Biology subjects

Thierer, J. H.

Publications and source records attributed to Thierer, J. H..

6 recordsLinked to original sources

bioRxiv2026

Fluorescently-labeled split-QF hemidrivers: simplifying and enhancing methods enabling intersectional targeting of discrete cell types

Single cell transcriptomics data predict greater cell type diversity than previously appreciated, defined by combinatorial gene expression "codes". These codes can enable intersectional targeting strategies for selectively labeling and manipulating predicted cell types, thereby facilitating functional tests to resolve cell-specific roles and refine cell-type definitions. One such approach cleaves driver components of binary expression systems (e.g., Gal4/UAS and QF/QUAS) into two co-dependent halves, an Activation Domain (AD) and a DNA Binding Domain (DBD). The transcriptional activity of these "split-drivers" (aka, hemidrivers) is only reconstituted in cells that co-express them. While widely used in invertebrates, these systems have yet to be systematically deployed in vertebrates. Here, we developed a series of fluorescently labeled split-QF and QF2 hemidrivers via direct reporter fusions or the 2A viral peptide co-expression system. Fluorescent labeling serves to 1) simplify hemidriver transgenic line creation and maintenance, 2) allow AD and DBD intersects to be visualized directly, and 3) enable robust quality control assessments of on-target efficacy and off-target activity. These resources can enhance transgenic targeting specificity, enabling functional dissections of cell types revealed by single cell transcriptomics. SUMMARYWe engineered fluorescently labeled split-QF and -QF2 hemidrivers in zebrafish to simplify intersectional targeting tool generation and enable robust quality control assessments of split-driver function.

genetics↗

bioRxiv2026

Mesenchymal-derived neural progenitors underlie local insulin production and neuronal transdifferentiation during retina regeneration

In humans, retinal-neuron death, optic-nerve injuries, and associated neurodegenerative diseases, such as glaucoma and age-related macular degeneration, often lead to permanent vision loss. While the capacity for regeneration is low in the human nervous system, including the retina, some non-mammalian vertebrate species, including zebrafish, are capable of endogenous neuronal regeneration after injury. Unlike mammals, zebrafish do not form a scar that inhibits axonal and neuronal regeneration after injury. Rather, they harbor neural progenitor and stem-cell populations allowing regeneration of entire parts of the nervous system and restoration of tissue integrity and function. In the zebrafish retina, cycling neural progenitor cells of the ciliary marginal zone and quiescent resident neural stem cells (the latter of which are also called Muller glial cells) participate in neuronal regeneration following different types of injury. In this study, we report the identification of a novel, additional cellular source participating in neuronal regeneration of neurons in the zebrafish retina after genetic ablation of retinal ganglion cells. Before injury, these progenitor cells express molecular markers of neural-crest-cell and/or fibroblast identity, such as sox10, pdgfrb, and eya2, while after neuronal ablation they also express proneural factors including the ascl1a and olig2 genes. Combining genetic ablation of neurons with photoconversion or Cre/Lox-dependent genetic lineage tracing of sox10-expressing cells, we demonstrated that these cells can differentiate into post-mitotic retinal neurons in the ganglion cell layer (GCL) in the absence of cell proliferation. We also showed, surprisingly, that this progenitor population locally produces insulin mRNA, and that insulin signaling is involved in the accumulation of mesenchymal-derived neural progenitors in the GCL and in their subsequent transdifferentiation into RGCs. This work reveals an unexpected and novel cellular mechanism of transdifferentiation, dependent on a neural-crest-derived mesenchymal cell population, participating in neuronal regeneration in the zebrafish retina. The discovery of this plastic cell population could potentially lead to new strategies to promote the formation of new neurons in the mammalian retina.

developmental biology↗

bioRxiv2024

A whole-animal phenotypic drug screen identifies suppressors of atherogenic lipoproteins.

