April 2024 paper from the Zeltner and Liu Labs: Parasympathetic Neurons Derived from Human Pluripotent Stem Cells

Autonomic parasympathetic neurons (parasymNs) control unconscious body responses, including “rest-and-digest.” ParasymN innervation is important for organ development, and parasymN dysfunction is a hallmark of autonomic neuropathy. However, parasymN function and dysfunction in humans are vastly understudied due to the lack of a model system. Human pluripotent stem cell (hPSC)-derived neurons can fill this void as a versatile platform. Here, we developed a differentiation paradigm detailing the derivation of functional human parasymNs from Schwann cell progenitors. We employ these neurons (1) to assess human autonomic nervous system (ANS) development, (2) to model neuropathy in the genetic disorder familial dysautonomia (FD), (3) to show parasymN dysfunction during SARS-CoV-2 infection, (4) to model the autoimmune disease Sjögren’s syndrome (SS), and (5) to show that parasymNs innervate white adipocytes (WATs) during development and promote WAT maturation. Our model system could become instrumental for future disease modeling and drug discovery studies, as well as for human developmental studies.

September 2023 paper from the Roberts-Galbraith Lab: Ets-1 transcription factor regulates glial cell regeneration and function in planarians

Glia play multifaceted roles in nervous systems in response to injury. Depending on the species, extent of injury and glial cell type in question, glia can help or hinder the regeneration of neurons. Studying glia in the context of successful regeneration could reveal features of pro-regenerative glia that could be exploited for new human therapies. Planarian flatworms completely regenerate their nervous systems after injury – including glia – and thus provide a strong model system for exploring glia in the context of regeneration. Here, we report that planarian glia regenerate after neurons, and that neurons are required for correct glial numbers and localization during regeneration. We also identify the planarian transcription factor-encoding gene ets-1 as a key regulator of glial cell maintenance and regeneration. Using ets-1 (RNAi) to perturb glia, we show that glial loss is associated with altered neuronal gene expression, impeded animal movement and impaired nervous system architecture – particularly within the neuropil. Importantly, our work reveals the inter-relationships of glia and neurons in the context of robust neural regeneration.

September 2023 paper from the Liu Lab: Taste papilla cell differentiation requires the regulation of secretory protein production by ALK3-BMP signaling in the tongue mesenchyme

Taste papillae are specialized organs, each of which comprises an epithelial wall hosting taste buds and a core of mesenchymal tissue. In the present study, we report that during early taste papilla development in mouse embryos, bone morphogenetic protein (BMP) signaling mediated by type 1 receptor ALK3 in the tongue mesenchyme is required for epithelial Wnt/β-catenin activity and taste papilla differentiation. Mesenchyme-specific knockout (cKO) of Alk3 using Wnt1-Cre and Sox10-Cre resulted in an absence of taste papillae at E12.0. Biochemical and cell differentiation analyses demonstrated that mesenchymal ALK3-BMP signaling governed the production of previously unappreciated secretory proteins, i.e. it suppressed those that inhibit and facilitated those that promote taste papilla differentiation. Bulk RNA-sequencing analysis revealed many more differentially expressed genes (DEGs) in the tongue epithelium than in the mesenchyme in Alk3 cKO versus control. Moreover, we detected downregulated epithelial Wnt/β-catenin signaling and found that taste papilla development in the Alk3 cKO was rescued by the GSK3β inhibitor LiCl, but not by Wnt3a. Our findings demonstrate for the first time the requirement of tongue mesenchyme in taste papilla cell differentiation.

