bluuebunny/crossref_metadata_2025_split huggingface dataset:
Foetal Intraperitoneal AAV8 Injections as a Valid and Promising Tool to Specifically Target Neuronal Subpopulations Within the Gastrointestinal (GI) Tract
Authors: Winckelmans Kathleen , Buckinx Roeland , Waddington Simon , Timmermans Jean‐Pierre 
Published: November 2023

Adeno‐associated viral vectors (AAV) are a versatile tool for gene transfer to manipulate neuronal function in preclinical research and are potential means for gene therapy. Recent studies showed successful in vivo transduction of the mouse enteric nervous system (ENS) by AAV after neonatal and postnatal injection of the viral vector (1–5). Although the full development of the mouse ENS also continues after birth, the prenatal phase starting from E9,5 till E14 spans the period of the start and complete colonization of the GI tract by neural crest cells. In humans the equivalent period corresponds to week 4–7 of gestation. Defects in ENS development result in congenital gastrointestinal phenotypes, such as Hirschsprung's disease. Therefore, in order to better understand the underlying mechanisms of ENS development and related dysfunctions, genetic studies performed at prenatal stage are essential. To test our transduction technique, AAV8 encoding eGFP driven by the immediately early human cytomegalovirus promoter was injected intraperitoneally into E14 mouse embryos. At postnatal day 35, the transduction of cell types in the gastrointestinal tract was evaluated using fluorescence microscopic imaging of the transgene‐encoded eGFP in combination with a set of neuronal and glial markers. In the distinct gastrointestinal segments, i.e. oesophagus, gastric corpus, ileum and distal colon, eGFP was exclusively found in neurons, stained with HuC/D, indicating a highly cell type specific transduction. Immunostaining revealed viral‐encoded eGFP neurons which were either nitrergic in nature, calbindin‐immunoreactive (ir), calretinin‐ir or CGRP‐ir, indicating that several functional neuronal subpopulations were transduced. A strong eGFP signal was also seen in neuronal fibers, either of intrinsic or possibly also of extrinsic origin as systemic administration of AAVs at foetal stage leads to extrinsic transduction of the nervous system as well (6). eGFP positive glial cells could not be observed. These results demonstrate the specific and successful transduction of the prenatal ENS. However, compared to earlier results from the neonatal and postnatal injections (2–4), the transduction efficiency in the myenteric plexus in prenatal injections is still significantly lower (6% versus 20–30%). Therefore, further research is still indicated to improve the technique (e.g. exploring other AAV serotypes and promoters), but the results presented in this study clearly show the enormous potential of this technique in addition to the transgenic animal breeding.Support or Funding InformationThis project is supported by BOF‐KP grant 32765 of the University of Antwerp.
Determining the Role of Natural Killer Cells in Immune‐Mediated Fetal Growth Restriction in Rats
Authors: Baines Kelly , Renaud Stephen 
Published: November 2023

Pregnancy is a unique state in which two genetically foreign individuals – mother and fetus – coexist in immunological harmony. The mechanisms facilitating this immunological tolerance are not well understood; however, the uterus contains a unique population of immune cells belonging to the natural killer (NK) cell lineage that likely have a key role in promoting tolerance. Uterine NK cells are purported to promote uterine vascular remodeling as well as facilitate placental and fetal development during pregnancy. We hypothesize that exposure to pathogenic stimuli early in pregnancy disrupts normal functioning of uterine NK cells, thereby impacting uterine vascular remodeling and fetal growth. To test this hypothesis, the viral mimetic polyinosinic:polycytidylic acid (polyI:C) was injected into pregnant rats under control conditions or following NK cell depletion using anti‐asialo GM1 antibodies, and changes in uterine cytokine production and fetal growth were assessed. Rats were injected with 1, 5, or 10 mg/kg polyI:C on gestational day 8.5, while control rats were injected with saline. Dams injected with 10 mg/kg polyI:C exhibited a 15% decrease in fetal weight and a 4% decrease in fetal crown‐rump length, without affecting fetal viability. Decreased fetal weight correlated with increased production of inflammatory cytokines (interferon‐gamma, tumour necrosis factor‐alpha, and interleukin‐6) in both spleen and implantation sites, and increased perforin expression within implantation sites. Interestingly, immunodepletion of NK cells prior to injection of 10 mg/kg polyI:C resulted in a 38% decrease in fetal weight compared to control animals, indicating that uterine NK cells have a role in preventing fetal growth restriction caused by polyI:C exposure. In conclusion, injection of polyI:C into pregnant rats resulted in elevated cytokine production and fetal growth restriction that was exacerbated by a lack of NK cells.Support or Funding InformationChildren's Health Research Institute, Preeclampsia Foundation of Canada, Schulich Medicine and Dentistry
Identification of a Novel Silencer within an Lmx1b Mediated Cis‐regulatory Module
Authors: Gray Austin L , Haro Endika , Spady Connor D , Pira Charmaine U , Oberg Kerby C 
Published: November 2023

