens Research Institute, Washington, DC, USA.
⁴ Dell Pediatric Research Institute, Department of Nutritional Sciences, University of Texas at Austin, Austin, TX, USA.
⁵ Department of Diagnostic & Biomedical Sciences, School of Dentistry, University of Texas Health Science Center at Houston, Houston, TX, USA.
⁶ Department of Human Genetics, Nagasaki University, Nagasaki, Japan.
⁷ Microbiology and Molecular Genetics.
⁸ Human Genetics Center, Division of Epidemiology, Human Genetics and Environmental Sciences, University of Texas School of Public Health, Houston, TX, USA.
⁹ Department of Pediatrics, Stanford University School of Medicine, Stanford, CA, USA.
¹⁰ Department of Human Genetics, Emory University School of Medicine, Atlanta, GA, USA.
¹¹ Departments of Pediatrics.
¹² Genetics PhD Program.
¹³ Department of Clinical Pathology, School of Medicine, University of Mansoura, Mansoura, Egypt.
¹⁴ Department of Population Health and Reproduction, School of Veterinary Medicine, University of California, Davis, CA, USA.
¹⁵ Anatomy and Cell Biology, University of Iowa, Iowa City, IA, USA.
¹⁶ Center for Regenerative Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA.
¹⁷ Harvard Stem Cell Institute, Harvard University, Cambridge, MA, USA.
¹⁸ Center for Statistical Training & Consulting, Michigan State University, East Lansing, MI, USA.
¹⁹ Department of Craniofacial Biology, University of Colorado Denver at Anschutz Medical Campus, Aurora, CO, USA.
²⁰ Center for Craniofacial Molecular Biology, Ostrow School of Dentistry, University of Southern California, Los Angeles, CA, USA.
²¹ Duke Molecular Physiology Institute, Duke University Medical Center, Durham, NC, USA.
²² Pediatrics and Human Development.

PMID: 30689861
PMCID: PMC6494790
DOI: 10.1093/hmg/ddz010

The TFAP2A-IRF6-GRHL3 genetic pathway is conserved in neurulation

Youssef A Kousa et al. Hum Mol Genet. 2019.

. 2019 May 15;28(10):1726-1737.

doi: 10.1093/hmg/ddz010.

Authors

Affiliations

¹ Departments of Biochemistry and Molecular Biology.
² Division of Neurology, Childrens National Health System.
³ Center for Neuroscience Research, The Childrens Research Institute, Washington, DC, USA.
⁴ Dell Pediatric Research Institute, Department of Nutritional Sciences, University of Texas at Austin, Austin, TX, USA.
⁵ Department of Diagnostic & Biomedical Sciences, School of Dentistry, University of Texas Health Science Center at Houston, Houston, TX, USA.
⁶ Department of Human Genetics, Nagasaki University, Nagasaki, Japan.
⁷ Microbiology and Molecular Genetics.
⁸ Human Genetics Center, Division of Epidemiology, Human Genetics and Environmental Sciences, University of Texas School of Public Health, Houston, TX, USA.
⁹ Department of Pediatrics, Stanford University School of Medicine, Stanford, CA, USA.
¹⁰ Department of Human Genetics, Emory University School of Medicine, Atlanta, GA, USA.
¹¹ Departments of Pediatrics.
¹² Genetics PhD Program.
¹³ Department of Clinical Pathology, School of Medicine, University of Mansoura, Mansoura, Egypt.
¹⁴ Department of Population Health and Reproduction, School of Veterinary Medicine, University of California, Davis, CA, USA.
¹⁵ Anatomy and Cell Biology, University of Iowa, Iowa City, IA, USA.
¹⁶ Center for Regenerative Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA.
¹⁷ Harvard Stem Cell Institute, Harvard University, Cambridge, MA, USA.
¹⁸ Center for Statistical Training & Consulting, Michigan State University, East Lansing, MI, USA.
¹⁹ Department of Craniofacial Biology, University of Colorado Denver at Anschutz Medical Campus, Aurora, CO, USA.
²⁰ Center for Craniofacial Molecular Biology, Ostrow School of Dentistry, University of Southern California, Los Angeles, CA, USA.
²¹ Duke Molecular Physiology Institute, Duke University Medical Center, Durham, NC, USA.
²² Pediatrics and Human Development.

