Biology subjects

Martin, B. L.

Publications and source records attributed to Martin, B. L..

11 recordsLinked to original sources

bioRxiv2026

A High-Affinity Nanobody Recognizing mNeonGreen Enables Versatile Biochemical, Cellular, and in vivo Applications.

mNeonGreen (mNG) is among the brightest and most photostable monomeric green fluorescent proteins and is widely used for protein tagging. Here, we present sdAb(mNG), a high-affinity single-domain antibody (sdAb) that enables biochemical capture, imaging, and manipulation of mNG-tagged proteins. A 1.26 [A] crystal structure reveals an extensive interaction surface between mNG and sdAb(mNG), accounting for its high affinity (KD = 0.39 nM) and robust target recognition across diverse experimental conditions. This allows a single sdAb to support applications that typically require multiple specialized tools. We demonstrate the utility of sdAb(mNG) in several example applications including highly specific immunoprecipitation, direct immunofluorescence, and super-resolution imaging. Importantly, sdAb(mNG) retains high-performance target recognition even in intracellular environments. When expressed as an intrabody in living mammalian cells, sdAb(mNG) enables relocalization of mNG-tagged proteins to defined compartments or visualization of synaptic vesicle transport in primary neurons. In zebrafish, fusion of sdAb(mNG) to an F-box degradation domain induces cell-autonomous depletion of an endogenous mNG-tagged transcription factor and produces a clear developmental phenotype. These findings establish sdAb(mNG) as a versatile and robust affinity reagent that converts mNG from a passive fluorescent reporter into a multifunctional handle for imaging, proteomics, and programmable manipulation of endogenous and engineered proteins.

biochemistry↗

bioRxiv2025

BOOPTHAT: An inexpensive and scalable system for spatiotemporal activation of heat-shock transgenes in zebrafish

We present the BOOPTHAT (Batch-Operating Optically Powered Targeted Heater for Activating Transgenes) as a low-cost system for activating heat-shock inducible transgenes with spatiotemporal control in multiple zebrafish embryos at a time. Gaining finer spatiotemporal control over gene expression is critical for unraveling the regulatory networks that coordinate embryogenesis. While the heat-shock inducible gene expression system is a widely used tool for controlling temporal transgene expression, its applicability in spatiotemporal control is limited. By adding a level of spatial control onto this well-established system, we take advantage of the existing infrastructure surrounding the HS induction system and introduce new ways to use the multitude of existing lines. The BOOPTHAT system is built from 3D printed components and inexpensive consumer parts. Independent 3D printed micromanipulators are used to position optical fiber probes. When coupled to a light source, the probes are heated photothermally and are used to perform targeted gene activation in multiple samples at a time. We demonstrate the capabilities of our system and highlight some areas of research that stand to benefit from this frugal and effective system.

developmental biology↗

bioRxiv2025

The ratio of Wnt signaling activity to Sox2 transcription factor levels predicts neuromesodermal fate potential

Neuromesodermal progenitors (NMPs) are a vertebrate cell type that contribute descendants to both the spinal cord and the mesoderm. The undifferentiated bipotential NMP state is maintained when both Wnt signaling is active and Sox2 is present. We used transgenic reporter lines to live-image both Wnt activity and Sox2 levels in NMPs and observed a unique cellular ratio in NMPs compared to NMP-derived mesoderm or neural tissue. We used this unique signature to identify the previously unknown anatomical position of a progenitor population that gives rise to the midline tissues of the floor plate of the spinal cord and the mesodermal notochord. Thus, quantification of the active Wnt signaling to Sox2 ratio can be used to predict and identify cells with neuromesodermal potential. We also developed the auxin inducible degron 2 system for use in zebrafish to test the temporal role that Sox2 plays during midline formation. We found ectopic Sox2 in the presence of Wnt activity holds cells in the undifferentiated floor plate/notochord progenitor state, and that degradation of the ectopic Sox2 is required for cells to adopt a notochord fate.

developmental biology↗

bioRxiv2023

Sclerotome is compartmentalized by parallel Shh and Bmp signaling downstream of CaMKII

