Biology subjects

Grainger, S.

Publications and source records attributed to Grainger, S..

9 recordsLinked to original sources

bioRxiv2026

Dishevelled 2 coordinates aortic Notch signaling through primitive myeloid and hemogenic endothelial cells during hematopoietic stem cell specification

Hematopoietic stem cells (HSCs) arise from the hemogenic endothelium in the dorsal aorta through the endothelial-to-hematopoietic transition, a tightly regulated developmental process requiring coordinated signaling from the developing hematopoietic niche. Wnt and Notch signaling are essential for HSC specification, with Wnt functioning upstream of Notch; however, the molecular mechanisms by which these pathways are coordinated during HSC development remain poorly understood. Here, we identify Disheveled 2 (Dvl2), a key intracellular Wnt scaffold protein, as an essential regulator of HSC identity specification in zebrafish. Loss of dvl2 impaired HSC specification and reduced Notch signaling in the dorsal aorta, despite normal canonical Wnt activity in the developing endothelium, suggesting that Dvl2 regulates HSC specification independently of endothelial beta-catenin signaling. Hemogenic endothelial-specific expression of the Notch intracellular domain rescued HSC defects in dvl2 morphants, demonstrating that Dvl2 functions upstream of Notch activation in the hemogenic endothelium during HSC specification. In addition, loss of dvl2 reduced primitive macrophages and neutrophil populations. Restoration of primitive myeloid cells through gata1a knockdown rescued both endothelial Notch signaling and HSC defects, demonstrating that Dvl2 is also required for the supportive immune cells that induce HSC identity. Together, these findings reveal that Dvl2 coordinates hemogenic endothelial and immune niche cues to control HSC identity specification.

developmental biology↗

bioRxiv2026

Kainate receptors coordinate primitive hematopoiesis and hemogenic niche organization to promote hematopoietic stem cell development

The adult hematopoietic system is established during embryogenesis through the generation of hematopoietic stem and progenitor cells (HSPCs) within specialized transient niches, yet the signaling mechanisms that coordinate this process remain poorly understood. Here, we identify a developmental role for kainate receptors (KARs), glutamate-gated ion channels classically associated with excitatory neurotransmission, in coordinating primitive hematopoiesis and hemogenic niche formation in zebrafish. Loss of the KAR subunits encoded by grik1b or grik5 depletes primitive blood cells and disrupts hemogenic niche organization. These defects are accompanied by endothelial oxidative stress and impaired Notch signaling, which together reduce HSPC emergence and limit the clonal diversity of the adult hematopoietic stem cell pool. Shifting primitive hematopoietic output away from erythropoiesis and toward myelopoiesis restored macrophage niche colonization, endothelial Notch activity, and HSPC formation in KAR-deficient embryos. Moreover, hemogenic cell-specific Notch reactivation rescued HSPC production, demonstrating that KAR-dependent primitive hematopoiesis regulates this process through endothelial Notch signaling. Lastly, conserved KAR expression in endothelial and hematopoietic populations across zebrafish, mouse, and human suggests that these receptors may regulate blood development across multiple cellular components. Together, these findings reveal that glutamate receptor signaling extends beyond its well-established functions in neuronal transmission to coordinate the developmental processes that establish the blood system.

developmental biology↗

bioRxiv2025

A zebrafish model of nicotinamide adenine dinucleotide (NAD+) deficiency-derived congenital disorders

Congenital NAD deficiency disorder (CNDD) is a multisystem condition in which cardiac, renal, vertebral, and limb anomalies are most common, but anomalies in all organ systems have been identified. Patients with this condition have biallelic pathogenic variants involving genes in the nicotinamide adenine dinucleotide (NAD+) synthesis pathway leading to decreased systemic NAD+ levels. CNDD anomalies mimic the clinical features described in vertebral-anal-cardiac-tracheoesophageal fistula-renal-limb (VACTERL) association raising the possibility that CNDD and VACTERL association possess similar underlying causes. However, the mechanism by which NAD+ deficiency causes CNDD developmental anomalies has not been determined, nor has NAD+ deficiency been definitively linked to VACTERL association. Therefore, additional animal models amenable to detailed observation of embryonic development are needed to address the causes and progression of congenital anomalies in both CNDD and VACTERL association. Here, we describe a zebrafish model of NAD+ disruption to begin to model CNDD and VACTERL association phenotypes, assessing developmental anomalies in real-time. Treatment of zebrafish embryos with 2-amino-1,3,4-thiadiazole (ATDA), a teratogen known to disrupt NAD+ metabolism, resulted in neural tube, craniofacial, cardiac, and tail defects. These defects were rescued by the administration of nicotinamide (NAM) in a dose-dependent manner. Our work establishes zebrafish as a useful model for investigating the mechanistic causes and developmental dynamics of CNDD and VACTERL association. Further, as VACTERL association has been linked to teratogens, our zebrafish model provides a platform to assess these agents. One sentence summaryNAD(H) deficiency causes multiple congenital anomalies in zebrafish.