PMID: 30689861
PMCID: PMC6494790
DOI: 10.1093/hmg/ddz010

Abstract

Mutations in IRF6, TFAP2A and GRHL3 cause orofacial clefting syndromes in humans. However, Tfap2a and Grhl3 are also required for neurulation in mice. Here, we found that homeostasis of Irf6 is also required for development of the neural tube and associated structures. Over-expression of Irf6 caused exencephaly, a rostral neural tube defect, through suppression of Tfap2a and Grhl3 expression. Conversely, loss of Irf6 function caused a curly tail and coincided with a reduction of Tfap2a and Grhl3 expression in tail tissues. To test whether Irf6 function in neurulation was conserved, we sequenced samples obtained from human cases of spina bifida and anencephaly. We found two likely disease-causing variants in two samples from patients with spina bifida. Overall, these data suggest that the Tfap2a-Irf6-Grhl3 genetic pathway is shared by two embryologically distinct morphogenetic events that previously were considered independent during mammalian development. In addition, these data suggest new candidates to delineate the genetic architecture of neural tube defects and new therapeutic targets to prevent this common birth defect.

Published by Oxford University Press 2019.

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Figures

**Figure 1**
Homeostasis of *Irf6* is required for neurulation. **(A–F′)**, E17.5 embryos from the *Irf6* allelic series, with increasing gene dosage from left to right. Compound heterozygotes for the *Irf6* hypomorphic and null alleles (A) have a completely penetrant kinked tail (N = 18). Embryos that are heterozygous for the *Irf6* hypomorphic allele (B) are not grossly different from wild-type embryos (C). Embryos that are wild type at the *Irf6* locus, but carry the *K14-Irf6* transgene, had one of three phenotypes; 86% (109/126) normal (D), 9% (11/126) with exencephaly (E) and 5% (6/126) with anencephaly, thoraco-abdominoschisis and kinked tail (F, lateral and F′, ventral view of same embryo). (G) Relative levels of *Irf6*, *Tfap2a* and *Grhl3* mRNA from dorsal back skin from embryos represented in (A–D and F) (N = 5, 4, 3, 5 and 5, respectively). Relative level of *Irf6* mRNA is higher in more severely affected embryos that carry the transgene (D versus F), suggesting variable *Irf6* expression from the *K14-Irf6* transgene. Relative level of *Tfap2a* mRNA decreases as *Irf6* mRNA levels increase. Relative level of *Ghrl3* mRNA decreases significantly for each group of embryos except between the embryos that carry the *K14-Irf6* transgene (D versus F). (**H and I)** 10% (7/68) of E17.5 embryos that are singly heterozygous for the *Tfap2a* null allele have exencephaly (H) while 0% (0/69) of embryos that are double heterozygotes for both *Irf6* and *Tfap2a* null alleles have exencephaly (I). (**J and K)** 55% (10/18) of E15.5 embryos that are heterozygous for the *Tfap2a* null allele and carry the *K14-Irf6* transgene have rostral neural tube defects and appear smaller than littermates (J), whereas 9% (1/11) of these embryos that also carry the *Irf6* null allele are less severely affected and had sub-epidermal blebbing (arrowhead; K). Sub-epidermal blebbing was also observed in embryos double heterozygotes for both *Irf6* and *Tfap2a* (arrowhead; I).