April 2023 paper from the Roberts-Galbraith Lab and recent doctoral graduate Dr. Jennifer Jenkins:  Heterotrimeric G proteins regulate planarian regeneration and behavior

G protein-coupled receptors play broad roles in development and stem cell biology, but few roles for G protein-coupled receptor signaling in complex tissue regeneration have been uncovered. Planarian flatworms robustly regenerate all tissues and provide a model with which to explore potential functions for G protein-coupled receptor signaling in somatic regeneration and pluripotent stem cell biology. As a first step toward exploring G protein-coupled receptor function in planarians, we investigated downstream signal transducers that work with G protein-coupled receptors, called heterotrimeric G proteins. Here, we characterized the complete heterotrimeric G protein complement in Schmidtea mediterranea for the first time and found that 7 heterotrimeric G protein subunits promote regeneration. We further characterized 2 subunits critical for regeneration, Gαq1 and Gβ1-4a, finding that they promote the late phase of anterior polarity reestablishment, likely through anterior pole-produced Follistatin. Incidentally, we also found that 5 G protein subunits modulate planarian behavior. We further identified a putative serotonin receptor, gcr052, that we propose works with Gαs2 and Gβx2 in planarian locomotion, demonstrating the utility of our strategy for identifying relevant G protein-coupled receptors. Our work provides foundational insight into roles of heterotrimeric G proteins in planarian biology and serves as a useful springboard toward broadening our understanding of G protein-coupled receptor signaling in adult tissue regeneration.

May 2023 paper from the Zeltner Lab: Isolation of human pluripotent stem cell-derived sensory neuron subtypes by immunopanning

Sensory neurons (SNs) detect a wide range of information from the body and the environment that is critical for homeostasis. There are three main subtypes of SNs: nociceptors, mechanoreceptors, and proprioceptors, which express different membrane proteins, such as TRKA, TRKB, or TRKC, respectively. Human pluripotent stem cell technology provides an ideal platform to study development and diseases of SNs, however there is not a viable method to isolate individual SN subtype for downstream analysis available. Here, we employ the method immunopanning to isolate each SN subtype. This method is very gentle and allows proper survival after the isolation. We use antibodies against TRKA, TRKB, and TRKC to isolate nociceptors, mechanoreceptors, and proprioceptors, respectively. We show that our cultures are enriched for each subtype and express their respective subtype markers. Furthermore, we show that the immunopanned SNs are electrically active and respond to specific stimuli. Thus, our method can be used to purify viable neuronal subtypes using respective membrane proteins for downstream studies.

May 2023 paper from the Zeltner and Lui Labs: O-GlcNAcylation is crucial for sympathetic neuron development, maintenance, functionality and contributes to peripheral neuropathy

O-GlcNAcylation is a post-translational modification (PTM) that regulates a wide range of cellular functions and has been associated with multiple metabolic diseases in various organs. The sympathetic nervous system (SNS) is the efferent portion of the autonomic nervous system that regulates metabolism of almost all organs in the body. How much the development and functionality of the SNS are influenced by O-GlcNAcylation, as well as how such regulation could contribute to sympathetic neuron (symN)-related neuropathy in diseased states, remains unknown. Here, we assessed the level of protein O-GlcNAcylation at various stages of symN development, using a human pluripotent stem cell (hPSC)-based symN differentiation paradigm. We found that pharmacological disruption of O-GlcNAcylation impaired both the growth and survival of hPSC-derived symNs. In the high glucose condition that mimics hyperglycemia, hPSC-derived symNs were hyperactive, and their regenerative capacity was impaired, which resembled typical neuronal defects in patients and animal models of diabetes mellitus. Using this model of sympathetic neuropathy, we discovered that O-GlcNAcylation increased in symNs under high glucose, which lead to hyperactivity. Pharmacological inhibition of O-GlcNAcylation rescued high glucose-induced symN hyperactivity and cell stress. This framework provides the first insight into the roles of O-GlcNAcylation in both healthy and diseased human symNs and may be used as a platform for therapeutic studies.

February 2023 article on the Menke Lab: CRISPR provides new understanding of reptile coloration

Snakes and mice might not look much alike, but much of what we know about skin coloration and patterning in vertebrates, including snakes, is based on research involving lab mice. There are limits, however, to what mice can tell us about other vertebrates because they don’t share all the same types of color-producing cells, known as chromatophores. For example, snakes have a type of chromatophore called iridophores that can generate iridescent colors by reflecting light.

