A small-molecule probe to decipher stress-induced ER microenvironments and ER-Golgi communication.

Cellular stress is a crucial factor in regulating and maintaining both organismal and microenvironmental homeostasis. It induces a response that also affects the micropolarity of specific cellular compartments, which is essential for early disease diagnosis. In this contribution, we present a quantitative study of micropolarity changes inside the endoplasmic reticulum (ER) during the G1/S and G2/M phases, using a biocompatible small-molecule fluorophore called ER-Oct. This probe is selectively driven to the ER by its hydrophobicity, and it has the fastest diffusion properties among a series of analogous probes. We found that induced ER stress caused cell cycle arrests leading to an increase in ER micropolarity which is well supported by lambda scanning experiments and fluorescence lifetime imaging microscopy (FLIM) as well. ER-Oct is a versatile staining agent that could effectively stain the ER in various living/fixed mammalian cells, isolated ER, Caenorhabditis elegans, and mice tissues. Furthermore, we used this probe to visualize a well-known biological event, ER to Golgi transport, by live-cell fluorescence microscopy. Our exhaustive investigation of micropolarity using ER-staining dye provides a new way to study ER stress, which could provide a deeper understanding of proteostasis in model systems and even in fixed patient samples.

RiTe conjugate mediated corneal collagen crosslinking, a novel therapeutic intervention for keratoconus - in vitro and in vivo study.

Corneal collagen crosslinking (CXL) is an effective method to halt the disease progression of keratoconus, a progressive corneal dystrophy leading to cone shaped cornea. Despite the efficacy of standard protocol, the concerning step of this procedure is epithelial debridement performed to facilitate the entry of riboflavin drug. Riboflavin, a key molecule in CXL protocol, is a sparsely permeable hydrophilic drug in corneal tissues. The present study has employed cell penetrating peptide (CPP), Tat2, to enhance the penetration of riboflavin molecule, and thereby improve currently followed CXL protocol. This study demonstrates approximately two-fold enhanced uptake of CPP riboflavin conjugate, Tat2riboflavin-5'Phosphate (RiTe conjugate), both in vitro and in vivo. Two different CXL protocols (Epi ON and Epi OFF) have been introduced and implemented in rabbit corneas using RiTe conjugate in the present study. The standard and RiTe conjugate mediated CXL procedures exhibited an equivalent extent of crosslinking in both the methods. Reduced keratocyte loss and no endothelial damage in RiTe conjugate mediated CXL further ascertains the safety of the proposed CXL protocols. Therefore, RiTe conjugate mediated CXL protocols present as potential alternatives to the standard keratoconus treatment in providing equally effective, less invasive and patient compliant treatment modality.

Characterization of Vps13b-mutant mice reveals neuroanatomical and behavioral phenotypes with females less affected.

The vacuolar protein sorting-associated protein 13B (VPS13B) is a large and highly conserved protein. Disruption of VPS13B causes the autosomal recessive Cohen syndrome, a rare disorder characterized by microcephaly and intellectual disability among other features, including developmental delay, hypotonia, and friendly-personality. However, the underlying mechanisms by which VPS13B disruption leads to brain dysfunction still remain unexplained. To gain insights into the neuropathogenesis of Cohen syndrome, we systematically characterized brain changes in Vps13b-mutant mice and compared murine findings to 235 previously published and 17 new patients diagnosed with VPS13B-related Cohen syndrome. We showed that Vps13b is differentially expressed across brain regions with the highest expression in the cerebellum, the hippocampus and the cortex with postnatal peak. Half of the Vps13b-/- mice die during the first week of life. The remaining mice have a normal lifespan and display the core phenotypes of the human disease, including microcephaly, growth delay, hypotonia, altered memory, and enhanced sociability. Systematic 2D and 3D brain histo-morphological analyses reveal specific structural changes in the brain starting after birth. The dentate gyrus is the brain region with the most prominent reduction in size, while the motor cortex is specifically thinner in layer VI. The fornix, the fasciculus retroflexus, and the cingulate cortex remain unaffected. Interestingly, these neuroanatomical changes implicate an increase of neuronal death during infantile stages with no progression in adulthood suggesting that VPS13B promotes neuronal survival early in life. Importantly, whilst both sexes were affected, some neuroanatomical and behavioral phenotypes were less pronounced or even absent in females. We evaluate sex differences in Cohen patients and conclude that females are less affected both in mice and patients. Our findings provide new insights about the neurobiology of VPS13B and highlight previously unreported brain phenotypes while defining Cohen syndrome as a likely new entity of non-progressive infantile neurodegeneration.

