Biology subjects

Lai, S.-L.

Publications and source records attributed to Lai, S.-L..

11 recordsLinked to original sources

bioRxiv2026

IFN-γ-Dependent Macrophage Reprogramming Coordinates Inflammatory Resolution and Matrix Remodeling in Heart Regeneration

Heart regeneration requires coordinated immune activation, timely inflammatory resolution, and dynamic extracellular matrix (ECM) remodeling in addition to cardiomyocyte (CM) proliferation. However, the cytokine signals that instruct immune cell functions during cardiac repair remain incompletely understood. Here, we identify interferon-gamma (IFN-{gamma}) as a critical regulator of macrophage plasticity in zebrafish heart regeneration. IFN-{gamma} signaling components are dynamically activated following cardiac injury, with early induction of ifng1 and temporally coordinated receptor expression. Genetic ablation of ifng1 impairs myocardial regeneration, resulting in reduced CM proliferation and persistent fibrotic scarring. Temporal transcriptional profiling reveals sustained inflammatory signatures, impaired efferocytosis, and abolished reparative programs, accompanied by aberrant immune cell dynamics and retention of injury-derived debris in mutant hearts. Transcriptomic analysis of cardiac macrophages further reveals that IFN-{gamma} deficiency disrupts the transition from an inflammatory state to a reparative, ECM-remodeling phenotype, leading to reduced collagen denaturation and diminished CM protrusion at the injury border zone. Inducible- and macrophage-specific blockade of IFN-{gamma} signaling phenocopies defects in global knockout, establishing a cell-autonomous requirement for IFN-{gamma} in coordinating regenerative immune function. Collectively, our findings define an IFN-{gamma}-dependent macrophage reprogramming axis that couples inflammatory resolution to ECM remodeling in heart regeneration, elucidating how cytokine signaling actively instructs tissue repair. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/712551v1_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@8cb384org.highwire.dtl.DTLVardef@15dd8d6org.highwire.dtl.DTLVardef@46dcc4org.highwire.dtl.DTLVardef@47e2ac_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2025

The Fd4 transcription factor translates transient spatial cues in progenitors into long-term lineage identity

Neural diversity is required for the brain to generate complex behaviors. During development, neural progenitors are exposed to different combination of transient spatial cues for their identity specification. This identity is then interpreted by their progeny to activate terminal selector genes to become lineage-specific neurons. After spatial cues fade, it remains unclear how progenitors maintain their unique identity so that their progeny express the accurate, lineage-specific terminal selector genes. Using single cell RNA sequencing in Drosophila, we identified a Forkhead domain transcription factor, Fd4, that is exclusively expressed in a single neural progenitor (neuroblast) and its new-born progeny. This neuroblast (NB), named NB7-1, forms at the intersection of the transient spatial cues Vnd (columnar expression) and En (row expression). We show that Fd4 expression overlaps spatial factor expression and terminal selector gene expression, thereby making Fd4 an excellent candidate for bridging transient spatial factors to lineage-specific terminal selector genes. We show that Fd4 is required for expression of terminal selector genes that maintain neuronal identity. Conversely, Fd4 misexpression generates ectopic NB7-1 progeny at the expense of Fd4-negative progenitor lineages. We conclude that Fd4 is continuously expressed in the NB7-1 and its new-born neuronal progeny where it activates terminal selector genes to produce lineage-specific neurons. We propose that Fd4 is a pioneering member of a class of "lineage identity genes" that translate transient spatial cues into a long-term lineage identity.

developmental biology↗

bioRxiv2025

Vnd and En are expressed in orthogonal stripes and act in a brief competence window to combinatorially specify NB7-1 and its early lineage

Understanding how neuronal diversity is generated is a major goal of neuroscience. Here we characterize the first step in generating neuronal diversity in the Drosophila embryo: spatial transcription factors (STFs) expressed in orthogonal rows and columns of neural progenitors. These factors give spatial identity to neural progenitors (neuroblasts, NBs), and are highly conserved in mammals. Here we investigate the roles of Engrailed (En+; posterior row) and Vnd+ (medial column) in specifying the well-characterized progenitor: neuroblast 7-1 (NB7-1). We show that NB7-1 is located at the intersection of Vnd and En, and we identify NB7-1 using a newly characterized gene, fd4, that we show is specifically expressed in NB7-1 and its progeny, giving us a specific assay for NB7-1 identity. We show that En and Vnd are both required for Fd4 expression, and that Vnd and En co-expression is sufficient to induce ectopic Fd4 expression in other NBs and their lineages. Finally, we show that NBs gradually lose competence to respond to En or Vnd. We conclude that En and Vnd are STFs that act combinatorially to specify the identity of an individual progenitor, NB7-1.

