Biology subjects

Mullins, M. C.

Publications and source records attributed to Mullins, M. C..

12 recordsLinked to original sources

bioRxiv2025

Acvr2b receptors transduce all BMP signaling in the zebrafish gastrula and restrict Fibrodysplasia Ossificans Progressiva ACVR1-R206H signaling in a dose-dependent manner

BMP signaling drives dorsoventral (DV) axial patterning in vertebrates and invertebrates, with BMP dimers recruiting tetrameric receptor complexes to phosphorylate SMADs that activate target gene expression. In zebrafish DV patterning, Bmp2/7 heterodimers exclusively signal, assembling a receptor complex of two distinct type I receptors, Bmpr1 and Acvr1l, that canonically bind Bmp2 and Bmp7 ligands, respectively. Here, we tested if the two distinct classes of BMP type II receptors, Bmpr2 and Acvr2, also act in the gastrula signaling complex. We mutated all four acvr2a and acvr2b genes in the zebrafish and found maternal-zygotic depletion of Acvr2b receptors causes severe embryonic dorsalization, while bmpr2 genes are not expressed in the gastrula, indicating Acvr2b is the primary type II receptor transducing the BMP signal. Further, while phosphorylated Smad5 (pSmad5) was reduced throughout the early gastrula, unexpectedly, pSmad5 was nearly abrogated at the embryonic margin, where Nodal signaling is active in specifying the mesendoderm. We found that inhibiting Nodal signaling by depleting its co-receptor, Oep, in MZ-Acvr2b deficient embryos restored marginal pSmad5 to non-marginal levels, suggesting that Nodal competes with BMP for Acvr2a receptors at the margin. Further, we find that while Bmpr2 is not expressed in the gastrula, it can signal in the absence of Acvr2b, indicating that, unlike the type I receptor classes, the type II receptor classes do not uniquely function in the Bmp2/7 signaling complex. We further demonstrate that the ACVR1-R206H Fibrodysplasia Ossificans Progressiva human disease-causing mutant receptor, ACVR1-R206H, displays an increased sensitivity to Acvr2b dosage compared to wild-type Acvr1l, indicating that Acvr2b receptor availability restricts multiple modes of signaling in the gastrula.

developmental biology↗

bioRxiv2025

Oocyte polarity is established independently of the Balbiani body

In most animals, oocyte polarity establishes the embryonic body plan by asymmetrically localizing axis-determining transcripts. These transcripts first localize in Xenopus and zebrafish oocytes to the Balbiani body (Bb), a large membrane-less organelle conserved from insects to humans. The Bb is transient, disassembling and anchoring at one pole the axis-determining transcripts that establish the vegetal pole of the oocyte. Aggregation of the Bb depends on the Bucky ball (Buc) protein, an intrinsically disordered protein with a prion-like self-aggregation domain. In zebrafish buc null mutants, the Bb fails to form and oocytes lack polarity. Here, we established buc hypomorphic mutants that fail to form the Bb, but remarkably Buc protein and vegetal mRNAs localized normally at the vegetal cortex of the oocyte. Thus, these buc hypomorphic mutants displayed normal oocyte polarity, demonstrating that the Bb is not required to establish oocyte polarity. We found that both a reduced Buc protein level and truncation of the N-terminal 10 amino acids contribute to Bb failure in the hypomorphic mutants.

developmental biology↗

bioRxiv2025

Zebrafish armc9 mutant reveals a link between ARM-Type fold domain loss, inflammation, and spinal curvature

