Small-molecule-induced epigenetic rejuvenation promotes SREBP condensation and overcomes barriers to CNS myelin regeneration.

Remyelination failure in diseases like multiple sclerosis (MS) was thought to involve suppressed maturation of oligodendrocyte precursors; however, oligodendrocytes are present in MS lesions yet lack myelin production. We found that oligodendrocytes in the lesions are epigenetically silenced. Developing a transgenic reporter labeling differentiated oligodendrocytes for phenotypic screening, we identified a small-molecule epigenetic-silencing-inhibitor (ESI1) that enhances myelin production and ensheathment. ESI1 promotes remyelination in animal models of demyelination and enables de novo myelinogenesis on regenerated CNS axons. ESI1 treatment lengthened myelin sheaths in human iPSC-derived organoids and augmented (re)myelination in aged mice while reversing age-related cognitive decline. Multi-omics revealed that ESI1 induces an active chromatin landscape that activates myelinogenic pathways and reprograms metabolism. Notably, ESI1 triggered nuclear condensate formation of master lipid-metabolic regulators SREBP1/2, concentrating transcriptional co-activators to drive lipid/cholesterol biosynthesis. Our study highlights the potential of targeting epigenetic silencing to enable CNS myelin regeneration in demyelinating diseases and aging.

Comparison of volumetric and 2D-based response methods in the PNOC-001 pediatric low-grade glioma clinical trial.

Background: Although response in pediatric low-grade glioma (pLGG) includes volumetric assessment, more simplified 2D-based methods are often used in clinical trials. The study's purpose was to compare volumetric to 2D methods. Methods: An expert neuroradiologist performed solid and whole tumor (including cyst and edema) volumetric measurements on MR images using a PACS-based manual segmentation tool in 43 pLGG participants (213 total follow-up images) from the Pacific Pediatric Neuro-Oncology Consortium (PNOC-001) trial. Classification based on changes in volumetric and 2D measurements of solid tumor were compared to neuroradiologist visual response assessment using the Brain Tumor Reporting and Data System (BT-RADS) criteria for a subset of 65 images using receiver operating characteristic (ROC) analysis. Longitudinal modeling of solid tumor volume was used to predict BT-RADS classification in 54 of the 65 images. Results: There was a significant difference in ROC area under the curve between 3D solid tumor volume and 2D area (0.96 vs 0.78, P = .005) and between 3D solid and 3D whole volume (0.96 vs 0.84, P = .006) when classifying BT-RADS progressive disease (PD). Thresholds of 15-25% increase in 3D solid tumor volume had an 80% sensitivity in classifying BT-RADS PD included in their 95% confidence intervals. The longitudinal model of solid volume response had a sensitivity of 82% and a positive predictive value of 67% for detecting BT-RADS PD. Conclusions: Volumetric analysis of solid tumor was significantly better than 2D measurements in classifying tumor progression as determined by BT-RADS criteria and will enable more comprehensive clinical management.

Effect of white mineral trioxide aggregate compared with biomimetic carbonated apatite on dentine bridge formation and inflammatory response in a dental pulp model.

AIM: To evaluate the effects of apatite precipitation on the biocompatibility and hard tissue induction properties of white mineral trioxide aggregate (WMTA) in a dental pulp model. METHODOLOGY: Pulp exposures were created on the axial walls of 32 sound canine teeth of eight dogs. Four additional sound teeth served as controls. The pulps were capped either with WMTA or apatite derivatives [biomimetic carbonated apatite (BCAp)] in the interaction of WMTA with a synthetic tissue fluid and restored with zinc oxide-eugenol cement. After 7 and 70 days, the animals were killed, and the histological specimens taken from the teeth were stained with haematoxylin and eosin for histomorphological evaluation. The Brown and Brenn technique was employed to stain bacteria. The data were subjected to nonparametric Kruskall-Wallis analysis and Mann-Whitney U_tests. RESULTS: Biomimetic carbonated apatite did not induce hard tissue bridge formation. WMTA performed significantly better than BCAp in this respect at both periods (P < 0.05). BCAp was associated with a significantly greater inflammatory response as compared with WMTA after 7 days (P < 0.05). Both materials were associated with similar reactions after 70 days (P >0.05). CONCLUSIONS: White mineral trioxide aggregate induced hard tissue formation via a mechanism other than that postulated via apatite formation.