November 2022 paper from the Zeltner Lab: Norepinephrine transporter defects lead to sympathetic hyperactivity in Familial Dysautonomia models

Familial dysautonomia (FD), a rare neurodevelopmental and neurodegenerative disorder affects the sympathetic and sensory nervous system. Although almost all patients harbor a mutation in ELP1, it remains unresolved exactly how function of sympathetic neurons (symNs) is affected; knowledge critical for understanding debilitating disease hallmarks, including cardiovascular instability or dysautonomic crises, that result from dysregulated sympathetic activity. Here, we employ the human pluripotent stem cell (hPSC) system to understand symN disease mechanisms and test candidate drugs. FD symNs are intrinsically hyperactive in vitro, in cardiomyocyte co-cultures, and in animal models. We report reduced norepinephrine transporter expression, decreased intracellular norepinephrine (NE), decreased NE re-uptake, and excessive extracellular NE in FD symNs. SymN hyperactivity is not a direct ELP1 mutation result, but may connect to NET via RAB proteins. We found that candidate drugs lowered hyperactivity independent of ELP1 modulation. Our findings may have implications for other symN disorders and may allow future drug testing and discovery.

October 2022 article on Genetics Professor, Pengpeng Bi, Receives Two NIH Awards

University of Georgia researcher Pengpeng Bi received a pair of National Institutes of Health grants in September: a Maximizing Investigators’ Research Award (MIRA, 2022–2027) and an Exploratory/Developmental Research Grant Award (R21, 2022–2024). The $2.3 million awards will support efforts to uncover the molecular mechanism of human muscle development and homeostasis.

May 2021 paper from the Roberts-Galbraith Lab: CBP/p300 homologs CBP2 and CBP3 play distinct roles in planarian stem cell function

Chromatin modifications function as critical regulators of gene expression and cellular identity, especially in the regulation and maintenance of the pluripotent state. However, many studies of chromatin modification in stem cells—and pluripotent stem cells in particular—are performed in mammalian stem cell culture, an in vitro condition mimicking a very transient state during mammalian development. Thus, new models for studying pluripotent stem cells in vivo could be helpful for understanding the roles of chromatin modification, for confirming prior in vitro studies, and for exploring evolution of the pluripotent state.

October 2018 paper from the Lechtreck Lab: In vivo analysis of outer arm dynein transport reveals cargo-specific intraflagellar transport properties


Outer dynein arms (ODAs) are multiprotein complexes that drive flagellar beating. Based on genetic and biochemical analyses, ODAs preassemble in the cell body and then move into the flagellum by intraflagellar transport (IFT). To study ODA transport in vivo, we expressed the essential intermediate chain 2 tagged with mNeonGreen (IC2-NG) to rescue the corresponding Chlamydomonas reinhardtii mutant oda6. IC2-NG moved by IFT; the transport was of low processivity and increased in frequency during flagellar growth. As expected, IFT of IC2-NG was diminished in oda16, lacking an ODA-specific IFT adapter, and in ift46 IFT46ΔN lacking the ODA16-interacting portion of IFT46. IFT loading appears to involve ODA16-dependent recruitment of ODAs to basal bodies followed by handover to IFT. Upon unloading from IFT, ODAs rapidly docked to the axoneme. Transient docking still occurred in the docking complex mutant oda3 indicating that the docking complex stabilizes rather than initiates ODA–microtubule interactions. In full-length flagella, ODAs continued to enter and move inside cilia by short-term bidirectional IFT and diffusion and the newly imported complexes frequently replaced axoneme-bound ODAs. We propose that the low processivity of ODA-IFT contributes to flagellar maintenance by ensuring the availability of replacement ODAs along the length of flagella.

