IN SITU LOCALIZATION OF GLOBIN MESSENGER RNA FORMATION

Abstract

Globin m R N A levels in 11-15-day mouse fetal liver cells have been estimated by in situ hybridization of a highly labeled D N A copy (cDNA) of adult globin messenger R N A s (mRNAs) (globin cDNA) to fixed preparations of cells. Under the conditions employed, no significant in situ hybridization occurred to lymphoma cells (L 51787), mouse L cells, or hepatocytes; whereas reticulocytes from phenyl hydrazine-treated mice showed extensive in situ hybridization. The proportion of fetal liver cells showing predominantly cytoplasmic in situ hybridization increased from about 30% at the 1 lth day of development to 80-85% by days 13-15. Unlike more mature cells, proerythroblasts did not show in situ hybridization, except to a slight extent at later stages of development. These studies therefore indicate that globin m R N A s begin to accumulate during or shortly after the proerythroblastbasophilic erythroblast transition.The fact that certain immature erythroid cells from 14-day fetal liver contain substantial amounts of globin m R N A s has been confirmed by comparing the hybridization in solution of globin c D N A to cytoplasmic R N A extracted from total fetal liver cells or from immature erythroid cells obtained by treatment of fetal liver cells with an antiserum raised against erythrocytes.During the llth-16th day of fetal mouse development, the liver becomes the main erythropoietic organ. At this time, the fetal liver consists of up to 70% erythroid cells, comprising both multipotential and intermediate "'stem cells" and all the recognizable erythroid cell types. Although erythroid cell development represents a continuous process, certain erythroid cell types have been classified, as summarized in the scheme below [see (1, 2) for review]:

Yolk-sac hematopoiesis

“…Receptor numbers subsequently decrease as terminal differentiation proceeds. While erythropoietin transcripts have been detected in the mouse embryo at E7.5 and in the fetal liver at E14.5, the low levels of erythropoietin expression have hampered the spatial and temporal analysis of its expression during development [36][37][38]. A small percentage (7%-10%) of a supraphysiologic dose of erythropoietin can cross the mouse placenta just prior to birth (E19) [39].…”

The Ets-related transcription factor PU.1 immortalizes erythroblasts.

“…When digested with HindIII, which cuts only once in the SFFV sequences but not within the PU.1 cDNA insert, a range of different-size DNAs that hybridized to the PU.1 cDNA probe was observed (Fig. 5, lane 1 small amounts in early hematopoietic progenitors (30) and in much larger amounts in late BFU-E (7, 15), whereas globin mRNA begins to accumulate later during the proerythroblast-normoblast transition (29,60). Although RNAs reactive with our EpoR and 3-globin probes were absent or relatively scarce in fibroblasts (Fig.…”

IN SITU LOCALIZATION OF GLOBIN MESSENGER RNA FORMATION

“…In the accompanying paper (1) in situ hybridization of globin DNA copy (cDNA) to fetal liver was used to elucidate the process of globin messenger RNA (mRNA) formation in erythroid precursor cells. The essential conclusions were also confirmed by conventional hybridization studies.…”