Fibroblast growth factor signaling and basement membrane assembly are connected during epithelial morphogenesis of the embryoid body.

Fibroblast growth factors and receptors are intimately connected to the extracellular matrix by their affinity to heparan sulfate proteoglycans. They mediate multiple processes during embryonic development and adult life. In this study, embryonic stem cell-derived embryoid bodies were used to model fibroblast growth factor signaling during early epithelial morphogenesis. To avoid redundancy caused by multiple receptors, we employed a dominant negative mutation of Fgfr2. Mutant-derived embryoid bodies failed to form endoderm, ectoderm, and basement membrane and did not cavitate. However, in mixed cultures they displayed complete differentiation induced by extracellular products of the normal cell. Evidence will be presented here that at least one of these products is the basement membrane or factors connected to it. It will be shown that in the mutant, collagen IV and laminin-1 synthesis is coordinately suppressed. We will demonstrate that the basement membrane is required for embryoid body differentiation by rescuing columnar ectoderm differentiation and cavitation in the mutant by externally added basement membrane proteins. This treatment induced transcription of Eomesodermin, an early developmental gene, suggesting that purified basement membrane proteins can activate inherent developmental programs. Our results provide a new paradigm for the role of fibroblast growth factor signaling in basement membrane formation and epithelial differentiation.

The PTPROt tyrosine phosphatase functions as an obligate haploinsufficient tumor suppressor in vivo in B-cell chronic lymphocytic leukemia.

The tyrosine phosphatase PTPROt is a suggested tumor suppressor (TS) in B-cell chronic lymphocytic leukemia (CLL), and its expression is reduced in this disease. In order to examine how reduced PTPROt expression affects CLL in vivo we induced CLL in PTPROt-targeted mice. Unexpectedly, loss of both Ptprot alleles delayed disease detection and progression and lengthened survival relative to mice carrying two intact alleles, indicating that PTPROt fulfills a novel tumor-promoting role in CLL. Tumor cells from mice lacking PTPROt exhibited reduced B-cell receptor (BCR)-induced signaling, as well as increased apoptosis and autophagy. Inhibition of BCR/Src signaling in CLL cells induced their apoptosis, indicating that these findings are linked causally. These results suggest a cell-autonomous mechanism for the weakened CLL phenotype of PTPROt-deficient mice and uncover non-redundant roles for PTPROt in support of BCR signaling and survival of CLL cells. In contrast, loss of only one Ptprot allele induced earlier detection and progression of CLL and reduced survival, consistent with a tumor-suppressing role for PTPROt. Tumor cells from mice lacking one or both Ptprot allele exhibited increased interleukin-10 (IL-10) expression and signaling, factors known to support CLL; cells lacking one Ptprot alleles exhibited normal BCR signaling and cell death rates. We conclude that loss of one Ptprot allele promotes CLL, most likely by activating IL-10 signaling. Loss of both Ptprot alleles also reduces BCR signaling and increases cell death rates, offsetting the IL-10 effects and reducing the severity of the disease. PTPROt thus functions as an obligate haploinsufficient TS in CLL, where its expression levels determine its role as a promoter or inhibitor of the tumorigenic process in mice. Partial loss of PTPROt generates the strongest disease phenotype, suggesting that its intermediate expression levels in CLL are selected for.

Common origin of transmissible venereal tumors (TVT) in dogs.

We determined the sequence of the 1.5-kb insert upstream to c-myc in the transmissible venereal tumor (TVT) of dogs. The sequence is highly homologous to the 3' region of the mammalian repetitive LINE element. The insert is bound by a 10-bp repeat indicating DNA transposition by a mechanism involving reverse transcriptase. We analyzed DNA of four TVT tumors from various geographical locations as well as normal canine DNA for the presence of the LINE insert. The results indicate that in all TVT tumors, but not in normal tissues, the same LINE insert was present upstream to c-myc. This result suggests that TVT tumors in various dogs may have a common cellular origin.

Analysis of the Hoxd-3 gene: structure and localization of its sense and natural antisense transcripts.

