Biology subjects

Gilliland, C.

Publications and source records attributed to Gilliland, C..

5 recordsLinked to original sources

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

Tet2 loss suppress α-synuclein pathology by stimulating ciliogenesis

There are no approved treatments that slow Parkinsons disease (PD) progression and therefore it is important to identify novel pathogenic mechanisms that can be targeted. Loss of the epigenetic marker, Tet2 appears to have some beneficial effects in PD models, but the underlying mechanism of action is not well understood. We performed an unbiased transcriptomic analysis of cortical neurons isolated from patients with PD to identify dysregulated pathways and determine their potential contributions to the disease process. We discovered that genes associated with primary cilia, non-synaptic sensory and signaling organelles, are upregulated in both early and late PD patients. Enhancing ciliogenesis in primary cortical neurons via sonic hedgehog signaling suppressed the accumulation of -synuclein pathology in vitro. Interestingly, deletion of Tet2 in mice also enhanced the expression of primary cilia and sonic hedgehog signaling genes and rescued the accumulation of -synuclein pathology and dopamine neuron degeneration in vivo. Our findings demonstrate the crucial role of Tet2 loss in regulating ciliogenesis and potentially affecting the progression of PD pathology.

neuroscience↗

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↗