A. Personal Statement

Highlighted publications:

Lewis A.E., Kuwahara A., Franzosi J., Bush, J.O.* (2022) Tracheal separation is driven by NKX2-1-mediated repression of Efnb2 and regulation of endodermal cell sorting. Cell Reports, 38(11):110510

Kindberg A.A., Srivastava, V., Muncie, J.M., Weaver V.M., Gartner, Z.J. and Bush, J.O.* (2021). EPH/EPHRIN regulates cellular organization by actomyosin contractility effects on cell contacts Journal of Cell Biology 220 (6): e202005216 PMCID: PMC8025214

Kuwahara, A., Lewis, A., Coombes, C., Leung, F.S., Percharde M., Bush J.O.* (2020) Delineating the early transcriptional specification of the mammalian trachea and esophagus. eLife, 9:e55526 PMCID: PMC7282815

Niethamer, T. K., Teng, T., Franco, M., Du, Y. X., Percival, C. J., Bush, J.O.* (2020). Aberrant cell segregation in the craniofacial primordium and the emergence of facial dysmorphology in craniofrontonasal syndrome. PLoS Genet. 16, e1008300.

Highlighted projects:
R35 DE031926-01 (PI: Bush) 4/1/2022-3/31/2030
NIH/NIDCR
Signaling control and cellular basis of craniofacial morphogenesis and congenital disease

R01 DE023337 (PI: Bush) 7/9/2013-3/31/2022
NIH/NIDCR
Mechanisms of Eph/Ephrin signaling in craniofacial morphogenesis and craniofrontonasal syndrome

R01DE028753 (PI: Selleri) 4/1/2019-3/31/2024
NIH/NIDCR
Phenotype-driven approach to understanding the function of craniofacial regulators using IMPC-generated mouse strains

UG3DE028872 (PI: Klein) 7/1/2019-6/30/2024
NIH/NIDCR
“Enamel atlas: systems-level amelogenesis tools at multiple scales”

B. Positions, Scientific Appointments and Honors
Positions and Employment:
2019-present Vice Chair, Department of Cell and Tissue Biology
2021-present Professor, Department of Cell and Tissue Biology and Program in Craniofacial Biology, University of California at San Francisco
2017-2021 Associate Professor, Department of Cell and Tissue Biology and Program in Craniofacial
Biology, University of California at San Francisco
2011-2017 Assistant Professor, Department of Cell and Tissue Biology and Program in
Craniofacial Biology, University of California at San Francisco
2005-2011 Postdoctoral research at FHCRC/MSSM
Advisor: Dr. Philippe Soriano
1999-2005 Graduate and postdoctoral research at University of Rochester
Advisor: Dr. Rulang Jiang

Awards and Honors:
2021 Marylou Buyse Distinguished Scientist in Craniofacial Research award from the Society for Craniofacial Genetics and Developmental Biology
2019 F1000 Faculty member for Developmental Biology
2019 David W. Smith workshop on malformations and morphogenesis keynote
2014 American Association of Anatomists Young Faculty Travel Award
2010-present F1000/F1000Prime has featured and highly ranked five of our publications
2010 NIH pathway to independence award from NIH/NIDCR K99/R00 (DE020855)
2006-2008 Ruth L Kirschstein NRSA Individual Fellowship from NIH/NIDCR F32 (DE17506)
2005 Basil Bibby award from the AADR
2004 Michael G. Buonocore award from the AADR