September 2018 paper from the Gaertig Lab: Proteins that control the geometry of microtubules at the ends of cilia


Cilia, essential motile and sensory organelles, have several compartments: the basal body, transition zone, and the middle and distal axoneme segments. The distal segment accommodates key functions, including cilium assembly and sensory activities. While the middle segment contains doublet microtubules (incomplete B-tubules fused to complete A-tubules), the distal segment contains only A-tubule extensions, and its existence requires coordination of microtubule length at the nanometer scale. We show that three conserved proteins, two of which are mutated in the ciliopathy Joubert syndrome, determine the geometry of the distal segment, by controlling the positions of specific microtubule ends. FAP256/CEP104 promotes A-tubule elongation. CHE-12/Crescerin and ARMC9 act as positive and negative regulators of B-tubule length, respectively. We show that defects in the distal segment dimensions are associated with motile and sensory deficiencies of cilia. Our observations suggest that abnormalities in distal segment organization cause a subset of Joubert syndrome cases.

August 2018 paper from the Haltiwanger Lab: Two novel protein O-glucosyltransferases that modify sites distinct from POGLUT1 and affect Notch trafficking and signaling


The Notch-signaling pathway is normally activated by receptor–ligand interactions. Extracellular domains (ECDs) of Notch receptors are heavily modified with O-linked glycans, such as O-glucose (O-Glc), O-fucose (O-Fuc), and O-GlcNAc. The significance of multiple types of O-glycans on Notch is not understood. NOTCH1 ECD interacts with ligands at multiple points, including an O-Glc monosaccharide on the 11th Epidermal Growth Factor (EGF) repeat (EGF11). Here, we identify two novel protein O-glucosyltransferases that modify NOTCH1 EGF11 with O-Glc. Combined deletion of the O-Glc site on EGF11 with O-Fuc modification sites on EGF8 or EGF12 markedly reduced NOTCH1 cell-surface expression or activation of NOTCH1 by Delta-like ligand 1, respectively. This study identifies a cooperative mechanism for fine-tuning the Notch-signaling pathway by different types of O-glycans.


The Notch-signaling pathway is normally activated by Notch–ligand interactions. A recent structural analysis suggested that a novel O-linked hexose modification on serine 435 of the mammalian NOTCH1 core ligand-binding domain lies at the interface with its ligands. This serine occurs between conserved cysteines 3 and 4 of Epidermal Growth Factor-like (EGF) repeat 11 of NOTCH1, a site distinct from those modified by protein O-glucosyltransferase 1 (POGLUT1), suggesting that a different enzyme is responsible. Here, we identify two novel protein O-glucosyltransferases, POGLUT2 and POGLUT3 (formerly KDELC1 and KDELC2, respectively), which transfer O-glucose (O-Glc) from UDP-Glc to serine 435. Mass spectrometric analysis of NOTCH1 produced in HEK293T cells lacking POGLUT2, POGLUT3, or both genes showed that either POGLUT2 or POGLUT3 can add this novel O-Glc modification. EGF11 of NOTCH2 does not have a serine residue in the same location for this O-glucosylation, but EGF10 of NOTCH3 (homologous to EGF11 in NOTCH1 and -2) is also modified at the same position. Comparison of the sites suggests a consensus sequence for modification. In vitro assays with POGLUT2 and POGLUT3 showed that both enzymes modified only properly folded EGF repeats and displayed distinct acceptor specificities toward NOTCH1 EGF11 and NOTCH3 EGF10. Mutation of the O-Glc modification site on EGF11 (serine 435) in combination with sensitizing O-fucose mutations in EGF8 or EGF12 affected cell-surface presentation of NOTCH1 or reduced activation of NOTCH1 by Delta-like1, respectively. This study identifies a previously undescribed mechanism for fine-tuning the Notch-signaling pathway in mammals.