**Figure 2**
*Tfap2a* and *Grhl3* interact genetically. (A) Levels of *Grhl3* and *Tfap2a* mRNA decrease in whole tail tissue when *Irf6* expression level is reduced, in contrast to dorsal skin where their levels increase as *Irf6* gene dosage is decreased. (**B–D**) Tails are normal in mice that are singly heterozygous for either *Tfap2a* (B) or *Grhl3* (C), but a curly tail was observed in mice that are double heterozygotes for *Grhl3* and *Tfap2a* null alleles (D). (E) Adult mice that are double heterozygotes for *Grhl3* and *Tfap2a* null alleles have a curly tail and are viable and fertile. (**F–N**), Representative embryos for the indicated genotypes. The three columns represent *Tfap2a^+/+* (*T^+/+*), *Tfap2a^+/−* (*T^+/−*) and *Tfap2a^−/−* (*T^−/−*), respectively. The three rows represent *Grhl3^+/+* (*G^+/+*), *Grhl3^+/−* (*G^+/−*) and *Grhl3^−/−* (*G^−/−*), respectively. Thus, the diagonal shows the embryos that are wild type (F), double heterozygotes (J) and double knockouts (N). Note that embryos with at least three null alleles (K, M and N) appear smaller and paler. Arrows point to neural tube defect (NTD), rostral NTD (red), curly tail (green) and lumbosacral spina bifida (black). Significantly, the double knockout embryos do not have lumbosacral spina bifida (blue arrow). Histogram adjacent to each embryo represents the distribution of phenotypes for each genotype. For example, fraction of embryos with wild-type phenotype (white), caudal NTD (green), rostral NTD (red), both rostral and caudal (yellow). The number of embryos for each genotype (N) is shown below each histogram.

**Figure 3**
IRF6, KRT14 and AP-2a expression during neurulation. (A) Illustration of E8.75 embryo with line indicating plane of section for Fig. 3B and C (A). (B) Immunostaining shows IRF6 (red) and AP-2a (green). (C) Magnified view of neural plate (arrow) and non-neural ectoderm at E8.75 showing IRF6 (red) co-localization with AP-2a (green) at the neural plate border (arrowhead). DAPI marks the nuclei (blue). (D) Illustration of E9.5 embryo with line indicating plane of section for Fig. 3E–L (D). (**E–J**) Immunostaining of wild-type (E, G–I) and mutant littermates (F, J). Compared with wild-type embryos (E), mutant littermates (F) have ectopic IRF6 expression (red) (arrow) and abnormal neural crest cell migration (arrowhead). IRF6 (red) and RHOB (green) are co-expressed in neural crest cells (G) (arrowhead). IRF6 (red) is co-expressed with KRT14 (green) in both the neural ectoderm (Supplementary Material, Fig. S2) and early migrating neural crest cells (H) (arrowhead) and with AP-2a (green) in the early migrating neural crest cells (I and J) (arrowhead). Relative to wild-type embryos (I), mutant littermates (J) have ectopic IRF6 expression (red) and loss of AP-2a staining (green) in the neural crest (arrowhead). In contrast, AP-2a expression in the non-neural ectoderm is unaffected (I versus J) (arrow). (G and H) Histological analysis of wild-type (K) and mutant embryos (L). Wild-type embryos have both rostral (arrow) and caudal (arrowhead) neural tube closure, with intact optic vesicle and facial mesenchyme (K). Mutant embryos had abnormal optic vesicles (op) and disorganized facial mesenchyme (fm) (H). Scale bar: B, E, F = 100 μm. Scale bar: C, G–J = 20 μm. Scale bar: K and L = 200 μm.