developmental biology↗

bioRxiv2024

Border-zone cardiomyocytes and macrophages contribute to remodeling of the extracellular matrix to promote cardiomyocyte invasion during zebrafish cardiac regeneration

Despite numerous advances in our understanding of zebrafish cardiac regeneration, an aspect that remains less studied is how regenerating cardiomyocytes invade, and eventually replace, the collagen-containing fibrotic tissue following injury. Here, we provide an in-depth analysis of the process of cardiomyocyte invasion using live-imaging and histological approaches. We observed close interactions between protruding cardiomyocytes and macrophages at the wound border zone, and macrophage-deficient irf8 mutant zebrafish exhibited defects in extracellular matrix (ECM) remodeling and cardiomyocyte protrusion into the injured area. Using a resident macrophage ablation model, we show that defects in ECM remodeling at the border zone and subsequent cardiomyocyte protrusion can be partly attributed to a population of resident macrophages. Single-cell RNA-sequencing analysis of cells at the wound border revealed a population of cardiomyocytes and macrophages with fibroblast-like gene expression signatures, including the expression of genes encoding ECM structural proteins and ECM-remodeling proteins. The expression of mmp14b, which encodes a membrane-anchored matrix metalloproteinase, was restricted to cells in the border zone, including cardiomyocytes, macrophages, fibroblasts, and endocardial/endothelial cells. Genetic deletion of mmp14b led to a decrease in 1) macrophage recruitment to the border zone, 2) collagen degradation at the border zone, and 3) subsequent cardiomyocyte invasion. Furthermore, cardiomyocyte-specific overexpression of mmp14b was sufficient to enhance cardiomyocyte invasion into the injured tissue and along the apical surface of the wound. Altogether, our data shed important insights into the process of cardiomyocyte invasion of the collagen-containing injured tissue during cardiac regeneration. They further suggest that cardiomyocytes and resident macrophages contribute to ECM remodeling at the border zone to promote cardiomyocyte replenishment of the fibrotic injured tissue.

cell biology↗

bioRxiv2023

Transcriptional complexity in the insect central complex: single nuclei RNA sequencing of adult brain neurons derived from type 2 neuroblasts

In both invertebrates such as Drosophila and vertebrates such as mouse or human, the brain contains the most diverse population of cell types of any tissue. It is generally accepted that transcriptional diversity is an early step in generating neuronal and glial diversity, followed by the establishment of a unique gene expression profile that determines morphology, connectivity, and function. In Drosophila, there are two types of neural stem cells, called Type 1 (T1) and Type 2 (T2) neuroblasts. In contrast to T1 neuroblasts, T2 neuroblasts generate intermediate neural progenitors (INPs) that expand the number and diversity of cell types. The diversity of T2-derived neurons contributes a large portion of the central complex (CX), a conserved brain region that plays a role in sensorimotor integration. Recent work has revealed much of the connectome of the CX, but how this connectome is assembled remains unclear. Mapping the transcriptional diversity of neurons derived from T2 neuroblasts is a necessary step in linking transcriptional profile to the assembly of the adult brain. Here we perform single nuclei RNA sequencing of T2 neuroblast-derived adult neurons and glia. We identify clusters containing all known classes of glia, clusters that are male/female enriched, and 161 neuron-specific clusters. We map neurotransmitter and neuropeptide expression and identify unique transcription factor combinatorial codes for each cluster (presumptive neuron subtype). This is a necessary step that directs functional studies to determine whether each transcription factor combinatorial code specifies a distinct neuron type within the CX. We map several columnar neuron subtypes to distinct clusters and identify two neuronal classes (NPF+ and AstA+) that both map to two closely related clusters. Our data support the hypothesis that each transcriptional cluster represents one or a few closely related neuron subtypes.