Lipoproteins are essential for lipid transport in all bilaterians. A single Apolipoprotein B (ApoB) molecule is the inseparable structural scaffold of each ApoB-containing lipoprotein (B-lps), which are responsible for transporting lipids to peripheral tissues. The cellular mechanisms that regulate ApoB and B-lp production, secretion, transport, and degradation remain to be fully defined. In humans, elevated levels of vascular B-lps play a causative role in cardiovascular disease. Previously, we have detailed that human B-lp biology is remarkably conserved in the zebrafish using an in vivo chemiluminescent reporter of ApoB (LipoGlo) that does not disrupt ApoB function. Thus, the LipoGlo model is an ideal system for identifying novel mechanisms of ApoB modulation and, due to the ability of zebrafish to generate many progeny, is particularly amenable to large-scale phenotypic drug screening. Here, we report a screen of roughly 3000 compounds that identified 49 unique ApoB-lowering hits. Nineteen hits passed orthogonal screening criteria and seven were subjected to extensive phenotyping. A licorice root component, enoxolone, significantly lowered B-lps only in animals that express a functional allele of the nuclear hormone receptor Hepatocyte Nuclear Factor 4 (HNF4). Consistent with this result, inhibitors of HNF4 also reduce B-lp levels. These data demonstrate that mechanism(s) of action can be rapidly determined from a whole animal zebrafish phenotypic screen. Given the well documented role of HNF4 in human B-lp biology, these data validate the LipoGlo screening platform for identifying small molecule modulators of B-lps that play a critical role in a leading cause of worldwide mortality.

physiology↗

bioRxiv2022

Pla2g12b is Essential for Expansion of Nascent Lipoprotein Particles

SUMMARYTriglyceride-rich lipoproteins (TRLs) are micelle-like particles that enable efficient transport of lipids throughout the bloodstream, but also promote atherosclerotic cardiovascular disease. Despite this central relevance to cardiovascular disease, very little is known about how lipids are loaded onto nascent TRLs prior to secretion. Here we show that Pla2g12b, a gene with no previously described function, concentrates components of the TRL biogenesis machinery along the ER membrane to ensure efficient delivery of lipids to nascent TRLs. We find that the lipid-poor TRLs secreted in PLA2G12B-/- mice and zebrafish support surprisingly normal growth and physiology while conferring profound resistance to atherosclerosis, and demonstrate that these same processes are conserved in human cells. Together these findings shed new light on the poorly understood process of TRL expansion, ascribe function to the previously uncharacterized gene Pla2g12b, and reveal a promising new strategy to remodel serum lipoproteins to prevent cardiovascular disease.

cell biology↗

bioRxiv2019

A missense mutation dissociates triglyceride and phospholipid transfer activities in zebrafish and human microsomal triglyceride transfer protein

Microsomal triglyceride transfer protein (MTP) transfers triglycerides and phospholipids and is essential for the assembly of Apolipoprotein B (ApoB)-containing lipoproteins in the endoplasmic reticulum. We have discovered a zebrafish mutant (mttpc655) expressing a C-terminal missense mutation (G863V) in Mttp, one of the two subunits of MTP, that is defective at transferring triglycerides, but retains phospholipid transfer activity. Mutagenesis of the conserved glycine in the human MTTP protein (G865V) also eliminates triglyceride but not phospholipid transfer activity. The G863V mutation reduces the production and size of ApoB-containing lipoproteins in zebrafish embryos and results in the accumulation of cytoplasmic lipid droplets in the yolk syncytial layer. However, mttpc655 mutants exhibit only mild intestinal lipid malabsorption and normal growth as adults. In contrast, zebrafish mutants bearing the previously identified mttpstl mutation (L475P) are deficient in transferring both triglycerides and phospholipids and exhibit gross intestinal lipid accumulation and defective growth. Thus, the G863V point mutation provides the first evidence that the triglyceride and phospholipid transfer functions of a vertebrate MTP protein can be separated, arguing that selective inhibition of the triglyceride transfer activity of MTP may be a feasible therapeutic approach for dyslipidemia.

physiology↗

bioRxiv2019

LipoGlo: A sensitive and specific reporter of atherogenic lipoproteins

Apolipoprotein-B (APOB) is the structural component of atherogenic lipoproteins, lipid-rich particles that drive atherosclerosis by accumulating in the vascular wall. As atherosclerotic cardiovascular disease is the leading cause of death worldwide, there is an urgent need to develop new strategies to prevent lipoproteins from causing vascular damage. Here we report the LipoGlo system, which uses a luciferase enzyme (NanoLuc) fused to ApoB to monitor several key determinants of lipoprotein atherogenicity including particle abundance, size, and localization. Using LipoGlo, we are able to comprehensively characterize the lipoprotein profile of individual larval zebrafish and collect the first images of atherogenic lipoprotein localization in an intact organism. We discover multiple unexpected extravascular lipoprotein localization patterns, as well as identify pla2g12b as a potent regulator of lipoprotein size. ApoB-fusion proteins thus represent a uniquely sensitive and specific approach to study atherogenic lipoproteins and their genetic and small molecule modifiers.

genetics↗