**Figure 4**
*Tfpa2a* is necessary for *MCS9.7* activity along the posterior spinal cord. (**A–K′**) *In vivo* activity of the *MCS9.7* enhancer is indicated by staining with X-gal (blue) using mice that carry the *Tg^MCS9.7-LacZ* transgene. (**A and B′**) *MCS9.7* is active at E10 in hindbrain neuro-epithelium. Lateral (A) and dorsal (B) views of a representative whole-mount stained embryo at E10. Coronal section, indicated by the dashed line (A), shows activity in the neuro-epithelium (B′). At E12.5, lateral (C) and dorsal (**D and E**) views of whole-mount stained embryos suggest *MCS9.7* activity along the neural plate border (arrowhead) and dorsal root ganglion (arrow) (N = 3) and confirmed using a transverse section (E′). At E14, dorsal (**F, H, I and K**) and ventral (**G and J**) views show that *MCS9.7* is active along the tail of wild-type embryos (F–H) (N = 4), but has a discontinuous, punctate pattern in *Tfap2a* knockout embryos (I–K) (N = 7). Sectioning reveals expression in the epithelium and the four tendons of the tail in wild-type embryos (H′) at the plane of section (H, arrowhead). In contrast, *Tfap2a* knockout embryos have a dysmorphic tail (K) with loss of MCS9.7 activity in both the dorsal epithelium and mesenchymal tail tissue (K′) at the plane of section (K, arrowhead). Scale bar: H′ and K′ = 200 μm.

**Figure 5**
A missense mutation identified in a patient with spina bifida. Genomic structure for *IRF6***(A)**. Exons 1 and 2 contain the 5′UTR (red). Exons 3 and 4 encode the DNA binding domain (blue). Exons 7 and 8 encode the protein binding domain (green). Exon 9 encodes the predicted auto-inhibitory domain (yellow) and the 3′UTR (red). Predicted protein structure of IRF6 **(B)**. The aspartic acid residue at 427 is highly conserved in vertebrates **(C)**. Based on the predicted protein structure of IRF6 **(B)**, this amino acid is located at the base of the C-terminal helix of IRF6. This ribbon structure is based on the crystal structures of IRF5. Sequencing in individuals with spina bifida identified a rare variant, D427Y **(D)**. *In vivo* analysis of this variant using a zebrafish model showed that D427Y, like R84C, did not rescue our zebrafish model at various concentrations of 25, 50 and 100 picograms of mRNA per embryo. V274I did rescue our zebrafish model. Zebrafish and human IRF6 are provided as baseline. Percent shown is the number of rescued embryos at 24 h post-fertilization, relative to the number of injected embryos. Three biological replicates were performed for reach concentration and variant tested. Errors bars represent standard error of the mean.

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References

1. Ingraham C.R., Kinoshita A., Kondo S., Yang B., Sajan S., Trout K.J., Malik M.I., Dunnwald M., Goudy S.L. and Lovett M. (2006) Abnormal skin, limb and craniofacial morphogenesis in mice deficient for interferon regulatory factor 6 (Irf6). Nat. Genet., 38, 1335–1340. - PMC - PubMed
1. Kondo S., Schutte B.C., Richardson R.J., Bjork B.C., Knight A.S., Watanabe Y., Howard E., de Lima R.L., Daack-Hirsch S., Sander A. et al. (2002) Mutations in IRF6 cause Van der Woude and popliteal pterygium syndromes. Nat. Genet., 32, 285–289. - PMC - PubMed
1. Milunsky J.M., Maher T.A., Zhao G., Roberts A.E., Stalker H.J., Zori R.T., Burch M.N., Clemens M., Mulliken J.B., Smith R. et al. (2008) TFAP2A mutations result in branchio-oculo-facial syndrome. Am. J. Hum. Genet., 82, 1171–1177. - PMC - PubMed
1. Peyrard-Janvid M., Leslie E.J., Kousa Y.A., Smith T.L., Dunnwald M., Magnusson M., Lentz B.A., Unneberg P., Fransson I., Koillinen H.K. et al. (2014) Dominant mutations in GRHL3 cause Van der Woude Syndrome and disrupt oral periderm development. Am. J. Hum. Genet., 94, 23–32. - PMC - PubMed
1. Richardson R.J., Dixon J., Malhotra S., Hardman M.J., Knowles L., Boot-Handford R.P., Shore P., Whitmarsh A. and Dixon M.J. (2006) Irf6 is a key determinant of the keratinocyte proliferation–differentiation switch. Nat. Genet., 38, 1329–1334. - PubMed