This study set out to investigate the structure and localized expression of the mouse homeobox-containing gene Hoxd-3. In addition to identifying a transcript of the type known from other Antennapedia (Antp)-like mammalian homeobox cDNAs, an antisense transcript was also detected. The antisense form of Hoxd-3 overlaps with 603 bp of the sense transcript including the homeobox. Active antisense transcription has been confirmed by RNA blot analysis with single-stranded probes and by the direction of splicing of an intron in the antisense transcript. The localized expression of sense and antisense transcripts was compared by in situ hybridization. Hoxd-3 expression was observed from 8.5 days p.c., in the neural tube with a sharp border in the hind brain at the level of rhombomeres 4-5. In contrast, the earliest antisense expression was detected at 10.5 days p.c. in cDNA libraries. At 12.5 days p.c., sense and antisense transcripts colocalized in the liver. The possible role of antisense homeobox transcripts during liver and the hematopoietic development is discussed.

Retention and loss of immunoglobulin heavy chain alleles in helper T cell hybridoma clones.

Retention or loss of immunoglobulin heavy chain genes was studied in 20 functional T cell hybridoma clones. DNA probes representing C mu, C alpha and JH genes, as well as VH subgroups II and III were hybridized with restriction enzyme fragments of hybridoma DNA by the Southern filter hybridization technique. Parental alleles of the hybridoma cells were distinguished on the basis of polymorphism of the lengths of restriction enzyme fragments. All clones retained the alleles of the lymphoma parent cell BW-5147 at all four loci. Thirteen clones lost both CH and VH alleles of the immune partner cell, whereas seven retained both VH alleles, and at least C alpha of the antigen-specific partner. Hence, T cell function in these cells is compatible with the loss of most immunoglobulin heavy chain alleles. This is interpreted to indicate either gene rearrangement and deletion, or chromosome loss. Accordingly, the T cell receptor is either controlled by two split gene loci in chromosome 12, at the two respective (5' and 3') ends of the mouse heavy chain gene family, or by a gene(s) outside chromosome 12.

Fgfr2 is required for limb outgrowth and lung-branching morphogenesis.

The aim of this study was to clarify the role of Fgfr2 during later stages of embryonic development. Of two previously reported gene-targeting experiments, the more extensive Fgfr2 deletion was lethal shortly after implantation, because of trophoblast defects, whereas the less extensive one survived until midgestation with placental insufficiency and defective limb outgrowth [Xu, X., Weinstein, M., Li, C., Naski, M., Cohen, R. I., Ornitz, D. M., Leder, P. & Deng, C. (1998) Development (Cambridge, U.K.) 125, 753-765]. Fgfr2 in the early embryo is expressed in the trophectoderm, and this extra-embryonic localization persists into mid- and late gestation, when Fgfr2 also is expressed in multiple developing organs. To gain insight into the later functions of Fgfr2, fusion chimeras were constructed from homozygous mutant embryonic stem cells and wild-type tetraploid embryos. This allowed survival until term and revealed that Fgfr2 is required for both limb outgrowth and branching lung morphogenesis. The use of fusion chimeras demonstrated that early lethality was indeed because of trophectoderm defects and indicated that in the embryonic cell lineages Fgfr2 activity manifests in limb and lung development. Highly similar lung and limb phenotypes were detected recently in the loss of function mutation of Fgf10, a ligand of Fgfr2. It is likely, therefore, that whereas during early development Fgfr2 interacts with Fgf4, in limb and lung development interactions between Fgf10 and Fgfr2 may be required. Possible epithelial-mesenchymal interactions between the splicing alternatives of Fgfr2 and their specific ligands will be discussed.

New murine homeoboxes: structure, chromosomal assignment, and differential expression in adult erythropoiesis.

The nucleotide sequence, chromosomal assignment, and preliminary transcriptional analysis of four murine homeoboxes is presented. Three of these are linked to the Hox-2 gene complex on chromosome 11, whereas the fourth, Hox-4, was assigned to mouse chromosome 12. A comparative analysis of homeobox sequences reveals that two of our sequences represent the previously described Hox-2.3 loci, whereas a third, mh19, could represent the predicted Hox-2.6 locus. Homeoboxes Hox-2.2 and Hox-2.3 are the cognates of two previously reported human homeoboxes that belong to a similar gene cluster on a closely related human chromosome (Chr 17), suggesting that homeoboxes may have been preserved as clusters during evolution. Moreover Hox-4, mh19, and the previously described Hox-1.5 homeobox form a separate subgroup of mammalian homeoboxes (90-92% amino acid and nucleotide homology). All four homeoboxes are expressed in the mouse embryo. Of special interest is the expression of mh19, a 4.2-kb transcript of which appears to be connected to the induced differentiation of Friend erythroleukemia cells.