Cancer plasticity: Investigating the causes for this agility.

Cancer is not a hard-wired phenomenon but an evolutionary disease. From the onset of carcinogenesis, cancer cells continuously adapt and evolve to satiate their ever-growing proliferation demands. This results in the formation of multiple subtypes of cancer cells with different phenotypes, cellular compositions, and consequently displaying varying degrees of tumorigenic identity and function. This phenomenon is referred to as cancer plasticity, during which the cancer cells exist in a plethora of cellular states having distinct phenotypes. With the advent of modern technologies equipped with enhanced resolution and depth, for example, single-cell RNA-sequencing and advanced computational tools, unbiased cancer profiling at a single-cell resolution are leading the way in understanding cancer cell rewiring both spatially and temporally. In this review, the processes and mechanisms that give rise to cancer plasticity include both intrinsic genetic factors such as epigenetic changes, differential expression due to changes in DNA, RNA, or protein content within the cancer cell, as well as extrinsic environmental factors such as tissue perfusion, extracellular milieu are detailed and their influence on key cancer plasticity hallmarks such as epithelial-mesenchymal transition (EMT) and cancer cell stemness (CSCs) are discussed. Due to therapy evasion and drug resistance, tumor heterogeneity caused by cancer plasticity has major therapeutic ramifications. Hence, it is crucial to comprehend all the cellular and molecular mechanisms that control cellular plasticity. How this process evades therapy, and the therapeutic avenue of targeting cancer plasticity must be diligently investigated.

A Comprehensive narrative review of transcriptomics and epigenomics of gallbladder cancer.

ABSTRACTS: Gallbladder cancer (GBC) is one of the quiet prevalent and aggressive biliary tract malignant neoplasms distinguished by significant cellular heterogeneity, metastatic activity, and a poor prognosis, with varied frequency worldwide. Most cases are detected incidentally while routine screening imaging or pathological investigation of cholecystectomy tissues and usually present with advanced disease. The surgical resection is usually done in the initial clinical stage having limited spread. Despite the surgical therapy, the death rate is significant. Furthermore, the molecular mechanisms affecting the clinical course of inflammatory gallbladder to carcinogenesis remain poorly understood. There is an impending need for developing diagnostic biomarkers and targeted approaches for GBC. The newer molecular platform, such as next-generation sequencing (NGS), such as RNA-sequencing (RNAseq), single-cell sequencing, and microarray technology, has revolutionized the field of genomics, opened a new perspective in defining genetic and epigenetic characteristics identifying molecules as possible therapeutic targets. Therefore, in this review, we would analyze transcriptomic and epigenomics profiles of GBC using already published high-throughput sequencing-based studies published between 2010 and 2023. The review would also analyze the possible impact of the technological advancement on the patient management strategy and overall survival. This may also help identify target genes and pathways linked to GBC, which may help establish molecular biomarkers, for early GBC diagnosis, personalized therapy, and management.

Cortical and Commissural Defects Upon HCF-1 Loss in Nkx2.1-Derived Embryonic Neurons and Glia.

Formation of the cerebral cortex and commissures involves a complex developmental process defined by multiple molecular mechanisms governing proliferation of neuronal and glial precursors, neuronal and glial migration, and patterning events. Failure in any of these processes can lead to malformations. Here, we study the role of HCF-1 in these processes. HCF-1 is a conserved metazoan transcriptional co-regulator long implicated in cell proliferation and more recently in human metabolic disorders and mental retardation. Loss of HCF-1 in a subset of ventral telencephalic Nkx2.1-positive progenitors leads to reduced numbers of GABAergic interneurons and glia, owing not to decreased proliferation but rather to increased apoptosis before cell migration. The loss of these cells leads to development of severe commissural and cortical defects in early postnatal mouse brains. These defects include mild and severe structural defects of the corpus callosum and anterior commissure, respectively, and increased folding of the cortex resembling polymicrogyria. Hence, in addition to its well-established role in cell proliferation, HCF-1 is important for organ development, here the brain.