Other Experience and Professional Memberships
2021-2025 Member NIH Skeletal Biology Development and Disease (SBDD)
2021 Cold Spring Harbor Labs Mouse Engineering Course lecturer and workshop leader
2021 American Association of Anatomists Program Committee
2020 Ad hoc reviewer NIH/NIDCR SEP ZDE1 Endogenous Regeneration of Dental, Oral and Craniofacial Tissue
2020 Ad hoc reviewer NIH CSR ZRG1 Fellowships: Cell Biology, Developmental Biology and Bioengineering
2020 Ad hoc reviewer Czech Science Foundation
2019 Ad hoc reviewer for NIH Skeletal Biology Development and Disease (SBDD)
2017 Ad hoc reviewer for NIH on Microphysiological Systems (MPS) for Disease Modeling and Efficacy Testing (UG3/UH3) “Tissue Chips and Disease Modeling”
2017-present International Association for Dental Research
2017 Guest Editor, Developmental Biology, special issue on signaling in development
2014 Ad hoc reviewer for NIH on SEP ZRG1 MOSS-D (02), “Bone, Cartilage and Tendon”
2013, 2014 Invited peer reviewer for UK MRC
2012 Ad hoc reviewer for NIH on Council ZES1, “Environmental influences on stem cells in development, health and disease”
2012 Organizer, session chair, Society of Craniofacial Genetics Annual Meeting, San Francisco, CA
2012-present Member, Society for Craniofacial Genetics and Development
2010-present Member, American Association of Anatomists
2000-present Member, Society for Developmental Biology
Ad hoc Reviewer for Development, Developmental Biology, Developmental Dynamics, Journal of Dental Research, Genesis, Immunobiology, Journal of Cell Biology, Science, PLOS Genetics, Immunobiology, PLOS One, Developmental Cell, PLOS Biology, Human Molecular Genetics, JOVE

C. Contributions to Science
1. Identified Eph/ephrin cellular mechanisms in morphogenesis
We have made major contributions to understanding EPH/EPHRIN signaling mechanisms in development in multiple contexts including the craniofacial, neural and neural crest systems. The EPHs compose the largest family of receptor tyrosine kinases in the vertebrate genome constituting one quarter of the total number. Though extensively studied in the nervous system, the signaling mechanisms utilized by this family in other contexts are complex and modestly understood, particularly in vivo. We have learned that a large number of these molecules exhibit distinct expression patterns in the developing craniofacial region. In a novel “conditional-rescue” approach we discovered that EPHRIN-B2 is required in the vascular endothelium for normal NCC development. EPH/EPHRIN signaling is widely known to regulate cellular organization, but the signaling mechanisms by which it does so are unclear. By studying a series of targeted and signaling mutations in EPHRIN-B1 and its receptors EPHB2 and EPHB3 in mice, we determined that EPH/EPHRIN cell segregation involves unidirectional forward signaling from EPHRIN-B1 and relies on kinase activity of the receptors. By live cell imaging we also observe that this unidirectional signaling results in changes in cortical actomyosin accumulation and leading us to interrogate the biophysical basis for EPH/EPHRIN cell segregation. We recently discovered that EPH/EPHRIN signaling modulates the strength of cell-cell contacts by regulating cell interfacial tension through increased heterotypic cortical actomyosin contractility. The minimization of heterotypic interfacial tension also informs how EPH/EPHRIN signaling regulates tissue shape changes relevant to Contributions number 2 and 4. Current efforts in this area focus on how EPH/EPHRIN signaling regulates mesenchymal cell polarity and cell position, forming the basis for part of “Focus one” of this proposal.

Agrawal, P., Wang, M., Kim, S., Lewis, A.E., Bush, J.O.* (2014) The embryonic expression of EphA receptor genes in mice supports their candidacy for involvement in cleft lip and palate. Developmental Dynamics, 243 (11): 1470-6. PMCID: PMC4404412
Lewis, A.E., Hwa, J., Wang, R., Soriano P., Bush, J.O.* (2015) Neural crest defects in ephrin-B2 mutant mice are non-autonomous and originate from defects in the vasculature. Developmental Biology, 406(2): 186-95. PMCID: PMC4639416
O’Neill, A.O., Kindberg, A.A., Niethamer, T.K. Larson, A.R., Ho, H.H., Greenberg, M.E., Bush, J.O.* (2016) Unidirectional Eph/ephrin signaling creates a cortical actomyosin differential to drive cell segregation. Journal of Cell Biology, 215 (2): 217 PMCID: PMC5984648
Kindberg A.A., Srivastava, V., Muncie, J.M., Weaver V.M., Gartner, Z.J. and Bush, J.O.* (2021). EPH/EPHRIN regulates cellular organization by actomyosin contractility effects on cell contacts Journal of Cell Biology 220 (6): e202005216 PMCID: PMC8025214