The sclerotome in vertebrates comprises an embryonic population of cellular progenitors that give rise to diverse adult tissues including the axial skeleton, ribs, intervertebral discs, connective tissue, and vascular smooth muscle. In the thorax, this cell population arises in the ventromedial region of each of the segmented tissue blocks known as somites. How and when sclerotome adult tissue fates are specified and how the gene signatures that predate those fates are regulated has not been well studied. We have identified a previously unknown role for Ca2+/calmodulin-dependent protein kinase II (CaMKII) in regulating sclerotome patterning in zebrafish. Mechanistically, CaMKII regulates the activity of parallel signaling inputs that pattern sclerotome gene expression. In one downstream arm, CaMKII regulates distribution of the established sclerotome-inductive morphogen sonic hedgehog (Shh), and thus Shh-dependent sclerotome genes. In the second downstream arm, we show a previously unappreciated inductive requirement for Bmp signaling, where CaMKII activates expression of bmp4 and consequently Bmp activity. Bmp activates expression of a second subset of stereotypical sclerotome genes, while simultaneously repressing Shh-dependent markers. Our work demonstrates that CaMKII promotes parallel Bmp and Shh signaling as a mechanism to first promote global sclerotome specification, and that these pathways subsequently regionally activate and refine discrete compartmental genetic programs. Our work establishes how the earliest unique gene signatures that likely drive distinct cell behaviors and adult fates arise within the sclerotome.

developmental biology↗

bioRxiv2023

Breaking the C. elegans invasion/proliferation dichotomy

The acquisition of the post-mitotic state is crucial for the execution of many terminally differentiated cell behaviors during organismal development. However, the mechanisms that maintain the post-mitotic state in this context remain poorly understood. To gain insight into these mechanisms, we used the genetically and visually accessible model of C. elegans anchor cell (AC) invasion into the vulval epithelium. The AC is a terminally differentiated uterine cell that normally exits the cell cycle and enters a post-mitotic state, initiating contact between the uterus and vulva through a cell invasion event. Here, we set out to identify the set of negative cell cycle regulators that maintain the AC in this post-mitotic, invasive state. Our findings revealed a critical role for CKI-1 (p21CIP1/p27KIP1) in redundantly maintaining the post-mitotic state of the AC, as loss of CKI-1 in combination with other negative cell cycle regulators--including CKI-2 (p21CIP1/p27KIP1), LIN-35 (pRb/p107/p130), FZR-1 (Cdh1/Hct1), and LIN-23 ({beta}-TrCP)--resulted in proliferating ACs. Remarkably, time-lapse imaging revealed that these ACs retain their ability to invade. Upon examination of a node in the gene regulatory network controlling AC invasion, we determined that proliferating, invasive ACs do so by maintaining aspects of pro-invasive gene expression. We therefore report that the requirement for a post-mitotic state for invasive cell behavior can be bypassed following direct cell cycle perturbation.

developmental biology↗

bioRxiv2022

A chemically inducible muscle ablation system for zebrafish

An effective method for tissue specific ablation in zebrafish is the nitroreductase/metronidazole system. Expressing bacterial nitroreductase (ntr) in the presence of nitroimidazole compounds causes apoptotic cell death, which can be useful for understanding many biological processes. However, this requires tissue specific expression of the ntr enzyme, and many tissues have yet to be targeted with transgenic lines that express ntr. We generated a transgenic zebrafish line expressing ntr in differentiated skeletal muscle. Treatment of embryos with metronidazole caused muscle specific cell ablation. We demonstrate this line can be used to monitor muscle regeneration in whole embryos and in transplanted transgenic cells. Summary statement

developmental biology↗

bioRxiv2022

Myogenic regulatory factors MyoD and Myf5 are required for dorsal aorta formation and angiogenic sprouting

The vertebrate embryonic midline vasculature forms in close proximity to the developing skeletal muscle, which originates in the somites. Angioblasts migrate from bilateral positions along the ventral edge of the somites until they meet at the midline, where they sort and differentiate into the dorsal aorta and the cardinal vein. This migration occurs at that the same time that myoblasts in the somites are beginning to differentiate into skeletal muscle, a process which requires the activity of the basic helix loop helix (bHLH) transcription factors Myod and Myf5. Here we examined vasculature formation in myod and myf5 mutant zebrafish. In the absence of skeletal myogenesis, angioblasts migrate normally to the midline but form only the cardinal vein and not the dorsal aorta. The phenotype is due to the failure to activate vascular endothelial growth factor ligand vegfaa expression in the somites, which in turn is required in the adjacent angioblasts for dorsal aorta specification. Myod and Myf5 cooperate with Hedgehog signaling to activate and later maintain vegfaa expression in the medial somites, which is required for angiogenic sprouting from the dorsal aorta. Our work reveals that the early embryonic skeletal musculature in teleosts evolved to organize the midline vasculature during development. Summary statementThe myogenic transcription factors MyoD and Myf5 have a novel function in inducing the artery through regulation of Vegf.

