c-Rel Regulates Inscuteable Gene Expression during Mouse Embryonic Stem Cell Differentiation

Abstract

Inscuteable (Insc) regulates cell fate decisions in several types of stem cells. Although it is recognized that the expression levels of mouse INSC govern the balance between symmetric and asymmetric stem cell division, regulation of mouse Insc gene expression remains poorly understood. Here, we showed that mouse Insc expression transiently increases at an early stage of differentiation, when mouse embryonic stem (mES) cells differentiate into bipotent mesendoderm capable of producing both endoderm and mesoderm in defined culture conditions. We identified the minimum transcriptional regulatory element (354 bases) that drives mouse Insc transcription in mES cells within a region >5 kb upstream of the mouse Insc transcription start site. We found that the transcription factor reticuloendotheliosis oncogene (c-Rel) bound to the minimum element and promoted mouse Insc expression in mES cells. In addition, short interfering RNA-mediated knockdown of either mouse INSC or c-Rel protein decreased mesodermal cell populations without affecting differentiation into the mesendoderm or endoderm. Furthermore, overexpression of mouse INSC rescued the mesoderm-reduced phenotype induced by knockdown of c-Rel. We propose that regulation of mouse Insc expression by c-Rel modulates cell fate decisions during mES cell differentiation.Insc was first identified as a novel neural precursor gene in Drosophila (1). Insc protein expression has been detected in embryonic areas where cell shape changes or movement occurs (i.e. neuroectoderm, midgut primordium, and muscle precursors) (1). More precise roles have emerged for Insc protein activity based on studies using neuroblasts, stem cells found in the central nervous system of Drosophila, which undergo asymmetric cell division (2-5). In neuroblasts, Insc localizes to the apical cell cortex by directly associating with bazooka-Par6-aPKC cell polarity protein complexes, whereas cell fate determinants, such as miranda (Mira), prospero (Pros), brain tumor (Brat), and Numb, localize to the basal cortex (6 -20). Alignment of the mitotic spindle along the apical-basal polarity axis drives asymmetric cell division to produce one self-renewing neuroblast and one ganglion mother cell fated for neural differentiation by asymmetrical inheritance of cell fate determinants (6,10,11,13,21,22). Insc plays a critical role in apical-basal spindle positioning by connecting spindle capturing machineries, consisting of partner of inscuteable (Pins) and mushroom body defect (Mud), with apical bazooka-Par6-aPKC cell polarity complexes (23-26).Similar molecular machineries are conserved in neural progenitors (27, 28) and skin basal cells of mice (29 -31), whereby mouse INSC (the mouse homologue of mammalian inscuteable) regulates spindle orientation and cell fate determination together with Par-3 (vertebrate homolog of Bazooka) and LGN (vertebrate counterpart of Pins) (27-31). Previous reports show that ectopic expression of mouse INSC promotes apical-basal spindle positioning in neuronal progenitors and keratin...

Transcriptional Control of Apical-Basal Polarity Regulators

“…Hereby, the NF-κB-familiy transcription factor reticuloendotheliosis oncogene (c-Rel) binds to the INSC promoter and induces INSC transcription. This leads to increased rates of asymmetric division, which ultimately promotes mesodermal cell fates [65].…”

Transcriptomic Analysis of Gene Expression and Effect of Electromagnetic Field in Brain Tissue after Traumatic Brain Injury

“…CFAP126 was also differentially expressed in single-cell profiling of induced pluripotent stem cell-derived cerebellar organoids [ 11 ]. Inscuteable (INSC) Spindle Orientation Adaptor Protein is another protein-coding gene that is involved in spindle orientation during mitosis, cell fate decision, regulates cell proliferation and differentiation, in the developing nervous system, and may function as an adapter linking the Par3 complex to the GPSM1/GPSM2 complex [ 12 , 13 ]. INSC expression significantly correlates negatively with the infiltration levels of various immune cells including B cells, CD4+ T cells, macrophages, and dendritic cells (DCs) [ 14 ].…”

Lowered Expression Level Of insc Predicts Poor Prognosis in Patients With Colon Cancer

“…To exemplify, reduced neurogenesis and defective cortical organization in association with loss of INSC expression are observed in mice. In contrast, INSC overexpression results in the expansion of the neuronal cell pool [29].…”