Rapid Recapitulation of Nonalcoholic Steatohepatitis upon Loss of Host Cell Factor 1 Function in Mouse Hepatocytes

Host-cell factor 1 (HCF-1), encoded by the ubiquitously expressed X-linked gene Hcfc1, is an epigenetic coregulator important for mouse development and cell proliferation, including during liver regeneration. We used a hepatocyte-specific inducible Hcfc1 knock-out allele (called Hcfc1hepKO), to induce HCF-1 loss in hepatocytes of hemizygous Hcfc1hepKO/Y males by four days. In heterozygous Hcfc1hepKO/+ females, owing to random X-chromosome inactivation, upon Hcfc1hepKO allele induction, a 50/50 mix of HCF-1 positive and negative hepatocyte clusters is engineered. The livers with Hcfc1hepKO/Y hepatocytes displayed a 21-24-day terminal non-alcoholic fatty liver (NAFL) followed by non-alcoholic steatohepatitis (NASH) disease progression typical of severe NAFL disease (NAFLD). In contrast, in livers with heterozygous Hcfc1hepKO/+ hepatocytes, HCF-1-positive hepatocytes replaced HCF-1-negative hepatocytes and revealed only mild-NAFL development. Loss of HCF-1 led to loss of PGC1α protein, probably owing to its destabilization, and deregulation of gene expression particularly of genes involved in mitochondrial structure and function, likely explaining the severe Hcfc1 hepKO/Y liver pathology. Thus, HCF-1 is essential for hepatocyte function, likely playing both transcriptional and non-transcriptional roles. These genetically-engineered loss-of-HCF-1 mice can be used to study NASH as well as NAFLD resolution.

Cycles of gene expression and genome response during mammalian tissue regeneration.

BACKGROUND: Compensatory liver hyperplasia-or regeneration-induced by two-thirds partial hepatectomy (PH) permits the study of synchronized activation of mammalian gene expression, particularly in relation to cell proliferation. Here, we measured genomic transcriptional responses and mRNA accumulation changes after PH and sham surgeries. RESULTS: During the first 10-20 h, the PH- and sham-surgery responses were very similar, including parallel early activation of cell-division-cycle genes. After 20 h, however, whereas post-PH livers continued with a robust and coordinate cell-division-cycle gene-expression response before returning to the resting state by 1 week, sham-surgery livers returned directly to a resting gene-expression state. Localization of RNA polymerase II (Pol II), and trimethylated histone H3 lysine 4 (H3K4me3) and 36 (H3K36me3) on genes dormant in the resting liver and activated during the PH response revealed a general de novo promoter Pol II recruitment and H3K4me3 increase during the early 10-20 h phase followed by Pol II elongation and H3K36me3 accumulation in gene bodies during the later proliferation phase. H3K36me3, generally appearing at the first internal exon, was preceded 5' by H3K36me2; 3' of the first internal exon, in about half of genes H3K36me3 predominated and in the other half H3K36me2 and H3K36me3 co-existed. Further, we observed some unusual gene profiles with abundant Pol II but little evident H3K4me3 or H3K36me3 modification, indicating that these modifications are neither universal nor essential partners to Pol II transcription. CONCLUSIONS: PH and sham surgical procedures on mice reveal striking early post-operatory gene expression similarities followed by synchronized mRNA accumulation and epigenetic histone mark changes specific to PH.

WD40-repeat 47, a microtubule-associated protein, is essential for brain development and autophagy.