developmental biology↗

bioRxiv2025

Secreted Frizzled-Related Protein 1a regulates hematopoietic development in a dose-dependent manner

Hematopoietic stem and progenitor cells (HSPCs) arise only during embryonic development, and their identity specification, emergence from the floor of the dorsal aorta, and proliferation are all tightly regulated by molecular mechanisms such as signaling cues. Among these, Wnt signaling plays an important role in HSPC specification, differentiation, and self-renewal, requiring precise modulation for proper development and homeostasis. Wnt signaling is initiated when a Wnt ligand binds to cell surface receptors such as those encoded by the frizzled gene family, activating intracellular signaling pathways that regulate gene expression. Secreted frizzled-related proteins (Sfrps) are known modulators of Wnt signaling, acting as both agonists and antagonists of this pathway. Yet, in vivo functions of Sfrps in HSPC development remain incompletely understood. Here, we demonstrate that Sfrp1a regulates zebrafish HSPC development and differentiation in a dose-dependent manner. In Sfrp1a loss of function animals, we observe an increase in HSPCs, an upregulation of canonical Wnt signaling, and a decrease in differentiation into both lymphoid and myeloid lineages. Conversely, at low-dose sfrp1a overexpression, there is a decrease in HSPCs and an increase in lymphoid differentiation. High-dose sfrp1a overexpression phenocopies the loss of function animals, with an increase in HSPCs, increased canonical Wnt signaling, and decreased lymphoid and myeloid differentiation. These findings highlight the importance of dose-dependent modulation of Sfrps, paralleling what is observed in hematopoietic cancers where SFRP1 loss-of-function and gain-of-function variants can drive tumorigenesis. One sentence summarySfrp1a is required for hematopoietic stem cell development.

developmental biology↗

bioRxiv2024

The E3 Ubiquitin Ligase Trip12 attenuates Wnt9a/Fzd9b signaling during hematopoietic stem cell development

Wnt signaling is essential for both the development and homeostasis of diverse cellular lineages, including hematopoietic stem cells. Organism-wide, Wnt signals are tightly regulated, as overactivation of the pathway can lead to tumorigenesis. Although numerous Wnt ligands and Frizzled (Fzd) receptors exist, how particular Wnt/Fzd pairings are established and how their signals are regulated is poorly understood. We have previously identified the requirements of the cognate pairing of Wnt9a and Fzd9b for early hematopoietic stem cell proliferation. However, the specific signals governing activation, but equally important, the molecular mechanisms required to turn the signal off, are unknown. Here, we show that the E3 ubiquitin ligase Trip12 (thyroid hormone receptor interactor 12) is specifically required to ubiquitinate the third intracellular loop of Fzd9b at K437, targeting it for lysosomal degradation. In contrast to other ubiquitin ligases described to regulate the cell surface availability of multiple Fzds broadly, our data indicate that Trip12 is selective for Fzd9b. We further demonstrate that this occurs through ubiquitination at K437 of Fzd9b in the third intracellular loop, ultimately leading to a decrease in Fzd9b receptor availability and in Wnt9a/Fzd9b signaling that impacts hematopoietic stem cell proliferation in zebrafish. Our results point to specific mechanisms driving the availability of different Fzd receptors. Determining how particular Fzd abundance is regulated at the membrane will be critical to developing specific therapies for human intervention. One sentence summaryTrip12 ubiquitinates Fzd9b

cell biology↗

bioRxiv2024

Endothelial cell Piezo1 promotes vascular smooth muscle cell differentiation on large arteries

Vascular stabilization is a mechanosensitive process, in part driven by blood flow. Here, we demonstrate the involvement of the mechanosensitive ion channel, Piezo1, in promoting arterial accumulation of vascular smooth muscle cells (vSMCs) during zebrafish development. Using a series of small molecule antagonists or agonists to temporally regulate Piezo1 activity, we identified a role for the Piezo1 channel in regulating klf2a levels and altered targeting of vSMCs between arteries and veins. Increasing Piezo1 activity suppressed klf2a and increased vSMC association with the cardinal vein, while inhibition of Piezo1 activity increased klf2a levels and decreased vSMC association with arteries. We supported the small molecule data with in vivo genetic suppression of piezo1 and 2 in zebrafish, resulting in loss of transgelin+ vSMCs on the dorsal aorta. Further, endothelial cell (EC)-specific Piezo1 knockout in mice was sufficient to decrease vSMC accumulation along the descending dorsal aorta during development, thus phenocopying our zebrafish data, and supporting functional conservation of Piezo1 in mammals. To determine mechanism, we used in vitro modeling assays to demonstrate that differential sensing of pulsatile versus laminar flow forces across endothelial cells changes the expression of mural cell differentiation genes. Together, our findings suggest a crucial role for EC Piezo1 in sensing force within large arteries to mediate mural cell differentiation and stabilization of the arterial vasculature.

