Biology subjects

Moody, S. A.

Publications and source records attributed to Moody, S. A..

5 recordsLinked to original sources

bioRxiv2024

Direct and Indirect Regulation of SIX1+EYA Transcriptional Activity by PA2G4, MCRS1, and SOBP

Branchio-oto-renal (BOR) syndrome is an autosomal dominant condition characterized by variable craniofacial malformations, hearing loss and in some patients, renal dysfunction. Recently, a clinical study showed that patients with mutations causative of BOR also present with craniosynostosis, indicating that this could be an undiagnosed feature of BOR. Approximately half of all patients presenting with BOR have a variant in either SIX1 or its activating co-factor EYA1. The genes underlying BOR in the other 50% of patients remain unknown. To date, most studies on the role of SIX1 in the inner ear have focused on its role in the placode-based development of the sensorineural epithelia (cochlear and vestibular organs), as sensorineural hearing loss has long term effects on human health and development. The role of SIX1 in the neural crest cells (NCCs) of the first pharyngeal (a.k.a., mandibular) arch, which will give rise to the upper and lower jaws as well as the middle ear ossicles which may be affected in patients with BOR, is less well understood. To that end, we examine herein the role of three proteins that have been identified in Xenopus laevis as SIX1 co-factors to determine whether their function as regulators of SIX1+EYA transcriptional activity is conserved in mouse and begin to elucidate their function in the NCCs of the mandibular arch. Our data reveal that SIX1 is co-expressed with PA2G4, MCRS1, and SOBP within the oral domain of the mandibular arch, although each has a very distinct expression pattern. We further demonstrate that MCRS1 and SOBP are bona fide SIX1 co-factors that can repress SIX1+EYA transcriptional activity. PA2G4, on the other hand, does not bind to SIX1 in the mouse but can indirectly enhance the transcriptional activity of the SIX1+EYA transcriptional complex. Further, we show that both MCRS1 and SOBP are also able to translocate EYA to the nucleus via direct (i.e., SOBP) or indirect (i.e., MCRS1) mechanisms. We also show that later, during development of the mandible and incisors that SOBP is co-expressed with SIX1 within these anlagen, confirming the biological relevance of these transcriptional complexes in craniofacial bone formation and within the derivatives of the mandibular NCCs. This study, therefore, highlights the complexity of SIX1+EYA transcriptional regulation suggesting that there are multiple, and potentially redundant, mechanisms in place to ensure precise levels of SIX1+EYA transcriptional activity during craniofacial development.

developmental biology↗

bioRxiv2022

Duplicated zebrafish (Danio rerio) inositol phosphatases inpp5ka and inpp5kb diverged in expression pattern and function

One hurdle in the development of zebrafish models of human disease is the presence of multiple zebrafish orthologs resulting from whole genome duplication in teleosts. Mutations in Inositol polyphosphate 5-phosphatase K (INPP5K) lead to a syndrome characterized by variable presentation of intellectual disability, brain abnormalities, cataracts, muscle disease, and short stature. INPP5K is a phosphatase acting at position 5 of phosphoinositides to control their homeostasis and is involved in insulin signaling, cytoskeletal regulation, and protein trafficking. Previously, our group and others have replicated the human phenotypes in zebrafish knockdown models by targeting both INPP5K orthologs inpp5ka and inpp5kb. Here, we show that inpp5ka is the more closely related orthologue to human INPP5K. While both inpp5ka and inpp5kb mRNA expression levels follow a similar trend in the developing head, eyes, and tail, inpp5ka is much more abundantly expressed in these tissues than inpp5kb. In situ hybridization revealed a similar trend, also showing unique localization of inpp5kb in the pineal gland indicating different transcriptional regulation. We also found that inpp5kb has lost its catalytic activity against its preferred substrate, PtdIns(4,5)P2. Since most human mutations are missense changes disrupting phosphatase activity, we propose that loss of inpp5ka alone can be targeted to recapitulate the human presentation. In addition, we show that the function of inpp5kb has diverged from inpp5ka and may play a novel role in the zebrafish.

