Biology subjects

Horb, M. E.

Publications and source records attributed to Horb, M. E..

5 recordsLinked to original sources

bioRxiv2024

Adeno-Associated Viral Tools to Trace Neural Development and Connectivity Across Amphibians

The development, evolution, and function of the vertebrate central nervous system (CNS) can be best studied using diverse model organisms. Amphibians, with their unique phylogenetic position at the transition between aquatic and terrestrial lifestyles, are valuable for understanding the origin and evolution of the tetrapod brain and spinal cord. Their metamorphic developmental transitions and unique regenerative abilities also facilitate the discovery of mechanisms for neural circuit remodeling and replacement. The genetic toolkit for amphibians, however, remains limited, with only a few species having sequenced genomes and a small number of transgenic lines available. In mammals, recombinant adeno-associated viral vectors (AAVs) have become a powerful alternative to genome modification for visualizing and perturbing the nervous system. AAVs are DNA viruses that enable neuronal transduction in both developing and adult animals with low toxicity and spatial, temporal, and cell-type specificity. However, AAVs have never been shown to transduce amphibian cells efficiently. To bridge this gap, we established a simple, scalable, and robust strategy to screen AAV serotypes in three distantly-related amphibian species: the frogs Xenopus laevis and Pelophylax bedriagae, and the salamander Pleurodeles waltl, in both developing larval tadpoles and post-metamorphic animals. For each species, we successfully identified at least two AAV serotypes capable of infecting the CNS; however, no pan-amphibian serotype was identified, indicating rapid evolution of AAV tropism. In addition, we developed an AAV-based strategy that targets isochronic cohorts of developing neurons - a critical tool for parsing neural circuit assembly. Finally, to enable visualization and manipulation of neural circuits, we identified AAV variants for retrograde tracing of neuronal projections in adult animals. Our findings expand the toolkit for amphibians to include AAVs, establish a generalizable workflow for AAV screening in non-canonical research organisms, generate testable hypotheses for the evolution of AAV tropism, and lay the foundation for modern cross-species comparisons of vertebrate CNS development, function, and evolution.

neuroscience↗

bioRxiv2023

Photoreceptor disc incisures form as an adaptive mechanism ensuring the completion of disc enclosure

The first steps of vision take place within a stack of tightly packed disc-shaped membranes, or "discs", located in the outer segment compartment of photoreceptor cells. In rod photoreceptors, discs are enclosed inside the outer segment and contain deep indentations in their rims called "incisures". The presence of incisures has been documented in a variety of species, yet their role remains elusive. In this study, we combined traditional electron microscopy with three-dimensional electron tomography to demonstrate that incisures are formed only after discs become completely enclosed. We also observed that, at the earliest stage of their formation, discs are not round as typically depicted but rather are highly irregular in shape and resemble expanding lamellipodia. Using genetically manipulated mice and frogs and measuring outer segment protein abundances by quantitative mass spectrometry, we further found that incisure size is determined by the molar ratio between peripherin-2, a disc rim protein critical for the process of disc enclosure, and rhodopsin, the major structural component of disc membranes. While a high perpherin-2 to rhodopsin ratio causes an increase in incisure size and structural complexity, a low ratio precludes incisure formation. Based on these data, we propose a model whereby normal rods express a modest excess of peripherin-2 over the amount required for complete disc enclosure in order to ensure that this important step of disc formation is accomplished. Once the disc is enclosed, the excess peripherin-2 incorporates into the rim to form an incisure.

cell biology↗

bioRxiv2022

Four novel, immortal cell lines derived from embryos of Xenopus tropicalis

The diploid anuran, Xenopus tropicalis, has emerged as a key research model in cell and developmental biology. To enhance the usefulness of this species, we developed methods for generating immortal cell lines from Nigerian strain (NXR_1018, RRID:SCR_013731) X. tropicalis embryos. We generated 14 cell lines that were propagated for several months. We selected four morphologically distinct lines, XTN-6, XTN-8, XTN-10, and XTN-12 for further characterization. Karyotype analysis revealed that three of the lines, XTN-8, XTN-10, and XTN-12 were primarily diploid. XTN-6 cultures showed a consistent mixed population of diploid cells, cells with chromosome 8 trisomy, and cells containing a tetraploid content of chromosomes. The lines were propagated using conventional culture methods as adherent cultures at 30{degrees}C in a simple, diluted L-15 medium containing fetal bovine serum without use of a high CO2 incubator. Transcriptome analysis indicated that the four lines were distinct lineages. These methods will be useful in the generation of cell lines from normal and mutant strains of X. tropicalis as well as other species of Xenopus.