developmental biology↗

bioRxiv2024

Regulation of angiogenesis by endocytic trafficking mediated by cytoplasmic dynein 1 light intermediate chain 1

Dynein cytoplasmic 1 light intermediate chain 1 (LIC1, DYNC1LI1) is a core subunit of the dynein motor complex. The LIC1 subunit also interacts with various cargo adaptors to regulate Rab-mediated endosomal recycling and lysosomal degradation. Defects in this gene are predicted to alter dynein motor function, Rab binding capabilities, and cytoplasmic cargo trafficking. Here, we have identified a dync1li1 zebrafish mutant, harboring a premature stop codon at the exon 12/13 splice acceptor site, that displays increased angiogenesis. In vitro, LIC1-deficient human endothelial cells display increases in cell surface levels of the pro-angiogenic receptor VEGFR2, SRC phosphorylation, and Rab11-mediated endosomal recycling. In vivo, endothelial-specific expression of constitutively active Rab11a leads to excessive angiogenesis, similar to the dync1li1 mutants. Increased angiogenesis is also evident in zebrafish harboring mutations in rilpl1/2, the adaptor proteins that promote Rab docking to Lic1 to mediate lysosomal targeting. These findings suggest that LIC1 and the Rab-adaptor proteins RILPL1 and 2 restrict angiogenesis by promoting degradation of VEGFR2-containing recycling endosomes. Disruption of LIC1- and RILPL1/2-mediated lysosomal targeting increases Rab11-mediated recycling endosome activity, promoting excessive SRC signaling and angiogenesis.

cell biology↗

bioRxiv2022

EGFR-initiated endocytosis of Wnt9a and Fzd9b is required for β-catenin signaling

Cell to cell communication through secreted ligands like those encoded by the Wnt gene family is critical for development and homeostasis during organismal life. One of the bottlenecks in the Wnt field has been identifying specific ligand/receptor pairings and decoding the mechanisms for their downstream signals. We previously discovered that the Wnt9a ligand signals through the cell surface receptors Fzd9b, LRP5/6 and EGFR to promote early proliferation of hematopoietic stem cells during development. We used this exquisitely specific ligand/receptor complex as a platform to determine if Wnt9a requires endocytosis for signaling. Using fluorescently labeled, biologically active Wnt9a and Fzd9b fusion proteins, we demonstrate here that the Wnt9a receptor complex is rapidly endocytosed within one minute of contact with Fzd9b. Following this, the Wnt9a/Fzd9b complex is trafficked through the cell to early and late endosomes, lysosomes, and the endoplasmic reticulum; it is also recycled back to the membrane. Using small molecule inhibitors, genetic and siRNA approaches, we identified that mechanistically this endocytosis requires EGFR-mediated phosphorylation of the Fzd9b tail, followed by endocytosis through a caveolin and EPS15 dependent pathway. Specific modes of endocytosis and trafficking may represent one of the ways in which Wnt/Fzd specificity is established, since other Wnt ligands do not require endocytosis for activity.

cell biology↗

bioRxiv2018

EGFR confers exquisite specificity of Wnt9a-Fzd9b signaling in hematopoietic stem cell development

The mechanisms of Wnt-Frizzled (Fzd) signaling selectivity and their biological implications remain unclear. We demonstrate for the first time that the epidermal growth factor receptor (EGFR) is required as a co-factor for Wnt signaling. Using genetic studies in zebrafish, paired with in vitro cell biology and biochemistry, we have determined that Fzd9b signals specifically with Wnt9a in vivo and in vitro to elicit {beta}-catenin dependent Wnt signals that regulate hematopoietic stem and progenitor cell (HSPC) development in the dorsal aorta. This requirement is conserved in the derivation of HSPCs from human embryonic stem cells. Wnt9a-Fzd9b specificity requires two intracellular domains in Fzd9b, which interact with EGFR as a required co-factor to promote signal transduction. EGFR phosphorylates one tyrosine residue on Fzd9b, a requirement for the Wnt signal. These findings indicate that Wnt signaling interactions can be exquisitely specific and inform protocols for derivation of HSPCs in vitro.\n\nHighlightsO_LIAn in vitro signaling screen identifies Fzd9b as a Wnt9a-specific receptor.\nC_LIO_LIFzd9b and Wnt9a regulate hematopoietic stem cell development as a cognate pair.\nC_LIO_LIWNT9A and FZD9 are required for HSPC derivation from human pluripotent cells in vitro.\nC_LIO_LIEGFR confers specificity to Wnt9a-Fzd9b signaling in zebrafish and human cells.\nC_LI

developmental biology↗