IntroductionLmx1b is a homeodomain transcription factor critical for limb dorsalization. Expression of Lmx1b is restricted to the dorsal limb mesoderm during development. ChIP‐seq analysis identified 2 closely associated Lmx1b bound intervals (LBI) that were highly conserved non‐coding regions located 60 kb upstream of the Lmx1b gene. Active chromatin regulatory marks were associated with these intervals supporting a role as regulatory elements. Furthermore, these LBIs contained 4 potential Lmx1b binding sites (3 in LBI1 and 1 in LBI2) that were conserved across divergent vertebrates. We hypothesized that these potential regulatory sequences worked in concert to autoregulate Lmx1b expression.MethodsWe isolated both LBIs, as individual fragments and a combined fragment. The fragments were inserted into a reporter construct containing a thymidine kinase (tk) basal promoter linked to a GFP reporter. The plasmids were then electroporated into the presumptive wing region of stage 14 chick embryos. After 48 hours, the chick embryos were harvested and photographed using UV and white light. Transfection quality was ensured by co‐electroporating a plasmid containing a β‐actin promoter linked to red fluorescent protein (RFP). The negative control consisted of the tk promoter without an insert. We also performed site directed mutagenesis of the conserved predicted Lmx1b binding sites to determine the role of Lmx1b binding in LBI activity.ResultsLBI1 demonstrated activity restricted to the limb but present in both the dorsal and ventral mesoderm. LBI2 showed no enhancer activity. Interestingly, the combined construct containing both LBI1/2 displayed activity that was restricted to the dorsal mesoderm. Site directed mutagenesis of the Lmx1b binding site in LBI2 permitted enhancer activity in the ventral mesoderm, while in a companion report, site directed mutagenesis of the Lmx1b binding sites in LBI1 ablated enhancer activity indicating a requirement for Lmx1b.ConclusionOur data indicate that these LBIs function collectively in the limb as an Lmx1b‐associated regulatory module (LARM) with both autoregulatory enhancer and ventral‐silencer sequences.Support or Funding InformationFunding in part by a grant from the National Organization for Rare Disorders (NORD).
Analysis of Sp6 and Sp8 Protein‐Protein Interactions and DNA Binding during Limb Development
Authors: Ros Marian , Pérez‐Gómez Rocío , Fernández‐Guerrero Marc , Zunzunegui Sandra , Bell Sheila , López‐Giménez Juan F 
Published: November 2023

Sp6 and Sp8, two members of the specificity protein (Sp) family of zinc‐finger transcription factors, are expressed in the limb ectoderm. Their importance in limb development was demonstrated by the analysis of double null mutants that exhibited a range of limb malformations that correlated with the dose of Sp6/Sp8 gene products remained (Haro et al., 2014). Thus, the progressive reduction led to predictable morphologies that transit from syndactyly, to split‐hand/foot malformation phenotype, oligodactyly, truncation and finally amelia. In addition, when digits form these show a double dorsal tip. The molecular study of these mutants revealed that Sp6 and Sp8 work together downstream to Wnt/bcatenin to activate Fgf8 and downstream of Bmp signaling, possible cooperating with Smads, to activate En1, the ventral determinant in limb patterning.Here, to further the understanding of Sp6/8 function, we have investigated Sp6/8 suspected protein‐protein interactions by co‐immunoprecipitation (CoIP) and by bimolecular fluorescence complementation (BiFC). To this end, Sp6, Sp8 and Smads were tagged with Myc or FLAG epitopes to their N‐terminal end and with YFP full length or YFP moieties to their C‐terminal end. Our results show that Sp6/8 form homo and heterodimers and that they form complexes with BMP R‐Smads. All these interactions require the C‐terminal domain of Sps. We are currently determining Sp6/8 DNA binding activity by luciferase reporter and EMSA assays.Support or Funding InformationGrant BFU2014‐57216‐P to MAR from the Spanish Government.
Chondrocyte Columns are Shorter in the Growth Plate of Collagen XXIV Null Mice
Authors: Gordon Marion , Zhou Peihong , Hahn Rita , Gerecke Donald R 
Published: November 2023