neuroscience↗

bioRxiv2022

Comparative single-cell profiling reveals distinct cardiac resident macrophages essential for zebrafish heart regeneration

Zebrafish exhibit a robust ability to regenerate their hearts following injury, and the immune system plays a key role in this process. We previously showed that delaying macrophage recruitment by clodronate liposome (CL) treatment compromises neutrophil resolution and heart regeneration, even when the infiltrating macrophage number was restored within the first-week post injury (Lai et al., 2017). Here, we examined the molecular mechanisms underlying the cardiac repair of regenerative PBS-control hearts vs. non-regenerative CL-treated hearts. Bulk transcriptomic analyses revealed that CL-treated hearts exhibited disrupted inflammatory resolution and energy metabolism during cardiac repair. Temporal single-cell profiling of inflammatory cells in regenerative vs. non-regenerative conditions further identified heterogenous macrophages and neutrophils with distinct infiltration dynamics, gene expression, and cellular crosstalk. Among them, two residential macrophage subpopulations were enriched in regenerative hearts and barely recovered in non-regenerative hearts. Early CL treatment at 8 days or even 1 month before cryoinjury led to the depletion of resident macrophages without affecting the circulating macrophage recruitment to the injured area. Strikingly, these resident macrophage-deficient zebrafish still exhibited compromised neovascularization and scar resolution. Our results characterized the inflammatory cells of the zebrafish injured hearts and identified key resident macrophage subpopulations prerequisite for successful heart regeneration.

developmental biology↗

bioRxiv2022

Transcriptional profiling from whole embryos to single neuroblast lineages in Drosophila

Embryonic development results in the production of distinct tissue types, and different cell types within each tissue. A major goal of developmental biology is to uncover the "parts list" of cell types that comprise each organ. Here we perform single cell RNA sequencing (scRNA-seq) of the Drosophila embryo to identify the genes that characterize different cell and tissue types during development. We assay three different timepoints, revealing a coordinated change in gene expression within each tissue. Interestingly, we find that the elav and mhc genes, whose protein products are widely used as markers for neurons and muscles, respectively, show broad pan-embryonic expression, indicating the importance of post-transcriptional regulation. We next focus on the central nervous system (CNS), where we identify genes characterizing each stage of neuronal differentiation: from neural progenitors, called neuroblasts, to their immediate progeny ganglion mother cells (GMCs), followed by new-born neurons, young neurons, and the most mature neurons. Finally, we ask whether the clonal progeny of a single neuroblast (NB7-1) share a similar transcriptional identity. Surprisingly, we find that clonal identity does not lead to transcriptional clustering, showing that neurons within a lineage are diverse, and that neurons with a similar transcriptional profile (e.g. motor neurons, glia) are distributed among multiple neuroblast lineages. Although each lineage consists of diverse progeny, we were able to identify a previously uncharacterized gene, Fer3, as an excellent marker for the NB7-1 lineage. Within the NB7-1 lineage, transcriptional clusters are identifiable in neuroblasts and neurons, and each cluster is composed of current temporal transcription factor (e.g. Hunchback, Kruppel, Pdm, and Castor), novel temporal factors, and/or targets of the temporal transcription factors. In conclusion, we have characterized the embryonic transcriptome for all major tissue types and for three stages of development, as well as the first transcriptomic analysis of a single, identified neuroblast lineage, finding a lineage-enriched transcription factor.

developmental biology↗

bioRxiv2020

A novel temporal identity window generates alternating cardinal motor neuron subtypes in a single progenitor lineage

Spatial patterning specifies neural progenitor identity, with further diversity generated by temporal patterning within individual progenitor lineages. These mechanisms generate cardinal classes of motor neurons (sharing a transcription factor identity and common muscle group targets). In Drosophila, two cardinal classes are Even-skipped (Eve)+ motor neurons projecting to dorsal muscles and Nkx6+ motor neurons projecting to ventral muscles. The Drosophila neuroblast 7-1 (NB7-1) lineage generates distinct Eve+ motor neurons via the temporal transcription factor (TTF) cascade Hunchback (Hb)-Kruppel (Kr)-Pdm-Castor (Cas). Here we show that a newly discovered Kr/Pdm temporal identity window gives rise to an Nkx6+ Eve-motor neuron projecting to ventral oblique muscles, resulting in alternation of cardinal motor neuron subtypes from a single progenitor (Eve>Nkx6>Eve). We show that co-overexpression of Kr/Pdm generates ectopic VO motor neurons within the NB7-1 lineage and that Kr/Pdm act via Nkx6, which itself is necessary and sufficient for VO motor neuron identity. Lastly, Nkx6 is required for ventral oblique muscle targeting, thereby linking temporal patterning to motor neuron morphology and synaptic target selection. In conclusion, we show that one neuroblast lineage generates interleaved cardinal motor neurons fates; that the Kr/Pdm TTFs form a novel temporal identity window that promotes expression of Nkx6; and that the Kr/Pdm>Nkx6 pathway is necessary and sufficient to specify VO motor neuron identity and morphology.

