Bando, H;Brinkmeier, ML;Castinetti, F;Fang, Q;Lee, MS;Saveanu, A;Albarel, F;Dupuis, C;Brue, T;Camper, SA;
PMID: 35951005 | DOI: 10.1093/hmg/ddac192
Congenital hypopituitarism is a genetically heterogeneous condition that is part of a spectrum disorder that can include holoprosencephaly. Heterozygous mutations in SIX3 cause variable holoprosencephaly in humans and mice. We identified two children with neonatal hypopituitarism and thin pituitary stalk who were doubly heterozygous for rare, likely deleterious variants in the transcription factors SIX3 and POU1F1. We used genetically engineered mice to understand the disease pathophysiology. Pou1f1 loss of function heterozygotes are unaffected; Six3 heterozygotes have pituitary gland dysmorphology and incompletely ossified palate; and the Six3+/-; Pou1f1+/dw double; heterozygote mice have a pronounced phenotype, including pituitary growth through the palate. The interaction of Pou1f1 and Six3 in mice supports the possibility of digenic pituitary disease in children. Disruption of Six3 expression in the oral ectoderm completely ablated anterior pituitary development, and deletion of Six3 in the neural ectoderm blocked development of the pituitary stalk and both anterior and posterior pituitary lobes. Six3 is required in both oral and neural ectodermal tissues for activation of signaling pathways and transcription factors necessary for pituitary cell fate. These studies clarify the mechanism of SIX3 action in pituitary development and provide support for a digenic basis for hypopituitarism.
Dai J, Yang L, Xu T, Si L, Cui C, Sheng X, Chi Z, Mao L, Lian B, Tang B, Bai X, Zhou L, Li S, Wang X, Yan X, Kong Y, Guo J
PMID: 32226509 | DOI: 10.7150/jca.43010
Purpose: Polymorphisms of genes in the platelet-derived growth factor (PDGF) signaling pathway have been found to predict cutaneous melanoma (CM) survival, but their clinical effects in acral melanoma (AM) patients have not been explored. The aim of this study was to characterize the functional effect of the tag single-nucleotide polymorphism (SNP) rs2228230:C>T and assess its association with clinical outcomes in AM patients. Methods: The effect of rs2228230:C>T on mRNA structures and codon usage values were evaluated using in silico analyses. PDGF receptor alpha (PDGFRA) expression vectors with the rs2228230:C or rs2228230:T allele were constructed to evaluate the expression and signaling activity of PDGFRA. The expression of PDGFRA in AM samples was measured using in situ RNAscope hybridization and immunohistochemical staining. The association of the rs2228230 genotype with survival was analyzed in two independent AM cohorts. Results: In silico analyses indicated that the rs2228230:T allele increases the minimum free energy and reduces synonymous codon usage. The rs2228230:T allele decreased the expression of PDGFRA by reducing the stability of its mRNA and protein as well as the signaling activity of the MAPK and PI3K/AKT pathways. PDGFRA mRNA and protein expression was significantly reduced in AM tissues with the rs2228230:T allele. The progression-free survival and overall survival of AM patients with the rs2228230:T allele were significantly longer than those of patients with the CC genotype. Conclusion: Our study indicated that rs2228230:T can reduce the expression of PDGFRA and downstream signaling activity and is associated with better survival in AM patients.
Invest Ophthalmol Vis Sci.
Chung DD, Frausto RF, Lin BR, Hanser EM, Cohen Z, Aldave AJ.
PMID: 28654985 | DOI: 10.1167/iovs.17-21423
Abstract
PURPOSE:
To investigate the molecular basis of posterior polymorphous corneal dystrophy (PPCD) by examining the PPCD transcriptome and the effect of decreased ZEB1 expression on corneal endothelial cell (CEnC) gene expression.
METHODS:
Next-generation RNA sequencing (RNA-seq) analyses of corneal endothelium from two PPCD-affected individuals (one with PPCD3 and one of unknown genetic cause) compared with two age-matched controls, and primary human CEnC (pHCEnC) transfected with siRNA-mediated ZEB1 knockdown. The expression of selected differentially expressed genes was validated by quantitative polymerase chain reaction (qPCR) and/or assessed by in situ hybridization in the corneal endothelium of four independent cases of PPCD (one with PPCD3 and three of unknown genetic cause).
RESULTS:
Expression of 16% and 46% of the 104 protein-coding genes specific to ex vivo corneal endothelium was lost in the endothelium of two individuals with PPCD. Thirty-two genes associated with ZEB1 and 3 genes (BMP4, CCND1, ZEB1) associated with OVOL2 were differentially expressed in the same direction in both individuals with PPCD. Immunohistochemistry staining and RNA-seq analyses demonstrated variable expression of type IV collagens in PPCD corneas. Decreasing ZEB1 expression in pHCEnC altered expression of 711 protein-coding genes, many of which are associated with canonical pathways regulating various cellular processes.