Collagen XXIV is fibrillar type of collagen that is a component of bone. Because its function is unknown, collagen XXIV knock out (KO) mice were generated, and their long bones were compared to those of age‐matched wild type (WT) mice. MicroCT analysis indicated that the KO mouse femurs were less mineralized. Histology of bone sections showed the growth plate to be smaller in the KO mice. At 5 months of age, the wild type mouse growth plate contained a physis of uniform thickness that stained light blue (amphophilic), and was composed of cartilage with regular columns of chondrocytes perpendicular to the physis. Secondary spongiosa continued from the primary as eosinophilic lamellar bone. However, in the KO mouse growth plate, the columns of chondrocytes were shorter, the cells within the columns appeared to be less organized, and fewer chondrocytes became hypertrophic. These features may correlate with their being unable to develop into osteoblasts. No detectable differences were seen in articular cartilage, cortical bone or the boney trabeculae in the marrow cavities.Support or Funding InformationNIEHS P30ES005022 and T32ES007148
The Back to Sleep Campaign, Positional Cranial Deformity, and the Foramen Magnum: New Lessons Learned from Old Pachacamac Crania
Authors: Zdilla Matthew J , Koons Aaron W , Russell Michelle L , Bliss Kaitlyn N , Mangus Kelsey R 
Published: November 2023

In 1992, the American Academy of Pediatrics discouraged prone sleeping positions because of its association with sudden infant death syndrome. The recommendations prompted the United States Public Health Service to begin the “Back to Sleep Campaign,” to promote the habitual practice of placing newborns to sleep in a supine or side‐lying positions. After the Back to Sleep Campaign was initiated, there was a sudden increase in the incidence of deformational plagiocephaly. Therefore, research is warranted regarding effects on the influence of extrinsic forces on the development of the cranium. This study utilized ‘traditional’ and geometric morphometric techniques to compare the foramen magnum size and shape of Peruvian crania from the Samuel George Morton Collection, largely from Pachacamac and likely to have undergone intentional cranial deformation, to those of craniofacially diverse cultures that were not reputed to practice intentional cranial deformation. The results demonstrate statistically significant differences in the sagittal dimension of the foramen magnum between groups. Likewise, geometric morphometric analysis revealed differences in foraminal shapes between groups. The results of this study suggest that extrinsic compressive forces likely alter the structure of the foramen magnum. These findings should be taken into consideration with regard to modern‐day newborn sleeping positions and positional cranial deformation.Support or Funding InformationWV Research Challenge Fund [HEPC.dsr.14.13]
Analysis of Craniofacial Variation in cleft secondary palate‐1 Heterozygous and Homozygous Newborn Mice
Authors: Bennett Holly , Moskal Russell , Holloway Jared , Jones Jennifer , Holton Nathan , Leslie Erin , Bjork Bryan 
Published: November 2023

Prdm16 is involved in normal human and mouse craniofacial development. The cleft secondary palate 1 (csp1) ENU‐induced mutation displays cleft palate, shortening of the snout, and micrognathia on a congenic FVB/NJ strain background due to reduced Prdm16 expression, which mirrors the mechanism of Pierre Robin Sequence (PRS) in human. However, overt cleft palate is not evident on a congenic C57BL6/J background (csp1‐B6). Adult heterozygous csp1‐B6 mice have significantly shortened snouts, palates, and right‐side mandibles compared to wt littermates along with variable incidence of malocclusion and hydrocephalus. Homozygous mutant csp1‐B6 mice die shortly after birth for unknown reasons. Our aim is to quantitatively assess variation in bony facial morphology in newborn heterozygous (het) and homozygous mutant (mut) csp1‐B6 and contrast this with the wild type morphology.We compared the size, shape, and volume of the facial bones in newborn wt, het, and mut csp1‐B6 mice. Anatomical landmarks, bone surface area, and bone volume data were collected from three‐dimensional microcomputed tomography (microCT) images.Homozygous mutant newborn mice have significantly longer left‐sided premaxillae and maxillae, but shorter left‐side mandibles than the wt and het mice. The mut mice also showed significantly reduced left‐ and right‐side premaxillae heights versus wt and het mice and smaller individual facial bone volumes and surface areas. The wt and het newborn mice did not differ significantly from each other in any contrasts.Reduced Prdm16 expression in newborn homozygous mutant csp1‐B6 mice is correlated with overall, and asymmetric, micrognathia and undergrowth of the snout compared to the newborn wt and het mice. These findings complement our concurrent research on adult heterozygous csp1‐B6 mice, and will provide insight on the downstream effects of Prdm16 expression on craniofacial development and integration among the bones of the face.Support or Funding InformationNIH/NIDCR (R15DE023982)Midwestern University, College of Dental Medicine Illinois Intramural Research Grant
Cleft Palate Case‐Derived ARHGAP29 Mutation Causes Lethality and Oral Adhesions in Mice
Authors: Paul Brian J , Palmer Kristina , Sharp Jocelyn C , Pratt C. Herbert , Murray Stephen A , Dunnwald Martine 
Published: November 2023

