No Two Faces Are Alike

Gene disruptions can cause severe dysmorphologies like cleft palate, but what causes the subtle shifts in facial morphology that make each face unique? Studying mice, Attanasio et al. (1241006) identified over 4000 candidate genetic enhancers around genes driving craniofacial development. To avoid the challenge of recognizing individual mouse faces, optical projection tomography was used to link changes in facial morphology with alterations in the function of specific enhancers.

Structured Abstract


The shape of the face is one of the most distinctive features among humans, and differences in facial morphology have substantial implications in areas such as social interaction, psychology, forensics, and clinical genetics. Craniofacial shape is highly heritable, including the normal spectrum of morphological variation as well as susceptibility to major craniofacial birth defects. In this study, we explored the role of transcriptional enhancers in the development of the craniofacial complex. Our study is based on the rationale that such enhancers, which can be hundreds of kilobases away from their target genes, regulate the spatial patterns, levels, and timing of gene expression in normal development. 


To identify distant-acting enhancers active during craniofacial development, we used chromatin immunoprecipitation on embryonic mouse face tissue followed by sequencing to identify noncoding genome regions bound by the enhancer-associated p300 protein. We used LacZ reporter assays in transgenic mice and optical projection tomography (OPT) to determine three-dimensional expression patterns of a subset of these candidate enhancers. Last, we deleted three of the craniofacial enhancers from the mouse genome to assess their effect on gene expression and craniofacial morphology during development.


We identified more than 4000 candidate enhancer sequences predicted to be active in the developing craniofacial complex. The majority of these sequences are at least partially conserved between humans and mice, and many are located in chromosomal regions associated with normal facial morphology or craniofacial birth defects. Characterization of more than 200 candidate enhancer sequences in transgenic mice revealed a remarkable spatial complexity of in vivo expression patterns. Targeted deletions of three craniofacial enhancers near genes with known roles in craniofacial development resulted in changes of expression of those genes as well as quantitatively subtle but definable alterations of craniofacial shape. 


Our analysis identifies enhancers that fine tune expression of genes during craniofacial development in mice. These results support that variation in the sequence or copy number of craniofacial enhancers may contribute to the spectrum of facial variation we find in human populations. Because many craniofacial enhancers are located in genome regions associated with craniofacial birth defects, such as clefts of the lip and palate, our results also offer a starting point for exploring the contribution of noncoding sequences to these disorders.


The shape of the human face and skull is largely genetically determined. However, the genomic basis of craniofacial morphology is incompletely understood and hypothesized to involve protein-coding genes, as well as gene regulatory sequences. We used a combination of epigenomic profiling, in vivo characterization of candidate enhancer sequences in transgenic mice, and targeted deletion experiments to examine the role of distant-acting enhancers in craniofacial development. We identified complex regulatory landscapes consisting of enhancers that drive spatially complex developmental expression patterns. Analysis of mouse lines in which individual craniofacial enhancers had been deleted revealed significant alterations of craniofacial shape, demonstrating the functional importance of enhancers in defining face and skull morphology. These results demonstrate that enhancers are involved in craniofacial development and suggest that enhancer sequence variation contributes to the diversity of human facial morphology.

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Supplementary Material


Materials and Methods
Supplementary Text
Figs. S1 to S6
Tables S1 to S6
References (6291)
Movies S1 to S20


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Published In

Volume 342 | Issue 6157
25 October 2013

Submission history

Received: 24 May 2013
Accepted: 20 August 2013
Published in print: 25 October 2013


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The authors thank J. Harkes and M. Satyanarayanan for development of the OPT viewer; S. Shen and H. Hochheiser for integration of the OPT viewer and data sets into FaceBase; and J. Murray, M. Marazita, J. Manak, B. Schutte, and all FaceBase members for help in the selection of relevant craniofacial intervals and comments on results. A.V. and L.A.P. were supported by NIDCR FaceBase grant U01DE020060 and by National Human Genome Research Institute grants R01HG003988 and U54HG006997. C.A. was supported by a Swiss National Science Foundation advanced researcher fellowship. A.S.N. was supported by a F32 NIH/National Institute of General Medical Sciences National Research Service Award fellowship GM105202. B.H. was supported by NIH 1R01DE021708, NIH 1R01DE01963, NIH 1U01DE020054, and Natural Sciences and Engineering Research Council of Canada #238992-11 grants. D.R.F. and H.M. were supported by a UK Medical Research Council core program grant. B.R. was supported by the Ludwig Institute for Cancer Research and NIH grants U54HG006997 and R01HG003991. B.H. was supported by NIH 1R01DE01963. Research was conducted at the E. O. Lawrence Berkeley National Laboratory and performed under Department of Energy contract DE-AC02-05CH11231, University of California. ChIP-Seq data are available through GEO (accession no. GSE49413) and In vivo reporter data are available through the Vista Enhancer Browser ( and OPT data, including raw images and interactive 3D viewing option, is available through All enhancer reporter vectors, as well as archived surplus LacZ-stained embryos for selected enhancers, are available from the authors. Craniofacial enhancer knockout lines are available through the Mutant Mouse Regional Resource Centers (Δhs1431, MMRRC 03895; Δhs746, MMRRC 03888; and Δhs586, MMRRCC 03894).



Catia Attanasio
Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
Alex S. Nord
Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
Yiwen Zhu
Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
Matthew J. Blow
U.S. Department of Energy Joint Genome Institute, Walnut Creek, CA 94598, USA.
Zirong Li*
Ludwig Institute for Cancer Research, and Department of Cellular and Molecular Medicine, University of California, San Diego School of Medicine, 9500 Gilman Drive, La Jolla, CA 92093, USA.
Denise K. Liberton
Department of Cell Biology and Anatomy, McCaig Bone and Joint Institute, University of Calgary, Calgary T2N 4N1, Canada.
Harris Morrison
MRC Human Genetics Unit, MRC Institute for Genetic and Molecular Medicine, University of Edinburgh, Edinburgh EH4 2XU, UK.
Ingrid Plajzer-Frick
Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
Amy Holt
Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
Roya Hosseini
Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
Sengthavy Phouanenavong
Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
Jennifer A. Akiyama
Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
Malak Shoukry
Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
Veena Afzal
Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
Edward M. Rubin
Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
U.S. Department of Energy Joint Genome Institute, Walnut Creek, CA 94598, USA.
David R. FitzPatrick
MRC Human Genetics Unit, MRC Institute for Genetic and Molecular Medicine, University of Edinburgh, Edinburgh EH4 2XU, UK.
Royal Hospital for Sick Children, Edinburgh EH9 1LF, UK.
Bing Ren
Ludwig Institute for Cancer Research, and Department of Cellular and Molecular Medicine, University of California, San Diego School of Medicine, 9500 Gilman Drive, La Jolla, CA 92093, USA.
Benedikt Hallgrímsson
Department of Cell Biology and Anatomy, McCaig Bone and Joint Institute, University of Calgary, Calgary T2N 4N1, Canada.
Alberta Children's Hospital Research Institute, University of Calgary, Calgary T2N 4N1, Canada.
Len A. Pennacchio
Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
U.S. Department of Energy Joint Genome Institute, Walnut Creek, CA 94598, USA.
Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
U.S. Department of Energy Joint Genome Institute, Walnut Creek, CA 94598, USA.


†Corresponding author. E-mail: [email protected]
Present address: EMD Millipore, 28820 Single Oak Drive, Temecula, CA 92590, USA.

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