2. Delineated Eph/ephrin signaling mechanisms underlying craniofrontonasal syndrome
We have focused on the role of EPH/EPHRIN signaling in craniofrontonasal syndrome (CFNS), an X-linked condition caused by mutations in EFNB1 characterized by hypertelorism, craniosynostosis, cleft lip and palate, agenesis of the corpus callosum, and particularly increased severity in female patients. By generating a series of signaling mutations in three different gene targeted mouse lines, we were able to determine the relevant modes of signaling for different EPHRIN-B1 phenotypes. We found that reverse signaling by a PDZ-dependent mechanism is critical for axon guidance, whereas it is dispensible for skeletal and craniofacial development. This work showed that different aspects of craniofrontonasal syndrome are caused by loss of function of distinct molecular EPHRIN-B1 signaling functions. Further, by integrating mouse genetics, phosphoproteomic and transcriptomic approaches we have investigated downstream signaling pathways utilized by EPHRIN-B1 in craniofacial development and disease. In this context, we were the first to show that EPH receptor expression is regulated by endocytosis in vivo, and that EPHRIN-B1 controls cell proliferation by the Ras/MAPK signaling pathway. We have also generated an hiPSC model for CFNS, which to our knowledge is the first hiPSC model of a congenital craniofacial syndrome, and used to it ask key questions about the cellular etiology underlying this perplexing disease. Recently, we coupled geometric morphometric techniques with temporal and spatial manipulation of EPHRIN-B1 signaling to elucidate how Efnb1 mutations result in stereotyped dysmorphology in CFNS. Current efforts in this area focus on delineating the proximal signal transduction mechanisms employed by EPH/EPHRIN-B1 signaling in craniofacial morphogenesis using CRISPR/CAS9 based methods.

Bush, J. O. and Soriano, P. (2010). Ephrin-B1 forward signaling regulates craniofacial morphogenesis by controlling cell proliferation across Eph-ephrin boundaries. Genes Dev. 24, 2048-60. PMCID: PMC2939368
Niethamer, T.K., Larson, A.R., O’Neill A.K., Bershteyn, M., Hsiao, E.C., Klein, O.D., Pomerantz, J.H., Bush J.O.* (2017) EPHRIN-B1 mosaicism drives cell segregation in craniofrontonasal syndrome hiPSC-derived neuroepithelial cells. Stem Cell Reports, 8(3): 529-537. PMCID: PMC5355632
Niethamer, T. K. and Bush, J. O.* (2019). Getting direction(s): The Eph/ephrin signaling system in cell positioning. Dev. Biol. 447, 42–57.
Niethamer, T. K., Teng, T., Franco, M., Du, Y. X., Percival, C. J., Bush, J. O. (2020). Aberrant cell segregation in the craniofacial primordium and the emergence of facial dysmorphology in craniofrontonasal syndrome. PLoS Genet. 16, e1008300.

3. Developed live imaging approaches to discover cellular mechanisms of craniofacial tissue fusions
Though tissue fusion is a critical final step of lip and palate development, we currently lack cellular resolution understanding of how these tissue fusion events occur, and how they are controlled. We have been examining the cellular and molecular mechanisms by which tissue fusion occurs by combining live imaging of mouse morphogenesis with mouse genetic and physical perturbations. We developed the first successful application of confocal live imaging technology to secondary palate development, and it has allowed us to make new discoveries on the cellular mechanisms at play. Our results indicate that tissue fusion proceeds by a progression of dynamic cell behaviors beginning with convergence of two independent epithelia and concomitant to orthogonal displacement of the resultant shared epithelium. Further, we have found that epithelial cell extrusion, in which cells are squeezed out of an epithelium, is a novel contributor to the removal of the midline epithelial seam (MES). A nearly completed manuscript presents a new live imaging approach to understanding secondary palate fusion, and demonstrates a unique and novel form of collective epithelial migration that is crucial for this process. Most recently, we have also established live imaging approaches for the study of upper lip/primary palate fusion, which we are using to study roles for actomyosin contractility and cell adhesion in this process.