The family of WD40-repeat (WDR) proteins is one of the largest in eukaryotes, but little is known about their function in brain development. Among 26 WDR genes assessed, we found 7 displaying a major impact in neuronal morphology when inactivated in mice. Remarkably, all seven genes showed corpus callosum defects, including thicker (Atg16l1, Coro1c, Dmxl2, and Herc1), thinner (Kif21b and Wdr89), or absent corpus callosum (Wdr47), revealing a common role for WDR genes in brain connectivity. We focused on the poorly studied WDR47 protein sharing structural homology with LIS1, which causes lissencephaly. In a dosage-dependent manner, mice lacking Wdr47 showed lethality, extensive fiber defects, microcephaly, thinner cortices, and sensory motor gating abnormalities. We showed that WDR47 shares functional characteristics with LIS1 and participates in key microtubule-mediated processes, including neural stem cell proliferation, radial migration, and growth cone dynamics. In absence of WDR47, the exhaustion of late cortical progenitors and the consequent decrease of neurogenesis together with the impaired survival of late-born neurons are likely yielding to the worsening of the microcephaly phenotype postnatally. Interestingly, the WDR47-specific C-terminal to LisH (CTLH) domain was associated with functions in autophagy described in mammals. Silencing WDR47 in hypothalamic GT1-7 neuronal cells and yeast models independently recapitulated these findings, showing conserved mechanisms. Finally, our data identified superior cervical ganglion-10 (SCG10) as an interacting partner of WDR47. Taken together, these results provide a starting point for studying the implications of WDR proteins in neuronal regulation of microtubules and autophagy.

Crucial roles of Pox neuro in the developing ellipsoid body and antennal lobes of the Drosophila brain.

The paired box gene Pox neuro (Poxn) is expressed in two bilaterally symmetric neuronal clusters of the developing adult Drosophila brain, a protocerebral dorsal cluster (DC) and a deutocerebral ventral cluster (VC). We show that all cells that express Poxn in the developing brain are postmitotic neurons. During embryogenesis, the DC and VC consist of only 20 and 12 neurons that express Poxn, designated embryonic Poxn-neurons. The number of Poxn-neurons increases only during the third larval instar, when the DC and VC increase dramatically to about 242 and 109 Poxn-neurons, respectively, virtually all of which survive to the adult stage, while no new Poxn-neurons are added during metamorphosis. Although the vast majority of Poxn-neurons express Poxn only during third instar, about half of them are born by the end of embryogenesis, as demonstrated by the absence of BrdU incorporation during larval stages. At late third instar, embryonic Poxn-neurons, which begin to express Poxn during embryogenesis, can be easily distinguished from embryonic-born and larval-born Poxn-neurons, which begin to express Poxn only during third instar, (i) by the absence of Pros, (ii) their overt differentiation of axons and neurites, and (iii) the strikingly larger diameter of their cell bodies still apparent in the adult brain. The embryonic Poxn-neurons are primary neurons that lay out the pioneering tracts for the secondary Poxn-neurons, which differentiate projections and axons that follow those of the primary neurons during metamorphosis. The DC and the VC participate only in two neuropils of the adult brain. The DC forms most, if not all, of the neurons that connect the bulb (lateral triangle) with the ellipsoid body, a prominent neuropil of the central complex, while the VC forms most of the ventral projection neurons of the antennal lobe, which connect it ipsilaterally to the lateral horn, bypassing the mushroom bodies. In addition, Poxn-neurons of the VC are ventral local interneurons of the antennal lobe. In the absence of Poxn protein in the developing brain, embryonic Poxn-neurons stall their projections and cannot find their proper target neuropils, the bulb and ellipsoid body in the case of the DC, or the antennal lobe and lateral horn in the case of the VC, whereby the absence of the ellipsoid body neuropil is particularly striking. Poxn is thus crucial for pathfinding both in the DC and VC. Additional implications of our results are discussed.

Nkx2.1 regulates the generation of telencephalic astrocytes during embryonic development.

The homeodomain transcription factor Nkx2.1 (NK2 homeobox 1) controls cell differentiation of telencephalic GABAergic interneurons and oligodendrocytes. Here we show that Nkx2.1 also regulates astrogliogenesis of the telencephalon from embryonic day (E) 14.5 to E16.5. Moreover we identify the different mechanisms by which Nkx2.1 controls the telencephalic astrogliogenesis. In Nkx2.1 knockout (Nkx2.1-/-) mice a drastic loss of astrocytes is observed that is not related to cell death. Further, in vivo analysis using BrdU incorporation reveals that Nkx2.1 affects the proliferation of the ventral neural stem cells that generate early astrocytes. Also, in vitro neurosphere assays showed reduced generation of astroglia upon loss of Nkx2.1, which could be due to decreased precursor proliferation and possibly defects in glial specification/differentiation. Chromatin immunoprecipitation analysis and in vitro co-transfection studies with an Nkx2.1-expressing plasmid indicate that Nkx2.1 binds to the promoter of glial fibrillary acidic protein (GFAP), primarily expressed in astrocytes, to regulate its expression. Hence, Nkx2.1 controls astroglial production spatiotemporally in embryos by regulating proliferation of the contributing Nkx2.1-positive precursors.