CONCLUSIONS:
Identification of the altered transcriptome in PPCD and in a cell-based model of PPCD provided insight into the molecular alterations characterizing PPCD. Further study of the differentially expressed genes associated with ZEB1 and OVOL2 is expected to identify candidate genes for individuals with PPCD and without a ZEB1 or OVOL2 mutation.
Losurdo M, Davidsson J, Sk�ld MK
PMID: 32290212 | DOI: 10.3390/brainsci10040229
Traumatic brain injury (TBI) commonly results in primary diffuse axonal injury (DAI) and associated secondary injuries that evolve through a cascade of pathological mechanisms. We aim at assessing how myelin and oligodendrocytes react to head angular-acceleration-induced TBI in a previously described model. This model induces axonal injuries visible by amyloid precursor protein (APP) expression, predominantly in the corpus callosum and its borders. Brain tissue from a total of 27 adult rats was collected at 24 h, 72 h and 7 d post-injury. Coronal sections were prepared for immunohistochemistry and RNAscope� to investigate DAI and myelin changes (APP, MBP, Rip), oligodendrocyte lineage cell loss (Olig2), oligodendrocyte progenitor cells (OPCs) (NG2, PDGFRa) and neuronal stress (HSP70, ATF3). Oligodendrocytes and OPCs numbers (expressed as percentage of positive cells out of total number of cells) were measured in areas with high APP expression. Results showed non-statistically significant trends with a decrease in oligodendrocyte lineage cells and an increase in OPCs. Levels of myelination were mostly unaltered, although Rip expression differed significantly between sham and injured animals in the frontal brain. Neuronal stress markers were induced at the dorsal cortex and habenular nuclei. We conclude that rotational injury induces DAI and neuronal stress in specific areas. We noticed indications of oligodendrocyte death and regeneration without statistically significant changes at the timepoints measured, despite indications of axonal injuries and neuronal stress. This might suggest that oligodendrocytes are robust enough to withstand this kind of trauma, knowledge important for the understanding of thresholds for cell injury and post-traumatic recovery potential
Nandadasa S, Kraft CM, Wang LW, O'Donnell A, Patel R, Gee HY, Grobe K, Cox TC, Hildebrandt F, Apte SS.
PMID: 30814516 | DOI: 10.1038/s41467-019-08520-7
Although hundreds of cytosolic or transmembrane molecules form the primary cilium, few secreted molecules are known to contribute to ciliogenesis. Here, homologous secreted metalloproteases ADAMTS9 and ADAMTS20 are identified as ciliogenesis regulators that act intracellularly. Secreted and furin-processed ADAMTS9 bound heparan sulfate and was internalized by LRP1, LRP2 and clathrin-mediated endocytosis to be gathered in Rab11 vesicles with a unique periciliary localization defined by super-resolution microscopy. CRISPR-Cas9 inactivation of ADAMTS9 impaired ciliogenesis in RPE-1 cells, which was restored by catalytically active ADAMTS9 or ADAMTS20 acting in trans, but not by their proteolytically inactive mutants. Their mutagenesis in mice impaired neural and yolk sac ciliogenesis, leading to morphogenetic anomalies resulting from impaired hedgehog signaling, which is transduced by primary cilia. In addition to their cognate extracellular proteolytic activity, ADAMTS9 and ADAMTS20 thus have an additional proteolytic role intracellularly, revealing an unexpected regulatory dimension in ciliogenesis.
EGF and BMPs govern differentiation and patterning in human gastric glands
Wölffling, S;Daddi, A;Imai-Matsushima, A;Fritsche, K;Goosmann, C;Traulsen, J;Lisle, R;Schmid, M;del Mar Reines-Benassar, M;Pfannkuch, L;Brinkmann, V;Bornschein, J;Peter Malfertheiner, ;Ordemann, J;Link, A;Meyer, T;Boccellato, F;
| DOI: 10.1053/j.gastro.2021.04.062
Background & Aims The homeostasis of the gastrointestinal epithelium relies on cell regeneration and differentiation into distinct lineages organised inside glands and crypts. Regeneration depends on WNT/β-Catenin pathway activation, but to understand homeostasis and its dysregulation in disease we need to identify the signalling microenvironment governing cell differentiation. By using gastric glands as a model, we have identified the signals inducing differentiation of surface mucus-, zymogen- and gastric acid- producing cells. Methods We generated mucosoid cultures from the human stomach and exposed them to different growth factors to obtain cells with features of differentiated foveolar, chief and parietal cells. We localised the source of the growth factors in the tissue of origin. Results We show that EGF is the major fate determinant distinguishing the surface and inner part of human gastric glands. In combination with BMP/NOGGIN signals, EGF controls the differentiation of foveolar cells vs. parietal or chief cells. We also show that EGF is likely to underlie alteration of the gastric mucosa in the pre-cancerous condition atrophic gastritis. Conclusions Use of our recently established mucosoid cultures in combination with analysis of the tissue-of-origin provided a robust strategy to understand differentiation and patterning of human tissue and allowed us to draw a new, detailed map of the signalling microenvironment in the human gastric glands.