developmental biology↗

bioRxiv2019

The role of lineage, hemilineage and temporal identity in establishing neuronal targeting and connectivity in the Drosophila embryo

The mechanisms specifying neuronal diversity are well-characterized, yet it remains unclear how or if these mechanisms regulate neural circuit assembly. To address this, we mapped the developmental origin of 160 interneurons from seven bilateral neural progenitors (neuroblasts), and identify them in a synapse-scale TEM reconstruction of the Drosophila larval CNS. We find that lineages concurrently build the sensory and motor neuropils by generating sensory and motor hemilineages in a Notch-dependent manner. Neurons in a hemilineage share common synaptic targeting within the neuropil, which is further refined based on neuronal temporal identity. Connectome analysis shows that hemilineage-temporal cohorts share common connectivity. Finally, we show that proximity alone cannot explain the observed connectivity structure, suggesting hemilineage/temporal identity confers an added layer of specificity. Thus, we demonstrate that the mechanisms specifying neuronal diversity also govern circuit formation and function, and that these principles are broadly applicable throughout the nervous system.

neuroscience↗

bioRxiv2018

A repressor-decay timer for robust temporal patterning in embryonic Drosophila neuroblast lineages

Biological timers synchronize patterning processes during embryonic development. In the Drosophila embryo, neural progenitors (neuroblasts; NBs) produce a sequence of unique neurons whose identities depend on the sequential expression of temporal transcription factors (TTFs). The stereotypy and precision of the NB lineages indicate reproducible temporal progression of the TTF timer. To examine the basis of this robustness, we combine theory and experiments. The TTF timer is commonly described as a relay of activators, but its regulatory circuit is also consistent with a repressor-decay timer, in which expression of each TTF begins once its repressor is sufficiently reduced. We find that repressor-decay timers are more robust to parameter variations compared to activator-relay timers. This suggests that the in-vivo TTF sequence progresses primarily by repressor-decay, a prediction that we support experimentally. Our results emphasize the role of robustness in the evolutionary design of patterning circuits.

bioinformatics↗

bioRxiv2018

Genetic compensation is triggered by mutant mRNA degradation

Genetic compensation by transcriptional modulation of related gene(s) (also known as transcriptional adaptation) has been reported in numerous systems 1-3; however, whether and how such a response can be activated in the absence of protein feedback loops is unknown. Here, we develop and analyze several models of transcriptional adaptation in zebrafish and mouse that we show are not caused by loss of protein function. We find that the increase in transcript levels is due to enhanced transcription, and observe a correlation between the levels of mutant mRNA decay and transcriptional upregulation of related genes. To assess the role of mutant mRNA degradation in triggering transcriptional adaptation, we use genetic and pharmacological approaches and find that mRNA degradation is indeed required for this process. Notably, uncapped RNAs, themselves subjected to rapid degradation, can also induce transcriptional adaptation. Next, we generate alleles that fail to transcribe the mutated gene and find that they do not show transcriptional adaptation, and exhibit more severe phenotypes than those observed in alleles displaying mutant mRNA decay. Transcriptome analysis of these different alleles reveals the upregulation of hundreds of genes with enrichment for those showing sequence similarity with the mutated genes mRNA, suggesting a model whereby mRNA degradation products induce the response via sequence similarity. These results expand the role of the mRNA surveillance machinery in buffering against mutations by triggering the transcriptional upregulation of related genes. Besides implications for our understanding of disease-causing mutations, our findings will help design mutant alleles with minimal transcriptional adaptation-derived compensation.

genetics↗