The vertebral column (VC) defines the central axis of vertebrates. Among its malformations, adolescent idiopathic scoliosis (AIS) is the most common, characterized by lateral spine curvature without congenital abnormalities. While its etiology remains elusive, genetic studies in zebrafish have linked AIS to skeletogenesis, cilia structure, cerebrospinal fluid flow, and inflammation. In this study, we characterized the zebrafish armc9 mutant, exhibiting AIS-like curvature without vertebral malformations, subcommissural organ, or Reissner fiber structure disruptions. We also detected an inflammatory response at the curvature origin. Structural bioinformatics and molecular docking analyses suggest that the mutant Armc9 protein partially lacks the functional ARM-type fold domain, which is crucial for its interaction with Togaram1, revealing that both proteins are relevant for ciliary signaling and spine curvature. Phylogenetic analyses indicate that the ARMC gene family encompasses 12 members, highlighting that the Armc9 gene has deep evolutionary roots. Our results demonstrate that the zebrafish armc9 mutant accurately represents the AIS phenotype, suggesting that the loss of Armc9s C-terminal ARM-type fold domain could affect the ciliary integrity and contribute to spine curvature by altering the skeletogenic environment. Taken together, this study proposes that the zebrafish armc9 mutant offers a unique opportunity to understand the etiology of AIS and provides new insights into the molecular interaction networks of Armc9 in ciliary signaling.

developmental biology↗

bioRxiv2025

Modeling Epithelial Morphogenesis and Cell Rearrangement during Zebrafish Epiboly: Tissue Deformation, Cell-Cell Coupling, and the Mechanical Response to Stress

Morphogenesis in early development involves complex and extreme deformations in response to intra- and intercellular forces. Zebrafish epiboly, the spreading of the blastoderm to cover and engulf the large yolk cell, is a key early event that sets the stage for the establishment of the body plan, but the way the forces driving expansion are generated and mediated is poorly understood. The enveloping layer (EVL), the thin squamous outer epithelium of the blastoderm, plays a central role. Forces generated in the yolk cell are transmitted through tight junctions to the marginal EVL cells, and then propagate through the rest of the EVL. To understand mechanisms of force generation and transduction during epiboly, we first need a mechanical model of the EVL capable of responding to such forces and undergoing the drastic deformation of epiboly. The expanding EVL more than doubles its surface area and experiences significant shear as it deforms from a thin cap at one pole to become a complete sphere, necessarily requiring extensive internal rearrangement. We constructed an agent-based model of the EVL and its response to exogenous forces using the center-based simulation framework, Tissue Forge. Our model captures the large viscoelastic deformation of the EVL by cell rearrangement, and incorporates algorithmic strategies to accommodate these dynamic changes while maintaining tissue cohesion. Features observed in living embryos, such as the straightening of the initially ragged leading edge, also emerge in the model. We identified two key components required for realistic epiboly in the model: first, a mechanism to enable tissue remodeling by cell rearrangement without tearing the tissue, and second, a negative feedback on the forces driving EVL expansion, to regulate and synchronize the advancement of the EVL margin. We discuss the implications of these findings for the behavior of living EVL and the mechanisms that drive epiboly.

developmental biology↗

bioRxiv2024

Identification of Fasnall as a therapeutically effective Complex I inhibitor

Proliferating cancer cells actively utilize anabolic processes for biomass production, including de novo biosynthesis of amino acids, nucleotides, and fatty acids. The key enzyme of the fatty acid biosynthesis pathway, fatty acid synthase (FASN), is widely recognized as a promising therapeutic target in cancer and other health conditions1,2. Here, we establish a metabolic signature of FASN inhibition using a panel of pharmacological inhibitors (GSK2194069, TVB-2640, TVB-3166, C75, cerulenin, and Fasnall). We find that the activity of commonly used FASN inhibitors is inconsistent with the metabolic signature of FASN inhibition (accumulation of malonate, succinate, malonyl coenzyme A, succinyl coenzyme A, and other metabolic perturbations). Moreover, we show that one of these putative FASN inhibitors, Fasnall, is a respiratory Complex I inhibitor that mimics FASN inhibition through NADH accumulation and consequent depletion of the tricarboxylic acid cycle metabolites. We demonstrate that Fasnall impairs tumor growth in several oxidative phosphorylation-dependent cancer models, including combination therapy-resistant melanoma patient-derived xenografts. Fasnall administration does not reproduce neurological side effects in mice reported for other Complex I inhibitors3,4. Our results have significant implications for understanding the FASN role in human health and disease and provide evidence of therapeutic potential for Complex I inhibitors with fast systemic clearance. Our findings also highlight the continuing need for validation of small molecule inhibitors to distinguish high-quality chemical probes and to expand the understanding of their application.