cell biology↗

bioRxiv2022

Maternal Wnt11b regulates cortical rotation during Xenopus axis formation: analysis of maternal-effect wnt11b mutants

Asymmetric signalling centres in the early embryo are essential for axis formation in vertebrates. These regions, namely the dorsal morula, yolk syncytial layer, and distal hypoblast/anterior visceral endoderm (in amphibians, teleosts and mammals, respectively), require the localised stabilisation of nuclear Beta-catenin (Ctnnb1), implying that localised Wnt/Beta-catenin signalling activity is critical in their establishment. However, it is becoming increasingly apparent that the stabilisation of Beta-catenin in this context may be initiated independently of secreted Wnt growth factor activity. In Xenopus, dorsal Beta-catenin stabilisation is initiated by a requisite microtubule-mediated symmetry-breaking event in the fertilised egg: "cortical rotation". Vegetally-localised wnt11b mRNA has been implicated upstream of Beta-catenin in this context, as has the dorsal enrichment of Wnt ligand-independent activators of Beta-catenin, but the extent that each of these processes contribute to axis formation in this paradigm remains unclear. Here we describe a maternal effect mutation in Xenopus laevis wnt11b.L, generated by CRISPR mutagenesis. We demonstrate a maternal requirement for timely and complete gastrulation morphogenesis and a zygotic requirement for proper left-right asymmetry. We also show that a subset of maternal wnt11b mutants have axis and dorsal gene expression defects, but that Wnt11b likely does not act through the Wnt coreceptor Lrp6 or through Dishevelled, which we additionally show (using exogenous constructs) do not exhibit patterns of activity consistent with roles in early Beta-catenin stabilisation. Instead, we find that microtubule assembly and cortical rotation are reduced in wnt11b mutant eggs, leading to less organised and directed vegetal microtubule arrays. In conclusion, we propose that Wnt11b signals to the cytoskeleton in the egg or early zygote to enable robust cortical rotation, and thus acts in the distribution of putative dorsal determinants rather than as a component or effector of the determinants themselves.

developmental biology↗

bioRxiv2019

Regulation of FXR1 by alternative splicing is required for muscle development and controls liquid-like condensates in muscle cells

Fragile-X mental retardation autosomal homolog-1 (FXR1) is a muscle-enriched RNA-binding protein. FXR1 depletion is perinatally lethal in mice, Xenopus, and zebrafish; however, the mechanisms driving these phenotypes remain unclear. The FXR1 gene undergoes alternative splicing, producing multiple protein isoforms and mis-splicing has been implicated in disease. Furthermore, mutations that cause frameshifts in muscle-specific isoforms result in congenital multi-minicore myopathy. We observed that FXR1 alternative splicing is pronounced in the serine and arginine-rich intrinsically-disordered domain; these domains are known to promote biomolecular condensation. Here, we show that tissue-specific splicing of fxr1 is required for Xenopus development and alters the disordered domain of FXR1. FXR1 isoforms vary in the formation of RNA-dependent biomolecular condensates in cells and in vitro. This work shows that regulation of tissue-specific splicing can influence FXR1 condensates in muscle development and how mis-splicing promotes disease.\n\nHIGHLIGHTSO_LIThe muscle-specific exon 15 impacts FXR1 functions\nC_LIO_LIAlternative splicing of FXR1 is tissue- and developmental stage specific\nC_LIO_LIFXR1 forms RNA-dependent condensates\nC_LIO_LISplicing regulation changes FXR1 condensate properties\nC_LI

cell biology↗