developmental biology↗

bioRxiv2021

Somite morphogenesis is required for axial blood vessel formation

Angioblasts that form the major axial blood vessels of the dorsal aorta and cardinal vein migrate towards the embryonic midline from distant lateral positions. Little is known about what controls the precise timing of angioblast migration and their final destination at the midline. Using zebrafish, we found that midline angioblast migration requires neighboring tissue rearrangements generated by somite morphogenesis. The somitic shape changes cause the adjacent notochord to separate from the underlying endoderm, creating a ventral midline cavity that provides a physical space for the angioblasts to migrate into. The anterior to posterior progression of midline angioblast migration is facilitated by retinoic acid induced anterior to posterior somite maturation and the subsequent progressive opening of the ventral midline cavity. Our work demonstrates a critical role for somite morphogenesis in organizing surrounding tissues to facilitate notochord positioning and angioblast migration, which is ultimately responsible for creating a functional cardiovascular system. Summary statementRetinoic acid induced somite morphogenesis generates a midline cavity that accommodates migrating angioblasts, which form the axial blood vessels.

developmental biology↗

bioRxiv2020

Mutations in MYLPF cause a novel segmental amyoplasia that manifests as distal arthrogryposis

We identified ten persons in six consanguineous families with Distal Arthrogryposis (DA) who had congenital contractures, scoliosis, and short stature. Exome sequencing revealed that each affected person was homozygous for one of two different rare variants (c.470G>T, p.(Cys157Phe) or c.469T>C, p.(Cys157Arg)) affecting the same residue of myosin light chain, phosphorylatable, fast skeletal muscle (MYLPF). In a seventh family, a c.487G>A, p.(Gly163Ser) variant in MYLPF arose de novo in a father, who transmitted it to his son. In an eighth family comprised of seven individuals with dominantly-inherited DA, a c.98C>T, p.(Ala33Val) variant segregated in all four persons tested. Variants in MYLPF underlie both dominant and recessively inherited DA. Mylpf protein models suggest that the residues associated with dominant DA interact with myosin whereas the residues altered in families with recessive DA only indirectly impair this interaction. Pathological and histological exam of a foot amputated from an affected child revealed complete absence of skeletal muscle (i.e., segmental amyoplasia). To investigate the mechanism for this finding, we generated an animal model for partial MYLPF impairment by knocking out zebrafish mylpfa. The mylpfa mutant had reduced trunk contractile force and complete pectoral fin paralysis, demonstrating that mylpf impairment most severely affects limb movement. mylpfa mutant muscle weakness was most pronounced in an appendicular muscle and was explained by reduced myosin activity and fiber degeneration. Collectively, our findings demonstrate that partial loss of MYLPF function can lead to congenital contractures, likely as a result of degeneration of skeletal muscle in the distal limb.

genetics↗

bioRxiv2020

Sox2 and canonical Wnt signaling interact to activate a developmental checkpoint coordinating morphogenesis with mesodermal fate acquisition

Animal embryogenesis requires a precise coordination between morphogenesis and cell fate specification. It is unclear if there are mechanisms that prevent uncoupling of these processes to ensure robust development. During mesoderm induction, mesodermal fate acquisition is tightly coordinated with the morphogenetic process of epithelial to mesenchymal transition (EMT). In zebrafish, cells exist transiently in a partial EMT state during mesoderm induction. Here we show that cells expressing the neural inducing transcription factor Sox2 are held in the partial EMT state, stopping them from completing the EMT and joining the mesodermal territory. This is critical for preventing ectopic neural tissue from forming. The mechanism involves specific interactions between Sox2 and the mesoderm inducing canonical Wnt signaling pathway. When Wnt signaling is inhibited in Sox2 expressing cells trapped in the partial EMT, cells are now able to exit into the mesodermal territory, but form an ectopic spinal cord instead of mesoderm. Our work identifies a critical developmental checkpoint that ensures that morphogenetic movements establishing the mesodermal germ layer are accompanied by robust mesodermal cell fate acquisition.

developmental biology↗

bioRxiv2018

Observing the Cell in Its Native State: Imaging Subcellular Dynamics in Multicellular Organisms

True physiological imaging of subcellular dynamics requires studying cells within their parent organisms, where all the environmental cues that drive gene expression, and hence the phenotypes we actually observe, are present. A complete understanding also requires volumetric imaging of the cell and its surroundings at high spatiotemporal resolution without inducing undue stress on either. We combined lattice light sheet microscopy with two-channel adaptive optics to achieve, across large multicellular volumes, noninvasive aberration-free imaging of subcellular processes, including endocytosis, organelle remodeling during mitosis, and the migration of axons, immune cells, and metastatic cancer cells in vivo. The technology reveals the phenotypic diversity within cells across different organisms and developmental stages, and may offer insights into how cells harness their intrinsic variability to adapt to different physiological environments.\n\nOne Sentence SummaryCombining lattice light sheet microscopy with adaptive optics enables high speed, high resolution in vivo 3D imaging of dynamic processes inside cells under physiological conditions within their parent organisms.

cell biology↗