Bush J.O., Jiang R. (2012) Palatogenesis: morphogenetic and molecular mechanisms of secondary palate development. Development. 139(2):231-43. PMCID: PMC3243091
Kim, S., Lewis A.E., Singh V., Ma, X., Adelstein, R., Bush, J.O.,* (2015) Convergence and extrusion are required for normal fusion of the mammalian secondary palate. PLOS Biology, 13(4) PMCID: 4388528
Kim S., Prochazka, J., Bush J.O.*, (2017) Live imaging of Mouse Secondary Palate Fusion. JoVE. July 2017 (125) PMCID: In process

4. Improved mouse genetics tools for the study of craniofacial and neural crest development and disease
In the course of our studies, we discovered that the Wnt1-Cre mouse line exhibits developmental phenotypes attributable to elevated and ectopic activation of Wnt signaling. This is a standard and widely used reagent in the fields of craniofacial development and neural crest stem cell biology and our careful analysis of this reagent has already had broad-reaching effects causing labs to re-examine previously published work. To provide a reagent that is devoid of these complications, we generated a Wnt1-Cre2 transgenic mouse line that exhibits the same pattern of activity as Wnt1-Cre but does not cause ectopic activation of Wnt signaling or developmental phenotypes. Over the past five years, I have also served as the faculty advisor for the UCSF mouse inventory database, which facilitates the sharing of genetically modified mouse lines. Most recently, my lab has successfully adopted the iGONAD electroporation method for rapid generation of new mouse lines which we are employing to generate new mouse models for the study of congenital disease.

Lewis, A., Vasudevan, H., O’Neill, A., Soriano, P., Bush, J.O.* (2013) The widely used Wnt1-Cre transgene causes developmental phenotypes by ectopic activation of Wnt signaling. Developmental Biology, 379(2):229-34. PMCID: PMC3804302
Wall, E., Scoles, J., Joo, A., Klein, O., Quinonez, C., Bush, J. O., Martin, G. R. and Laird, D. J. (2020). The UCSF Mouse Inventory Database Application, an Open-Source Web App for Sharing Mutant Mice within a Research Community. G3 (Bethesda).

5. Understanding how cell fate and morphogenesis are coupled in the developing foregut
Based initially on phenotypes that we discovered in an Efnb2 mutant mouse line that we generated, we have now established a robust research focus on understanding the specification and morphogenesis of the trachea and esophagus. Classical developmental biology experiments hypothesize that two transcription factors, NKX2.1 and SOX2 act as master regulators of these fates, but this remains untested at the transcriptome level and their targets were unknown. we combine state-of-the-art transcriptomic experiments in embryonic tissues with functional mouse genetic experiments to expose the fundamental process of fate specification of the trachea and esophagus. We performed single cell RNA sequencing in mice to establish a transcriptome-wide understanding of the early steps of trachea and esophagus development and used these data, combined with RNA-sequencing of mutants and embryonic ChIP-seq to interrogate the transcriptome-wide function of NKX2.1 in tracheal and esophageal development. The results of these experiments provide a new understanding of how the trachea and esophagus are initially specified at the genome-wide level. Our studies also reveal that NKX2.1 directly represses Efnb2 to regulate the site of tracheoesophageal separation. These discoveries may be impactful to the study of tracheal, lung, and esophageal developmental biology and related structural anomalies in humans, as well as for the fields of embryonic stem cell biology and for lung and esophageal cancer for which these are keystone transcriptional pathways.

Kuwahara, A., Lewis, A., Coombes, C., Leung, F.S., Percharde M., Bush J.O.* (2020) Delineating the early transcriptional specification of the mammalian trachea and esophagus. eLife, 9:e55526 PMCID: PMC7282815
Lewis A.E., Kuwahara A., Franzosi J., Bush, J.O.* (2022) Tracheal separation is driven by NKX2-1-mediated repression of Efnb2 and regulation of endodermal cell sorting. Cell Reports, 38(11):110510