Segregated hepatocyte proliferation and metabolic states within the regenerating mouse liver.

Mammalian partial hepatectomy (PH) induces an orchestrated compensatory hyperplasia, or regeneration, in remaining tissue to restore liver mass; during this process, liver functions are maintained. We probed this process in mice with feeding- and light/dark-entrained animals subjected to sham or PH surgery. Early on (i.e., 10 hours), irrespective of sham or PH surgery, hepatocytes equidistant from the portal and central veins (i.e., midlobular) accumulated the G1-phase cell-division-cycle marker cyclin D1. By 24 hours, however, cyclin D1 disappeared absent PH but was reinforced in midlobular hepatocytes after PH. At 48 hours after PH and 2 hours fasting, synchronously mitotic hepatocytes possessed less glycogen than surrounding nonproliferating hepatocytes. The differential glycogen content generated a conspicuous entangled pattern of proliferating midlobular and nonproliferating periportal and pericentral hepatocytes. The nonproliferating hepatocytes maintained aspects of normal liver properties. Conclusion: In the post-PH regenerating mouse liver, a binary switch segregates midlobular cells to proliferate side-by-side with nonproliferating periportal and pericentral cells, which maintain metabolic functions. Our results also indicate that mechanisms of liver regeneration display evolutionary flexibility. (Hepatology Communications 2017;1:871-885).

Epiblast-specific loss of HCF-1 leads to failure in anterior-posterior axis specification.

Mammalian Host-Cell Factor 1 (HCF-1), a transcriptional co-regulator, plays important roles during the cell-division cycle in cell culture, embryogenesis as well as adult tissue. In mice, HCF-1 is encoded by the X-chromosome-linked Hcfc1 gene. Induced Hcfc1(cKO/+) heterozygosity with a conditional knockout (cKO) allele in the epiblast of female embryos leads to a mixture of HCF-1-positive and -deficient cells owing to random X-chromosome inactivation. These embryos survive owing to the replacement of all HCF-1-deficient cells by HCF-1-positive cells during E5.5 to E8.5 of development. In contrast, complete epiblast-specific loss of HCF-1 in male embryos, Hcfc1(epiKO/Y), leads to embryonic lethality. Here, we characterize this lethality. We show that male epiblast-specific loss of Hcfc1 leads to a developmental arrest at E6.5 with a rapid progressive cell-cycle exit and an associated failure of anterior visceral endoderm migration and primitive streak formation. Subsequently, gastrulation does not take place. We note that the pattern of Hcfc1(epiKO/Y) lethality displays many similarities to loss of ß-catenin function. These results reveal essential new roles for HCF-1 in early embryonic cell proliferation and development.

Compensatory embryonic response to allele-specific inactivation of the murine X-linked gene Hcfc1.

Early in female mammalian embryonic development, cells randomly inactivate one of the two X chromosomes to achieve overall equal inactivation of parental X-linked alleles. Hcfc1 is a highly conserved X-linked mouse gene that encodes HCF-1 - a transcriptional co-regulator implicated in cell proliferation in tissue culture cells. By generating a Cre-recombinase inducible Hcfc1 knock-out (Hcfc1(lox)) allele in mice, we have probed the role of HCF-1 in actively proliferating embryonic cells and in cell-cycle re-entry of resting differentiated adult cells using a liver regeneration model. HCF-1 function is required for both extraembryonic and embryonic development. In heterozygous Hcfc1(lox/+) female embryos, however, embryonic epiblast-specific Cre-induced Hcfc1 deletion (creating an Hcfc1(epiKO) allele) around E5.5 is well tolerated; it leads to a mixture of HCF-1-positive and -negative epiblast cells owing to random X-chromosome inactivation of the wild-type or Hcfc1(epiKO) mutant allele. At E6.5 and E7.5, both HCF-1-positive and -negative epiblast cells proliferate, but gradually by E8.5, HCF-1-negative cells disappear owing to cell-cycle exit and apoptosis. Although generating a temporary developmental retardation, the loss of HCF-1-negative cells is tolerated, leading to viable heterozygous offspring with 100% skewed inactivation of the X-linked Hcfc1(epiKO) allele. In resting adult liver cells, the requirement for HCF-1 in cell proliferation was more evident as hepatocytes lacking HCF-1 fail to re-enter the cell cycle and thus to proliferate during liver regeneration. The survival of the heterozygous Hcfc1(epiKO/+) female embryos, even with half the cells genetically compromised, illustrates the developmental plasticity of the post-implantation mouse embryo - in this instance, permitting survival of females heterozygous for an X-linked embryonic lethal allele.