Non‐syndromic cleft lip and/or palate (NSCLP) is among the most frequent major birth defects. The etiology is complex, drawing influence from environmental and genetic factors. Previous studies have identified mutations affecting ARHGAP29 on chromosome 1p22 as a factor for increased risk of NSCLP. To investigate the effects of altered ARHGAP29 in vivo, we generated a novel murine allele by replacing Arhgap29 with a mutated sequence from a human NSLCLP patient. This single‐nucleotide variation of ARHGAP29 led to a nonsense mutation (K326X) in the 11th exon of 31 total, presumably resulting in a nonfunctional protein product. Arhgap29K326X/+ mice were crossed and embryos were harvested for analysis at various time points. We hypothesize that this allele will lead to altered craniofacial development, supporting a critical role for ARHGAP29 in this process. Our results show the absence of homozygous ArhgapK326X animals in all the analyzed litters (N=36), assessed as early as embryonic day 9.5 (e9.5). At e14.5, 54.8% of Arhgap29K326X/+ mice (N=31) exhibited the presence of oral adhesions between the maxilla and mandible or maxilla and tongue, compared to 12.5% of wild type animals (N=8) (P=0.0489). Further quantification of the oral adhesions revealed that 15.94% of the length of the oral epithelium was in improper contact with another oral epithelium in Arhgap29K326X/+ compared with only 3.26% in wild type embryos (P=0.0029). Previous reports have implicated alterations in the periderm as the potential cause of oral adhesions. We performed immunofluorescent analyses at sites of adhesions and detected the presence of p63‐negative, Keratin 17‐positive cells at those sites. This suggests that periderm cells are still present, yet whether or not they are functioning properly remains unknown. Evaluation of E‐cadherin expression at adhesion sites shows proper location at cell‐cell boundaries. Oral adhesions did not appear to impair palatogenesis, as all analyzed embryos show confluent palatal mesenchyme and epithelium at e18.5 (N=22). Collectively, our data demonstrate that ARHGAP29 is required for embryonic survival, and that heterozygosity for Arhgap29 loss of function variants increases the incidence and length of oral adhesions at a critical time point during palatogenesis. In conclusion, we validate the damaging function of the human K326X mutation in vivo and reveal a previously unknown effect of altered Arhgap29 in murine embryonic and craniofacial development.Support or Funding InformationPartial financial support was provided by a grant from the National Institute of Health R37DE08559, R01AR067739, and by the FaceBase consortium (grants DE020052 and DE020057). Brian Paul was a recipient of a fellowship from the Iowa Center for Research by Undergraduates.
Homeostatic Neurogenesis in the Lizard Brain
Authors: McDonald Rebecca Paula , Vickaryous Matthew Kenneth 
Published: November 2023

