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Sydney thesis 20250505 V43.70 MB
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Dissertation
Functional analysis of human orofacial clefting genes in Xenopus laevis
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Spring 2025
DOI: 10.25820/etd.007862
Abstract
Orofacial clefting (OFC) is one of the most common congenital anomalies and is accompanied by medical, economic, and social burdens on the affected individuals and their families. A significant proportion of OFC cases have a genetic basis, but the majority of genetic variation underlying orofacial clefting remains unknown. Our lab previously conducted a large copy-number variation study of OFC patients aimed at identifying previously unknown OFC loci. Three of the candidate genes identified in this study were ARHGEF38, COBLL1, and RIC1, all members of the Rho/Rab GTPase signaling pathway. Other members of this pathway have been implicated in OFC, suggesting that these candidates are likely to be important in proper facial development. To test this hypothesis, we knocked down these genes in zebrafish (Danio rerio) and frogs (Xenopus laevis) and assessed how their facial tissue developed. In this work, we show that CRISPR-Cas9 and morpholino knockdown of arhgef38, cobll1, and ric1 in zebrafish results in defects in cranial cartilage development. We also show that arhgef38, cobll1, and ric1 are expressed in the developing head in frogs, and that knockdown of these genes also leads to reduced or absent cartilage development. Together, this data supports the validation of ARHGEF38, COBLL1, and RIC1 as human OFC genes, expanding our knowledge of the genetic regulation of mouth development.
Additionally, our lab previously found that heterozygous deletion of ISM1 contributes to human OFC, an idea supported by loss-of-function studies in Xenopus embryos. ISM1 is expressed in tissues important for facial development, including in cranial neural crest cells (CNCC), branchial arches, and the midbrain-hindbrain boundary. ISM1 is in the same synexpression group as FGF8 and is thought to interact with the integrin signaling pathway, which has also been implicated in craniofacial and palate development. However, the extent that ISM1 modulates this or other pathways is not known. Here, we show that ISM1 regulates the development of cranial neural crest cells (CNCCs), a key cell population that undergoes coordinated migration and differentiation to properly pattern the face and mouth. We show that morpholino knockdown of ism1 in Xenopus embryos results in defects in craniofacial cartilage, which is derived from CNCCs, and in craniofacial muscle, which develops in close association with CNCCs. We also show that ism1 is required for CNCC migration, but not for the initial specification of neural crest. Knockdown of ism1 leads to persistence of snai2 expression in CNCCs beyond the stage at which it is typically downregulated, suggesting that ism1 function is key for the transition from a pre- to post-migratory state. This is also supported by a pilot single- cell RNA sequencing experiment we conducted in wildtype heads, showing that ism1 is enriched in a separate neural crest population than snai2 is enriched. Finally, we utilized co-immunoprecipitation and co-knockdown functional studies to assess whether ism1’s role in craniofacial development was mediated by integrinβ1. We saw no synergistic effect of the co-knockdown of ism1 and integrinβ1 and did not detect any direct interaction in our co- immunoprecipitation, suggesting that ism1 is targeting a different receptor, possibly another integrin subunit.
In conclusion, this thesis reports functional analysis of human OFC genes identified in our lab’s copy-number variation study. We show that members of the Rho/Rab GTPase signaling pathway have conserved functions in the regulation of human, zebrafish, and frog facial development. We also identify an essential role of ism1 signaling in neural crest cell development, likely by regulation of the transition to a migratory fate. This new data expands our understanding of genetic loci associated with OFC and identifies a new function of ism1 signaling in the regulation of neural crest development.
Details
- Title: Subtitle
- Functional analysis of human orofacial clefting genes in Xenopus laevis
- Creators
- Sydney Ellen Arlis
- Contributors
- John R Manak (Advisor)Douglas W Houston (Committee Member)Bryan Phillips (Committee Member)Daniel Summers (Committee Member)Eric Van Otterloo (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Integrated Biology
- Date degree season
- Spring 2025
- DOI
- 10.25820/etd.007862
- Publisher
- University of Iowa
- Number of pages
- xvi, 120 pages
- Copyright
- Copyright 2025 Sydney Ellen Arlis
- Language
- English
- Date submitted
- 04/25/2025
- Description illustrations
- illustrations, tables, graphs
- Description bibliographic
- Includes bibliographical references (pages 85-107).
- Public Abstract (ETD)
- Cleft lip and/or palate is one of the most common birth disorders, affecting 1 in every 1000 babies, and is accompanied by medical, economic, and social/emotional burdens on patients and their families. For many of the individuals affected by this disorder, we do not know the underlying cause, emphasizing the importance of identifying new genes that may be regulating the development of the face. Our lab previously collected DNA samples from patients with cleft lip and/or palate and identified candidate genes that may be associated with developing a cleft. We then employed knockdown strategies to reduce expression of these candidate genes in zebrafish and frog model organisms to investigate how loss of these genes affects formation of the mouth. We first show that reducing expression of three genes, ARHGEF38, COBLL1, and RIC1, disrupts facial cartilage development, validating their role in craniofacial patterning. We next show evidence that another candidate gene, ISTHMIN1, is important in the development of neural crest cells, which make up most of the tissue in the face. We show that loss of isthmin1 disrupts the migration of these neural crest cells, resulting in disruptions to facial cartilage and mouth structure. Taken together, the data generated in this thesis identifies ARHGEF38, COBLL1, RIC1, and ISTHMIN1 as important genes for the development of the mouth and targets for screening to detect the potential development of cleft lip and/or palate.
- Academic Unit
- Biology; Craniofacial Anomalies Research Center
- Record Identifier
- 9984830924602771
Metrics
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