cancer biology↗

bioRxiv2024

EpicTope: narrating protein sequence features to identify non-disruptive epitope tagging sites

Epitope tagging is a valuable technique enabling the in vivo identification, tracking, and purification of proteins. We developed a tool, EpicTope, to facilitate this method by identifying amino acid positions most suitable for epitope insertion. Our method uses a scoring function that considers protein sequence secondary and tertiary structural features, solvent accessibility, and disordered binding regions to determine locations least disruptive to the proteins function. We validated our approach on the zebrafish Smad5 and Hdac1 proteins. We show that multiple predicted internally tagged Smad5 proteins rescue zebrafish smad5 mutant embryos, while the N- and C-terminal tagged variants do not, as predicted. Similarly, we found that optimally-predicted internal and C-terminal Hdac1 tags rescued hdac1 mutant embryos, while a less-optimal N-terminal tag did not. We further show that these functionally tagged Smad5 and Hdac1 proteins are accessible to antibodies in wholemount zebrafish embryo immunohistochemistry, by western blot, and by immunoprecipitation from embryo extracts. Our work demonstrates that EpicTope is an accessible and effective tool for designing epitope tag insertion sites.

developmental biology↗

bioRxiv2023

Maternal regulation of the vertebrate egg-to-embryo transition

Egg activation and early embryonic development are complex and highly regulated processes that involve a series of coordinated cellular and molecular events after fertilization. While significant progress has been made in unraveling the mechanisms underlying these processes, there remains a need to comprehensively understand the precise molecular pathways and regulatory factors involved. We characterized four recessive maternal-effect mutants identified from a zebrafish forward genetic screen that function during the egg-to-embryo transition. We found that these genes encompass distinct aspects of egg activation, including cortical granule biology, cytoplasmic segregation, and the suppression of microtubule organizing center (MTOC) assembly and ectopic aster-like microtubule formation. These genes are essential to the development of the early embryo and the establishment of the basic body plan. Notably, we discovered a novel gene that we named krang, which is highly conserved across metazoans. Maternal Krang was found to be associated with the function of cortical granules during egg activation. This collection of mutants represents valuable tools to understand the genetic architecture underlying phenotypic traits shaping the egg-to-embryo transition. Furthermore, these results highlight the evolutionary conservation of maternal functions in diverse species. By deepening our understanding of these findings, we will improve our knowledge of reproductive traits and potentially develop new diagnostic tools to address human reproductive disorders. Author SummaryDuring egg activation, the egg undergoes a series of biochemical and cellular modifications that activate its dormant metabolism, preparing it to initiate embryonic development. Egg activation initiates the completion of meiosis, and the newly formed zygote undergoes a series of additional changes, including the cell cycle establishment. However, our knowledge of the molecular mechanisms controlling these processes remain limited. We identified four recessive maternal-effect mutants in zebrafish that exhibit a range of developmental alterations during egg activation and early embryogenesis. We found that these maternally acting mutant genes are associated with defects in cortical granule exocytosis, yolk-cytoplasm segregation, microtubule nucleation and dynamics that are critical for the egg-to-embryo transition. Our results suggest that the proper regulation of these processes is essential for successful egg development and embryogenesis. We characterized the Krang factor, which regulates aspects of egg activation likely by modulating the secretory pathway. Further studies on this gene may provide new insights into the molecular mechanisms underlying oocyte development and egg quality acquisition. Overall, our collection of maternal-effect mutants sheds light on the proper regulation of key molecular and cellular events for successful egg development, with important implications for reproductive medicine and assisted reproductive technologies.