developmental biology↗

bioRxiv2021

Generation of a new six1-null line in Xenopus tropicalis for study of development and congenital disease

The vertebrate Six (Sine oculis homeobox) family of homeodomain transcription factors play critical roles in the development of several organs. Six1 plays a central role in cranial placode development, including the precursor tissues of the inner ear, as well as other cranial sensory organs and the kidney. In humans, mutations in SIX1 underlie some cases of branchio-oto-renal syndrome (BOR), which is characterized by moderate to severe hearing loss. We utilized CRISPR/Cas9 technology to establish a six1 mutant line in Xenopus tropicalis that is available to the research community. We demonstrate that at larval stages, the six1-null animals show severe disruptions in gene expression of putative Six1 target genes in the otic vesicle, cranial ganglia, branchial arch and neural tube. At tadpole stages, six1-null animals display dysmorphic Meckels, ceratohyal and otic capsule cartilage morphology. This mutant line will be of value for the study of the development of several organs as well as congenital syndromes that involve these tissues.

developmental biology↗

bioRxiv2021

Mutations in SIX1 associated with Branchio-oto-renal Syndrome (BOR) differentially affect otic expression of putative target genes

Single nucleotide mutations in SIX1 are causative in some individuals diagnosed with branchio-otic/branchio-oto-renal (BOR) syndrome. To test whether these mutations have differential effects on otic gene expression, we engineered four BOR mutations in Xenopus six1 and targeted mutant protein expression to the neural crest and cranial placode precursor cells in wild-type embryos. Changes in the otic expression of putative Six1 targets and/or co-factors were monitored by qRT-PCR and in situ hybridization. We found that each mutant had a different combination of effects. The V17E mutant reduced eya2, tspan13, zbtb16 and pa2g4 otic vesicle expression at a frequency indistinguishable from wild-type Six1, but reduced prdm1 more and spry1 less compared to wild-type Six1. For most of these genes, the R110W, W122R and Y129C mutants were significantly less repressive compared to wild-type Six1. Their individual effects varied according to the level at which they were expressed. The R110W, W122R and Y129C mutants also often expanded prdm1 otic expression. Since previous studies showed that all four mutants are transcriptionally deficient and differ in their ability to interact with co-factors such as Eya1, we propose that altered co-factor interactions at the mutated sites differentially interfere with their ability to drive otic gene expression.

developmental biology↗

bioRxiv2021

Sobp modulates Six1 transcriptional activation and is required during craniofacial development

Branchio-oto-renal syndrome (BOR) is a disorder characterized by hearing loss, craniofacial and/or renal defects. Mutations in the transcription factor Six1 and its cofactor Eya1, both required for otic development, are linked to BOR. We previously identified Sobp as a potential Six1 cofactor and SOBP mutations in mouse and humans cause otic phenotypes; therefore, we asked whether Sobp interacts with Six1 and thereby may contribute to BOR. Co-IP and immunofluorescence experiments demonstrate that Sobp binds to and co-localizes with Six1 in the cell nucleus. Luciferase assays show that Sobp represses Six1+Eya1 transcriptional activation. Experiments in Xenopus embryos that either knockdown or increase expression show that Sobp is required for formation of ectodermal domains at neural plate stages. In addition, altering Sobp levels disrupts otic vesicle development and causes craniofacial cartilage defects. Expression of Xenopus Sobp containing the human mutation disrupts the pre-placodal ectoderm similar to full-length Sobp, but other changes are distinct. These results indicate that Sobp modifies Six1 function, is required for vertebrate craniofacial development, and identifies Sobp as a potential candidate gene for BOR and other deafness syndromes. Summary statementSobp interacts with Six1 in the cell nucleus and represses the Six1+Eya1 transcriptional activation. In Xenopus embryos, Sobp functions during early stages of inner ear development.

developmental biology↗