November 2017 paper from the Haltiwanger Lab: Inhibition of Delta-induced Notch signaling using fucose analogs

Notch is a cell-surface receptor that controls cell-fate decisions and is regulated by O-glycans attached to epidermal growth factor-like (EGF) repeats in its extracellular domain. Protein O-fucosyltransferase 1 (Pofut1) modifies EGF repeats with O-fucose and is essential for Notch signaling. Constitutive activation of Notch signaling has been associated with a variety of human malignancies. Therefore, tools that inhibit Notch activity are being developed as cancer therapeutics. To this end, we screened L-fucose analogs for their effects on Notch signaling. Two analogs, 6-alkynyl and 6-alkenyl fucose, were substrates of Pofut1 and were incorporated directly into Notch EGF repeats in cells. Both analogs were potent inhibitors of binding to and activation of Notch1 by Notch ligands Dll1 and Dll4, but not by Jag1. Mutagenesis and modeling studies suggest that incorporation of the analogs into EGF8 of Notch1 markedly reduces the ability of Delta ligands to bind and activate Notch1.

December 2017 paper from the Lechtreck Lab: The Bardet–Biedl syndrome protein complex is an adapter expanding the cargo range of intraflagellar transport trains for ciliary export


Bardet–Biedl syndrome (BBS) is a rare disease caused by dysfunctional cilia. In bbs mutants, the composition of the ciliary membrane is altered due to defects in the BBSome, a conserved complex of BBS proteins. To determine the molecular function of the BBSome, we used single particle in vivo imaging. Transport of the ciliary membrane protein phospholipase D (PLD) is BBSome-dependent, and PLD comigrates with BBSomes on intraflagellar transport (IFT) trains. PLD accumulates inside cilia after removal of its ciliary export sequence (CES) or in the absence of BBSomes. In conclusion, the BBSome participates directly in ciliary protein transport by serving as an adapter allowing proteins that alone are unable to bind to IFT to be exported from cilia on IFT trains.


Bardet–Biedl syndrome (BBS) is a ciliopathy resulting from defects in the BBSome, a conserved protein complex. BBSome mutations affect ciliary membrane composition, impairing cilia-based signaling. The mechanism by which the BBSome regulates ciliary membrane content remains unknown. Chlamydomonas bbs mutants lack phototaxis and accumulate phospholipase D (PLD) in the ciliary membrane. Single particle imaging revealed that PLD comigrates with BBS4 by intraflagellar transport (IFT) while IFT of PLD is abolished in bbs mutants. BBSome deficiency did not alter the rate of PLD entry into cilia. Membrane association and the N-terminal 58 residues of PLD are sufficient and necessary for BBSome-dependent transport and ciliary export. The replacement of PLD’s ciliary export sequence (CES) caused PLD to accumulate in cilia of cells with intact BBSomes and IFT. The buildup of PLD inside cilia impaired phototaxis, revealing that PLD is a negative regulator of phototactic behavior. We conclude that the BBSome is a cargo adapter ensuring ciliary export of PLD on IFT trains to regulate phototaxis.

December 2017 paper from the Lechtreck Lab: In vivo imaging of radial spoke proteins reveals independent assembly and turnover of the spoke head and stalk


Radial spokes (RSs) are multiprotein complexes regulating dynein activity. In the cell body and ciliary matrix, RS proteins are present in a 12S precursor, which is converted into axonemal 20S spokes consisting of a head and stalk. To study RS assembly in vivo, we expressed fluorescent protein (FP)-tagged versions of the head protein RSP4 and the stalk protein RSP3 to rescue the corresponding Chlamydomonas mutants pf1, lacking spoke heads, and pf14, lacking RSs entirely. RSP3 and RSP4 mostly co-migrated by intraflagellar transport (IFT). Transport was elevated during ciliary assembly. IFT of RSP4-FP depended on RSP3. To study RS assembly independently of ciliogenesis, strains expressing FP-tagged RS proteins were mated to untagged cells with, without, or with partial RSs. RSP4-FP is added a tip-to-base fashion to preexisting pf1 spoke stalks while de novo RS assembly occurred lengthwise. In wild-type cilia, the exchange rate of head protein RSP4 exceeded that of the stalk protein RSP3 suggesting increased turnover of spoke heads. The data indicate that RSP3 and RSP4 while transported together separate inside cilia during RS repair and maintenance. The 12S RS precursor encompassing both proteins could represent transport form of the RS ensuring stoichiometric delivery by IFT.