developmental biology↗

bioRxiv2023

Maternal regulation of the vertebrate oocyte-to-egg transition

Maternally-loaded factors in the egg accumulate during oogenesis and are essential for the oocyte and egg to acquire developmental competence and ensure the production of viable embryos. The oocyte-to-egg transition consists of the regulation of multiple molecular processes both cytoplasmic and nuclear acting in the late oocyte during a process called oocyte maturation. However, the molecular nature and functional importance of factors acting at this stage remain poorly understood. Here, we present a collection of 5 recessive maternal-effect mutants identified in a zebrafish forward genetic screen that reveal unique molecular insights into the mechanisms controlling the vertebrate oviparous oocyte-to-egg transition. We identified critical cytoplasmic regulators of yolk globule formation and maturation that are essential for egg development and embryogenesis. Specifically, the maternal-effect genes, over easy, poached, p33bjta, and black caviar control yolk globule sizing and/or protein cleavage during oogenesis, likely through endolysosomal organization independent of nuclear oocyte maturation. Furthermore, we cloned one of the mutant genes, identifying a subunit of the Adaptor Protein complex 5, which regulates intracellular trafficking, and yolk vesicle formation. Together, these mutant genes represent novel genetic entry points to decipher the molecular mechanisms functioning in the oocyte-to-egg transition, fertility, and human disease. Additionally, our genetic screen provides valuable functional tools for exploring the evolutionary fates of maternal factors and their contribution to developmental strategies for reproductive success in metazoans. Author SummaryThe oocyte-to-egg transition consists of the coordinated regulation of multiple molecular processes acting in the late oocyte. This transcriptionally silent period requires the precisely timed function of maternally-supplied gene products during oogenesis. However, knowledge of their molecular nature and in vivo function remains incomplete. The mutants reported here provide access to maternal factors regulating the processes that prepare an oocyte for reproductive competence and embryogenesis. We have identified essential regulators of yolk granule formation and protein processing. Specifically, we found that the highly conserved maternal Ap5m1 protein regulates yolk granule maturation, which generate essential nutrients and immunity for growth and development in oviparous animals. The mutants presented here represent attractive genetic models to investigate the molecular and cell biological mechanisms that control the oocyte-to-egg transition, as well as reveal a collection of genetic factors indispensable for reproduction and survival. Importantly, knowledge of their genetic underpinnings and biological importance in reproduction will also pave the way to investigate the evolution of maternal genes during vertebrate development.

developmental biology↗

bioRxiv2022

Reduced GS domain serine/threonine requirements of Fibrodysplasia Ossificans Progressiva mutant type I BMP receptor ACVR1 in the zebrafish

Fibrodysplasia ossificans progressiva (FOP) is a rare human genetic condition characterized by altered skeletal development and extra-skeletal bone formation. All cases of FOP are caused by mutations in the type I BMP receptor gene ACVR1 that result in over-activation of the BMP signaling pathway. Activation of the wild-type ACVR1 kinase requires assembly of a tetrameric type I and II BMP receptor complex followed by phosphorylation of the ACVR1 GS domain by type II BMP receptors. Previous studies showed that the FOP mutant ACVR1-R206H requires type II BMP receptors and presumptive GS domain phosphorylation for over-active signaling. Structural modeling of the FOP-ACVR1 mutant kinase domain supports that FOP mutations alter the conformation of the GS domain, but it is unclear how this leads to overactive signaling. Here we show using a developing zebrafish embryo BMP signaling assay that the FOP mutant receptors ACVR1-R206H and -G328R have reduced requirements for GS domain phosphorylation sites to signal compared to wild-type ACVR1. Further, ligandindependent and ligand-dependent signaling through the FOP ACVR1 receptors have distinct GS domain phosphorylation site requirements. Moreover, ACVR1-G328 showed increased GS domain serine/threonine requirements for ligand-independent signaling compared to ACVR1-R206H, whereas it exhibited reduced serine/threonine requirements for ligand-dependent signaling. Remarkably, while ACVR1-R206H does not require the type I BMP receptor partner, Bmpr1, to signal, a ligand-dependent GS domain mutant of ACVR1-R206H could signal independently of Bmpr1 only when Bmp7 ligand was overexpressed. Of note, unlike human ACVR1-R206H, the zebrafish paralog Acvr1l-R203H does not show increased signaling activity. However, in domain-swapping studies the human kinase domain, but not the human GS domain, was sufficient to confer overactive signaling to the Acvr1l-R203H receptor. Together these results reflect the importance of GS domain activation and kinase domain functions in regulating ACVR1 signaling and identify mechanisms of reduced regulatory constraints conferred by FOP mutations.