Targeted disruption of fibroblast growth factor (FGF) receptor 2 suggests a role for FGF signaling in pregastrulation mammalian development.

We disrupted the fibroblast growth factor (FGF) receptor 2 (FGFR2) gene by introducing a neo cassette into the IIIc ligand binding exon and by deleting a genomic DNA fragment encoding its transmembrane domain and part of its kinase I domain. A recessive embryonic lethal mutation was obtained. Preimplantation development was normal until the blastocyst stage. Homozygous mutant embryos died a few hours after implantation at a random position in the uterine crypt, with collapsed yolk cavity. Mutant blastocysts hatched, adhered, and formed a layer of trophoblast giant cells in vitro, but after prolonged culture, the growth of the inner cell mass stopped, no visceral endoderm formed, and finally the egg cylinder disintegrated. It follows that FGFR2 is required for early postimplantation development between implantation and the formation of the egg cylinder. We suggest that FGFR2 contributes to the outgrowth, differentiation, and maintenance of the inner cell mass and raise the possibility that this activity is mediated by FGF4 signals transmitted by FGFR2. The role of early FGF signaling in pregastrulation development as a possible adaptation to mammalian (amniote) embryogenesis is discussed.

Akt/PKB regulates laminin and collagen IV isotypes of the basement membrane.

Basement membranes are important for epithelial differentiation, cell survival, and normal and metastatic cell migration. Much is known about their breakdown and remodeling, yet their positive regulation is poorly understood. Our previous analysis of a fibroblast growth factor (FGF) receptor mutation raised the possibility that protein kinase B (Akt/PKB) activated by FGF is connected to the expression of certain laminin and type IV collagen isotypes. Here we test this hypothesis and demonstrate that constitutively active Akt/PKB, an important downstream element of phosphoinositide 3'-kinase signaling, induces the synthesis of laminin-1 and collagen IV isotypes and causes their translocation to the basement membrane. By using promoter-reporter constructs, we show that constitutively active phosphoinositide 3'-kinase-p110 or Akt/PKB activates, whereas dominant negative Akt/PKB inhibits, transcription of laminin beta1 and collagen IV alpha1 in differentiating C2 myoblast- and insulin-induced Chinese hamster ovary-T cell cultures. These results suggest that Akt/PKB activated by receptor tyrosine kinases is involved in the positive regulation of basement membrane formation. The possible role of Akt/PKB-induced laminin and collagen IV synthesis in cell survival and differentiation will be discussed.

Developmental localization of the splicing alternatives of fibroblast growth factor receptor-2 (FGFR2).

The gene for fibroblast growth factor receptor-2 (FGFR2) encodes two splice variants designated here as keratinocyte growth factor (KGFR) and bek. Their ligand-binding specificity is markedly different due to mutually exclusive alternative splicing. We asked whether alternative exon usage, in addition to influencing receptor specificity, could be correlated with transcriptional localization. This problem was studied by in situ hybridization and PCR, using probes and primers specific for the alternative exons of FGFR2. Transcripts of both variants were detected in all three germ layers within the embryonic and the extraembryonic areas of the primitive-streak embryo. The overall level of KGFR expression surpassed that of bek. The localized expression of both variant receptors was, however, more diffuse in the gastrula than later during organogenesis, when KGFR transcripts were evident mainly in epithelia, whereas bek was present in the corresponding mesenchymes. Our findings show the following: (1) Expression of both FGFR2 variants is concordant with their involvement in murine gastrulation. They may endow competence to multiple areas, which may be restricted by their more confined ligands. (2) KGFR and bek seem to have unique roles in the development of the skin and its derivatives, whereas bek is preferentially expressed during osteogenesis. The two variants share potential regions of trans regulation in the genome; hence, we suggest that alternative splicing is jointly responsible for ligand binding and spatial specificity. (3) Finally, we defined the binding specificity of KGFR and bek to various FGF. The possibility of identifying specific functional areas for certain ligand-receptor pairs is discussed.