February 2018 paper from the Eggenschwiler Lab: Cell cycle-related kinase regulates mammalian eye development through positive and negative regulation of the Hedgehog pathway

Cell cycle-related kinase (CCRK) is a conserved regulator of ciliogenesis whose loss in mice leads to a wide range of developmental defects, including exencephaly, preaxial polydactyly, skeletal abnormalities, and microphthalmia. Here, we investigate the role of CCRK in mouse eye development. Ccrk mutants show dramatic patterning defects, with an expansion of the optic stalk domain into the optic cup, as well as an expansion of the retinal pigment epithelium (RPE) into neural retina (NR) territory. In addition, Ccrk mutants display a shortened optic stalk. These defects are associated with bimodal changes in Hedgehog (Hh) pathway activity within the eye, including the loss of proximal, high level responses but a gain in distal, low level responses. We simultaneously removed the Hh activator GLI2 in Ccrk mutants (Ccrk-/-;Gli2-/-), which resulted in rescue of optic cup patterning and exacerbation of optic stalk length defects. Next, we disrupted the Hh pathway antagonist GLI3 in mutants lacking CCRK (Ccrk-/-;Gli3-/-), which lead to even greater expansion of the RPE markers into the NR domain and a complete loss of NR specification within the optic cup. These results indicate that CCRK functions in eye development by both positively and negatively regulating the Hh pathway, and they reveal distinct requirements for Hh signaling in patterning and morphogenesis of the eyes.

February 2018 paper from the Menke Lab: PITX1 promotes chondrogenesis and myogenesis in mouse hindlimbs through conserved regulatory targets

The PITX1 transcription factor is expressed during hindlimb development, where it plays a critical role in directing hindlimb growth and the specification of hindlimb morphology. While it is known that PITX1 regulates hindlimb formation, in part, through activation of the Tbx4 gene, other transcriptional targets remain to be elucidated. We have used a combination of ChIP-seq and RNA-seq to investigate enhancer regions and target genes that are directly regulated by PITX1 in embryonic mouse hindlimbs.

April 2016 article on Esther van der Knaap has been named as one of the Presidential Extraordinary Research Faculty Hiring Initiative builds on UGA’s Signature Themes

Esther van der Knaap, a professor of horticulture in the Institute of Plant Breeding, Genetics and Genomics, is exploring the regulation of fruit shape and size in tomatoes as well as in peppers. Much of her research focuses on the molecular genetic mechanisms of cell division and cell size underlying fruit formation, and her work seeks to help boost the yield and quality of fruit and vegetable crops for the agricultural industry.

October 2015 article on Doug Menke’s lab “Snake Genomes Reveal Shared Plans for Making Legs, Penises”

Penises and limbs are clearly very different (exaggerated references to third legs aside), but they develop in similar ways. They both involve long bits of tissue that grow out from a small embryonic bud, under the direction of very similar proteins, and molecules. Carlos Infante and Douglas Menke from the University of Georgia has shown that similar enhancers—sequences that switch genes on or off—are also at work in both organs.

August 2014 article on Nancy Manley’s Lab: “Scientists grow fully functional organ from transplanted cells”

The researchers created a thymus, a butterfly-shaped gland and vital component of the human immune system. Located beneath the breastbone in the upper chest, the thymus is responsible for producing T-lymphocytes, or T-cells, which help organize and lead the body’s fighting forces against threats like bacteria, viruses and even cancerous cells.

“We were all surprised by how well this works,” said Nancy Manley, professor of genetics in UGA’s Franklin College of Arts and Sciences and co-author of the paper describing their finding in Nature Cell Biology.

March 2014 article on Nancy Manley’s Lab: “Franklin College researchers discover origin of unusual glands in the body”

The thymus gland is a critical component of the human immune system. It is responsible for the development of T-lymphocytes, or T-cells, which help organize and lead the body’s fighting forces against harmful organisms like bacteria and viruses. Nancy Manley, a professor of genetics in UGA’s Franklin College of Arts and Sciences and principal investigator for the project.