molecular biology↗

bioRxiv2021

Bmper is required for morphogenesis of the anterior and posterior semicircular canal ducts in the developing zebrafish inner ear

BMP signalling is known to have a conserved function in development of the semicircular canal system of the vertebrate inner ear, but its regulation, target genes and effects on cell behaviour during otic morphogenesis are not fully understood. We have characterised the effects of mutations in the zebrafish gene bmper, which codes for a regulator of BMP signalling with both pro- and anti-BMP roles in different developmental contexts. The inner ears of bmper mutant embryos develop with truncations of the anterior and posterior semicircular canal ducts. To image the developing ear in live embryos, we have exploited a new transgenic line, Tg(smad6b:EGFP), which exhibits strong GFP expression in the otic epithelium. Morphometric analysis indicates defects in the bmper mutant ear from early stages of semicircular canal formation, correlating with a specific reduction in BMP signalling activity and specific loss of dlx5a expression in dorsal otic epithelium. Subsequent changes to cell shape occur at the truncation site and the dorsolateral septum. The bmper mutations that we describe are adult viable; truncation of the anterior and posterior semicircular canal ducts persists into adulthood. Our results argue against a major role for Bmper in specification of the pre-placodal region, induction of the otic placode, or development of the neural crest, processes in which Bmper function has previously been implicated. Instead, we conclude that a key requirement for Bmper function in the zebrafish is to promote BMP signalling during patterning and morphogenesis of the semicircular canal system.

developmental biology↗

bioRxiv2019

Evaluation of BMP-mediated patterning in zebrafish embryos using a growing finite difference embryo model

Bone Morphogenetic Proteins (BMPs) play an important role in dorsal-ventral (DV) patterning of the early zebrafish embryo. BMP signaling is regulated by a network of extracellular and intracellular factors that impact the range and signaling of BMP ligands. Recent advances in understanding the mechanism of pattern formation support a source-sink mechanism, however it is not clear how the source-sink mechanism shapes patterns in 3D, nor how sensitive the pattern is to biophysical rates and boundary conditions along both the anteroposterior (AP) and DV axes of the embryo. We propose a new three-dimensional growing Partial Differential Equation (PDE)-based model to simulate the BMP patterning process during the blastula stage. This model provides a starting point to elucidate how different mechanisms and components work together in 3D to create and maintain the BMP gradient in the embryo. We also show how the 3D model fits the BMP signaling gradient data at multiple time points along both axes. Furthermore, sensitivity analysis of the model suggests that the spatiotemporal patterns of Chordin and BMP ligand gene expression are dominant drivers of shape in 3D and more work is needed to quantify the spatiotemporal profiles of gene and protein expression to further refine the models.

systems biology↗

bioRxiv2018

The Hippo pathway effector Taz is required for cell fate specification and fertilization in zebrafish

In the last decade, Hippo signaling has emerged as a critical pathway integrating extrinsic and intrinsic mechanical cues to regulate cell proliferation and survival, tissue morphology and organ size in vivo. Despite its essential role in organogenesis, surprisingly much less is known about how it connects biomechanical signals to control of cell fate and cell size during development. Here we unravel a novel and unexpected role of the Hippo pathway effector Taz (wwtr1) in the control of cell size and cell fate specification. In teleosts, fertilization occurs through a specific structure at the animal pole, called the micropyle. This opening in the chorion is formed during oogenesis by a specialized somatic follicle cell, the micropylar cell (MC). The MC has a peculiar shape and is much larger than its neighboring follicle cells but the mechanisms underlying its specification and cell shape acquisition are not known. Here we show that Taz is essential for the specification of the MC and subsequent micropyle formation in zebrafish. We identify Taz as the first bona fide MC marker and show that Taz is specifically and strongly enriched in the MC precursor before the cell can be identified morphologically. Altogether, our genetic data and molecular characterization of the MC lead us to propose that Taz is a key regulator of the MC fate activated by physical cues emanating from the oocyte to initiate the MC morphogenetic program. We describe here for the first time the mechanism underlying the specification of the MC fate.

developmental biology↗