Neurogenesis – the generation of new neurons from a precursor population – has been documented as a normal or homeostatic process within various regions of the adult brain. In most instances, these precursors or endogenous neural stem/progenitor cells (NSPCs) reside in neurogenic zones located adjacent to the ventricular system. Across vertebrates, the distribution of NSPCs, and the capacity of homeostatic neurogenesis, varies greatly. Among mammals, NSPCs are restricted to only two constitutively active regions of the brain. In contrast, among teleost fish NSPCs, and constitutive neurogenesis, are widespread throughout the brain. Although less is known about reptiles, available evidence indicates that NSPCs are present within multiple continuously active regions. Here we investigate neurogenesis in an emerging amniote model of central nervous system regeneration, the leopard gecko. We have identified populations of NSPCs within various regions of the brain, including the ventricular zone (VZ) of the lateral ventricles. VZ cells of the lateral ventricle characteristically express the stem cell markers SOX2 and Musashi‐1. In addition, they share features in common with radial glia, including a lengthy radial process that contacts the pial surface, and the expression of the glial markers Vimentin and glial fibrillary associated protein. As evidenced by immunostaining for both proliferating cell nuclear antigen and phosphorylated histone H3, VZ cells of the lateral ventricles are mitotically active. Utilizing a bromodeoxyuridine (BrdU) pulse‐chase experiment, we labelled VZ cells for short‐term cell tracking. Immediately following the pulse period, BrdU+ cells were confined to the VZ. Following a 30‐day chase, we observed BrdU+ cells within the cellular layer of the cortex, a region dominated by mature neurons (immunopositive for the neuronal markers HuCD and NeuN). We conclude that the VZ of the lateral ventricles is a neurogenic zone, and provide compelling evidence of homeostatic neurogenesis in the leopard gecko brain.Support or Funding InformationNatural Sciences and Engineering Research Council (NSERC) Discovery grant 400358
Expression of Glial Fibrillary Acidic Protein (GFAP) and Histopathological Changes in Brains of Trypanosoma brucei brucei Experimentally Infected Rats
Authors: Alawa Judith Nkiruka , Maliki Joseph Simpa , Tanko Yusuf , Alawa Clement Innocent 
Published: November 2023

African trypanosomiasis, also known as sleeping sickness in humans which affects the brain, is characterized by alterations in the functions of brain regions. It is unknown if the disease affects the expression of Glial Fibrillary Acidic Protein (GFAP) which is a marker for astroglia activation. Therefore, this study was aimed at investigating the astrocytic response and histopathological changes in the striatum and hippocampus of rats experimentally infected with Trypanosoma brucei brucei (Tbb). Immunohistochemical evaluation, demonstrated reactive astrocytosis characterized by dense staining of enlarged cell bodies (astrocyte hypertrophy) with distinct cell processes in the striatum and hippocampus. Histopathological changes in the striatum of infected rats showed neuronal degeneration characterized by chromatolysis and satellitosis. Hippocampus showed gliosis in the molecular layer while axonal spheroids were observed in the white matter indicative of degenerating neuronal process. Control rats did not show any of these signs. These results demonstrate that the disease alters the expression of GFAP indicative of astroglia activation thus suggests that the astroglia cells may mediate immune response during trypanosome infection.Support or Funding InformationAhmadu Bello University
Serum enkephalin levels correlate with EAE disease severity
Authors: Ludwig Michael D. , Zagon Ian S. , McLaughlin Patricia J 
Published: November 2023

Multiple sclerosis, and the animal model experimental autoimmune encephalomyelitis (EAE), are characterized by elevated immune cell replication. The endogenous peptide, opioid growth factor (OGF), chemically termed [Met5]‐enkephalin has been shown to inhibit proliferation of activated T and B cells in vitro, and to reduce the number of T and B cells in peripheral tissues spleen, nodes) and CNS (spinal cord) following EAE immunization. Systemic OGF injection of mice with chronic EAE or relapse‐remitting EAE results in diminishment of clinical disease, reductions in activated astrocytes, and fewer relapses. The hypothesis of this research is that serum levels of OGF decline during the course of EAE, and that treatment with exogenous OGF is associated with severity of disease. The chronic mouse model of MS (Ch‐EAE) was established, with one group of mice receiving daily OGF injections beginning at the time of clinical disease onset. Clinical behavior was monitored daily; locomotor activity and thermal sensitivity were measured periodically. Blood samples were collected and enkephalin (OGF) levels measured by ELISA (MyBiosource). EAE disease onset occurred on day 9 post immunization when the mean clinical score was 1.5. Peak disease scores for saline‐injected EAE mice reached a mean of 5.7 on day 18, in comparison to a peak clinical score of 2.5 for EAE‐mice receiving OGF. Serum OGF levels in non‐diseased mice were approximately 149 pg/ml, whereas EAE mice had serum OGF levels that ranged between 8 and 128 pg/ml, suggesting that EAE corresponded to a decrease in enkephalins. Following exogenous OGF therapy, serum levels increased; higher serum OGF levels were related to reduce disease severity, as well as changes in open field behavior and hot‐plate sensitivity. These data suggest that there is a dysregulation of endogenous OGF in the disease state and that treatment with exogenous OGF restores serum OGF levels to normal. Moreover, the non‐invasive tracking of serum OGF may be useful as a marker for disease progression and response to therapy.Support or Funding InformationSupported in part by the generous gifts from The Paul K. and Anna E. Shockey Family Foundation