Sakagami N, Matsushita Y, Syklawer-Howle S, Kronenberg HM, Ono W, Ono N.
PMID: 29746183 | DOI: 10.1177/0022034518771014
Craniofacial development requires a set of patterning codes that define the identities of postmigratory mesenchymal cells in a region-specific manner, in which locally expressed morphogens, including fibroblast growth factors (FGFs) and bone morphogenetic proteins (BMPs), provide instructive cues. Msx2, a bona fide target of BMP signaling, is a transcription factor regulating Runx2 and osterix (Osx), whose mutations are associated with cranial deformities in humans. Here we show that Msx2 defines osteo-chondro precursor cells in specific regions of the craniofacial mesenchyme at the postmigratory stage, particularly in the mandibular process and the posterior cranial vault. Analysis of Msx2-creER mice revealed that early mesenchymal cells in proximity to the BMP4-expressing mesenchyme were marked upon tamoxifen injection, and their descendants contributed to diverse types of mesenchymal cells in the later stage, such as chondrocytes and perichondrial cells of the transient cartilage, as well as osteoblasts and suture mesenchymal cells. By contrast, Osx-creER marked osteoblast precursors at the later stage, and their descendants continued to become osteoblasts well into the postnatal stage. Therefore, Msx2 marks spatially restricted populations of mesenchymal precursor cells with diverse differentiation potential, suggesting that extrinsic molecular cues can dictate the nature of postmigratory mesenchymal cells in craniofacial development.
Kapalczynska, M;Lin, M;Maertzdorf, J;Heuberger, J;Muellerke, S;Zuo, X;Vidal, R;Shureiqi, I;Fischer, AS;Sauer, S;Berger, H;Kidess, E;Mollenkopf, HJ;Tacke, F;Meyer, TF;Sigal, M;
PMID: 35332152 | DOI: 10.1038/s41467-022-29176-w
Helicobacter pylori causes gastric inflammation, gland hyperplasia and is linked to gastric cancer. Here, we studied the interplay between gastric epithelial stem cells and their stromal niche under homeostasis and upon H. pylori infection. We find that gastric epithelial stem cell differentiation is orchestrated by subsets of stromal cells that either produce BMP inhibitors in the gland base, or BMP ligands at the surface. Exposure to BMP ligands promotes a feed-forward loop by inducing Bmp2 expression in the epithelial cells themselves, enforcing rapid lineage commitment to terminally differentiated mucous pit cells. H. pylori leads to a loss of stromal and epithelial Bmp2 expression and increases expression of BMP inhibitors, promoting self-renewal of stem cells and accumulation of gland base cells, which we mechanistically link to IFN-γ signaling. Mice that lack IFN-γ signaling show no alterations of BMP gradient upon infection, while exposure to IFN-γ resembles H. pylori-driven mucosal responses.
Gupta K, Levinsohn J, Linderman G, Chen D, Sun TY, Dong D, Taketo MM, Bosenberg M, Kluger Y, Choate K, Myung P.
PMID: 30595533 | DOI: 10.1016/j.devcel.2018.11.032
Delineating molecular and cellular events that precede appendage morphogenesis has been challenging due to the inability to distinguish quantitative molecular differences between cells that lack histological distinction. The hair follicle (HF) dermal condensate (DC) is a cluster of cells critical for HF development and regeneration. Events that presage emergence of this distinctive population are poorly understood. Using unbiased single-cell RNA sequencing and in vivo methods, we infer a sequence of transcriptional states through which DC cells pass that begins prior to HF morphogenesis. Our data indicate that Wnt/β-catenin signaling is required to progress into an intermediate stage that precedes quiescence and differentiation. Further, we provide evidence that quiescent DC cells are recent progeny of selectively proliferating cells present prior to morphogenesis and that are later identified in the peri-DC zone during DC expansion. Together, these findings provide an inferred path of molecular states that lead to DC cell differentiation.