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[1] Ingraham C.R., Kinoshita A., Kondo S., Yang B., Sajan S., Trout K.J., Malik M.I., Dunnwald M., Goudy S.L. and Lovett M. (2006) Abnormal skin, limb and craniofacial morphogenesis in mice deficient for interferon regulatory factor 6 (Irf6). Nat. Genet., 38, 1335–1340. - PMC - PubMed

[2] Ingraham C.R., Kinoshita A., Kondo S., Yang B., Sajan S., Trout K.J., Malik M.I., Dunnwald M., Goudy S.L. and Lovett M. (2006) Abnormal skin, limb and craniofacial morphogenesis in mice deficient for interferon regulatory factor 6 (Irf6). Nat. Genet., 38, 1335–1340. - PMC - PubMed

[3] Kondo S., Schutte B.C., Richardson R.J., Bjork B.C., Knight A.S., Watanabe Y., Howard E., de Lima R.L., Daack-Hirsch S., Sander A. et al. (2002) Mutations in IRF6 cause Van der Woude and popliteal pterygium syndromes. Nat. Genet., 32, 285–289. - PMC - PubMed

[4] Kondo S., Schutte B.C., Richardson R.J., Bjork B.C., Knight A.S., Watanabe Y., Howard E., de Lima R.L., Daack-Hirsch S., Sander A. et al. (2002) Mutations in IRF6 cause Van der Woude and popliteal pterygium syndromes. Nat. Genet., 32, 285–289. - PMC - PubMed

[5] Milunsky J.M., Maher T.A., Zhao G., Roberts A.E., Stalker H.J., Zori R.T., Burch M.N., Clemens M., Mulliken J.B., Smith R. et al. (2008) TFAP2A mutations result in branchio-oculo-facial syndrome. Am. J. Hum. Genet., 82, 1171–1177. - PMC - PubMed

[6] Milunsky J.M., Maher T.A., Zhao G., Roberts A.E., Stalker H.J., Zori R.T., Burch M.N., Clemens M., Mulliken J.B., Smith R. et al. (2008) TFAP2A mutations result in branchio-oculo-facial syndrome. Am. J. Hum. Genet., 82, 1171–1177. - PMC - PubMed

[7] Peyrard-Janvid M., Leslie E.J., Kousa Y.A., Smith T.L., Dunnwald M., Magnusson M., Lentz B.A., Unneberg P., Fransson I., Koillinen H.K. et al. (2014) Dominant mutations in GRHL3 cause Van der Woude Syndrome and disrupt oral periderm development. Am. J. Hum. Genet., 94, 23–32. - PMC - PubMed

[8] Peyrard-Janvid M., Leslie E.J., Kousa Y.A., Smith T.L., Dunnwald M., Magnusson M., Lentz B.A., Unneberg P., Fransson I., Koillinen H.K. et al. (2014) Dominant mutations in GRHL3 cause Van der Woude Syndrome and disrupt oral periderm development. Am. J. Hum. Genet., 94, 23–32. - PMC - PubMed

[9] Richardson R.J., Dixon J., Malhotra S., Hardman M.J., Knowles L., Boot-Handford R.P., Shore P., Whitmarsh A. and Dixon M.J. (2006) Irf6 is a key determinant of the keratinocyte proliferation–differentiation switch. Nat. Genet., 38, 1329–1334. - PubMed

[10] Richardson R.J., Dixon J., Malhotra S., Hardman M.J., Knowles L., Boot-Handford R.P., Shore P., Whitmarsh A. and Dixon M.J. (2006) Irf6 is a key determinant of the keratinocyte proliferation–differentiation switch. Nat. Genet., 38, 1329–1334. - PubMed

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The TFAP2A-IRF6-GRHL3 genetic pathway is conserved in neurulation

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The TFAP2A-IRF6-GRHL3 genetic pathway is conserved in neurulation

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