NG2 glia are required for vessel network formation during embryonic development.

The NG2(+) glia, also known as polydendrocytes or oligodendrocyte precursor cells, represent a new entity among glial cell populations in the central nervous system. However, the complete repertoire of their roles is not yet identified. The embryonic NG2(+) glia originate from the Nkx2.1(+) progenitors of the ventral telencephalon. Our analysis unravels that, beginning from E12.5 until E16.5, the NG2(+) glia populate the entire dorsal telencephalon. Interestingly, their appearance temporally coincides with the establishment of blood vessel network in the embryonic brain. NG2(+) glia are closely apposed to developing cerebral vessels by being either positioned at the sprouting tip cells or tethered along the vessel walls. Absence of NG2(+) glia drastically affects the vascular development leading to severe reduction of ramifications and connections by E18.5. By revealing a novel and fundamental role for NG2(+) glia, our study brings new perspectives to mechanisms underlying proper vessels network formation in embryonic brains.

Nkx2.1-derived astrocytes and neurons together with Slit2 are indispensable for anterior commissure formation.

Guidepost cells present at and surrounding the midline provide guidance cues that orient the growing axons through commissures. Here we show that the transcription factor Nkx2.1 known to control the specification of GABAergic interneurons also regulates the differentiation of astroglia and polydendrocytes within the mouse anterior commissure (AC). Nkx2.1-positive glia were found to originate from three germinal regions of the ventral telencephalon. Nkx2.1-derived glia were observed in and around the AC region by E14.5. Thereafter, a selective cell ablation strategy showed a synergistic role of Nkx2.1-derived cells, both GABAergic interneurons and astroglia, towards the proper formation of the AC. Finally, our results reveal that the Nkx2.1-regulated cells mediate AC axon guidance through the expression of the repellent cue, Slit2. These results bring forth interesting insights about the spatial and temporal origin of midline telencephalic glia, and highlight the importance of neurons and astroglia towards the formation of midline commissures.

Two specific populations of GABAergic neurons originating from the medial and the caudal ganglionic eminences aid in proper navigation of callosal axons.

The corpus callosum (CC) plays a crucial role in interhemispheric communication. It has been shown that CC formation relies on the guidepost cells located in the midline region that include glutamatergic and GABAergic neurons as well as glial cells. However, the origin of these guidepost GABAergic neurons and their precise function in callosal axon pathfinding remain to be investigated. Here, we show that two distinct GABAergic neuronal subpopulations converge toward the midline prior to the arrival of callosal axons. Using in vivo and ex vivo fate mapping we show that CC GABAergic neurons originate in the caudal and medial ganglionic eminences (CGE and MGE) but not in the lateral ganglionic eminence (LGE). Time lapse imaging on organotypic slices and in vivo analyses further revealed that CC GABAergic neurons contribute to the normal navigation of callosal axons. The use of Nkx2.1 knockout (KO) mice confirmed a role of these neurons in the maintenance of proper behavior of callosal axons while growing through the CC. Indeed, using in vitro transplantation assays, we demonstrated that both MGE- and CGE-derived GABAergic neurons exert an attractive activity on callosal axons. Furthermore, by combining a sensitive RT-PCR technique with in situ hybridization, we demonstrate that CC neurons express multiple short and long range guidance cues. This study strongly suggests that MGE- and CGE-derived interneurons may guide CC axons by multiple guidance mechanisms and signaling pathways.