Temporally regulated expression of Lin-28 in diverse tissues of the developing mouse

The gene lin-28 was originally identified through a mutant of the nematode Caenorhabditis elegans displaying defects in developmental timing. It is expressed stage-specifically in tissues throughout the animal and is required for cell fates to be expressed at the appropriate stage of larval development. lin-28 encodes a cytoplasmic protein with a unique pairing of RNA-binding motifs. Diverse animals possess Lin-28 homologues and mouse Lin-28 is expressed in embryos, embryonic stem cells and embryonal carcinoma cells, but not in some differentiated cell types. To assess whether mammalian Lin-28 may function as a developmental timing regulator, we examined adult and embryonic tissues of the mouse for its expression. We observed Lin-28 protein in many diverse tissues of the embryo through the period of organogenesis and that it persists in some tissues in the adult. In addition to an overall down-regulation during embryogenesis, in at least two tissues Lin-28 expression shows temporal regulation, as opposed to cell type or tissue-specific regulation: in the developing bronchial epithelium, where it is present in the developing lung and absent in the adult, and in a subset of cells developing along the crypt-villus axis of the intestine. Interestingly, unlike epithelia, cardiac and skeletal muscle continuously express Lin-28, suggesting an ongoing need for its activity there. We also observed that Lin-28 expression is repressed during the retinoic acid-induced differentiation of mouse P19 cells into neuronal cells, suggesting that down-regulation of Lin-28 in some tissues may occur in response to hormonal signals that govern development.     

Involvement of EAT/mcl-1, an anti-apoptotic bcl-2-related gene, in murine embryogenesis and human development

Apoptosis plays an important regulatory role in mammalian embryogenesis and development. EAT/mcl-1 (EAT), an anti-apoptotic bcl-2-related gene, was isolated during the early differentiation of a human embryonal carcinoma cell line, an event which serves as a model of early embryogenesis. EAT is involved in apoptotic regulation and is believed to also function as an immediate-early gene. Thus it was hypothesized that EAT would be expressed during early embryogenesis and would be involved in the regulation of apoptosis during this critical period. To clarify this early expression, two antibodies to EAT were generated by use of immunizing oligopeptide (aa 37-55) and recombinant protein (aa 31-229) for use in immunohistochemistry and immunoblotting, respectively. With these antibodies, we then determined EAT expression during murine embryogenesis and in human development, using human fetal tissue of 6 to 23 gestational weeks. During murine embryogenesis, the EAT protein was found to be rapidly induced after fertilization, to peak at the 2-cell stage, to remain constant until the 8-cell stage, and then to decrease to below unfertilized egg levels in blastocysts. EAT expression patterns in early human development were found to essentially overlap those observed in adult tissues which suggest that EAT expression continues until adulthood in terminally differentiated tissues. Among tissues distinct to fetal development, EAT was detected in the mesonephric (Wolffian) duct and paramesonephric (Müllerian) duct. It is also noteworthy that prominent EAT immunoreactivity was also observed in large primary oocytes in 21-week fetal ovary, but was not detected in primordial germ cells in 23-week fetal testis. In summary, EAT expression was detected in hematopoietic, epithelial, neural, endocrine, and urogenital cells; this provides evidence that EAT, as an anti-apoptotic molecule, possibly functions to regulate apoptosis during development in these systems.     

Developmentally regulated expression of hemoglobin subunits in avascular tissues

We investigated the spatio-temporal profile of hemoglobin subunit expression in developing avascular tissues. Significant up-regulation of hemoglobin subunits was identified in microarray experiments comparing blastocyst inner cell masses with undifferentiated embryonic stem (ES) cells. Hemoglobin expression changes were confirmed using embryoid bodies (derived from in vitro differentiation of ES cells) to model very early development at pre-vascular stages of embryogenesis; i.e. prior to hematopoiesis. We also demonstrate, using RT-PCR, Western blotting and immunocytochemistry, expression of adult and fetal mouse hemoglobin subunits in the avascular ocular lens at various stages of development and maturation. Hemoglobin proteins were expressed in lens epithelial cells (cytoplasmic) and cortical lens fiber cells (nuclear and cell-surface-associated); however, a sensitive heme assay demonstrated negligible levels of heme in the developing lens postnatally. Hemoglobin expression was also observed in the developing eye in corneal endothelium and retinal ganglion cells. Gut sections showed, in addition to erythrocytes, hemoglobin protein staining in rare, individual villus epithelial cells. These results suggest a paradigm shift: hemoglobin subunits are expressed in the avascular lens and cornea and in pre-hematopoietic embryos. It is likely, therefore, that hemoglobin subunits have novel developmental roles; the absence of the heme group from the lens would indicate that at least some of these functions may be independent of oxygen metabolism. The pattern of expression of hemoglobin subunits in the perinuclear region during lens fiber cell differentiation, when denucleation is taking place, may indicate involvement in the apoptosis-like signaling processes occurring in differentiating lens fiber cells.     

Unfolded Protein Response (UPR) is activated during normal lens development

The lens of the eye is a transparent structure responsible for focusing light onto the retina. It is composed of two morphologically different cell types, epithelial cells found on the anterior surface and the fiber cells that are continuously formed by the differentiation of epithelial cells at the lens equator. The differentiation of an epithelial precursor cell into a fiber cell is associated with a dramatic increase in membrane protein synthesis. How the terminally differentiating fiber cells cope with the increased demand on the endoplasmic reticulum for this membrane protein synthesis is not known. In the present study, we have found evidence of Unfolded Protein Response (UPR) activation during normal lens development and differentiation in the mouse. The ER-resident chaperones, immunoglobulin heavy chain binding protein (BiP) and protein disulfide isomerase (PDI), were expressed at high levels in the newly forming fiber cells of embryonic lenses. These fiber cells also expressed the UPR-associated molecules; XBP1, ATF6, phospho-PERK and ATF4 during embryogenesis. Moreover, spliced XBP1, cleaved ATF6, and phospho-eIF2α were detected in embryonic mouse lenses suggesting that UPR pathways are active in this tissue. These results propose a role for UPR activation in lens fiber cell differentiation during embryogenesis.     

Embryonic expression profile of phospholipid hydroperoxide glutathione peroxidase

The selenoenzyme phospholipid hydroperoxide glutathione peroxidase (PHGPx) is indispensable for murine embryonic development; yet, the cellular mechanisms leading to embryonic death around gastrulation are still unclear. To investigate PHGPx expression patterns during embryogenesis, we performed a detailed analysis that revealed a complex expression profile. Up to embryonic day 9.5, PHGPx was ubiquitously expressed, which was, albeit to a lower extent, maintained throughout later stages of embryogenesis. Notably, strong expression was frequently observed in epithelial tissue. A transient increase in PHGPx expression was detected in developing tissues, suggesting a crucial role for PHGPx in proliferation and differentiation. By semi-quantitative RT-PCR analysis we observed that the cytosolic form of PHGPx was present in embryonic and somatic tissues whereas the mitochondrial and nuclear forms were detectable only in testicular tissue. This strongly suggests that it is the cytosolic form of PHGPx that is indispensable for embryonic development.