Received 22 _Ma~ch 1973 ]ntroduclion MethodsAn important aspect of th,e control of cel]~ax differ.enfiafi~on Js the sequenfi~d activation of genes, .]ea,d. ~.mg to synthesis of specific me~.~er~ger RNA's ,(mRNA's) spe~L~cing proteins characteristic of a parfi.cUla~ id~ffer~ntiated cell. The p;oblems revolved ~n ducidafing 'these complex b~ological changes are basically two-fo]d: !) t,o detect in a ~ven ceB type rnmut.e ~mmunts of specific rnRNA's and ii) ~o correlate changes m amolm ts ,of specific mRNA's with changes ~n the differentiated sta'te.This repolt ,describes a general rne~_,od which allows ,det, e,efion of minute traces ofz specific mRNA ha cytological specimens .of cells. "lhe method is app]ied to detect ~obin naRNA in ery~roJd ce]].s. We have ~own [1; 2] that a reasonably fai~ftfl DNA copy (eDNA) of globin rnRNA can be obtained by ~anseribing mouse reficulocy, t~ 9-8 RNA by a'everse t,ran.sefiptase, Using ',this eDNA as a probe, the conventional in sin hybfidisa..fion "technique is extended to ~eteet globin xnRNA'z an the developing foetal ,~vex and in F~rien.d-vLrus ti.masformed Cells treated with .,di-:

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]:

No abstract

We have analysed the transcriptional regulation of the murine alpha 1 and beta maj globin genes and the glutathione peroxidase (GSHPx) gene, which are all highly expressed during erythropoiesis. The levels of minor RNAs compared to the major message were monitored throughout differentiation within the erythroid lineage. For each gene, upstream transcripts arise from distinct clusters of sites which are regulated differently during differentiation: some occur only during early erythropoiesis, some occur early and persist to the terminal stages, while others accumulate later and roughly in parallel with the main RNA transcript. In addition, opposite strand transcripts from the GSHPx gene were found in increasing amounts during later stages of erythropoiesis. The initiation sites for specific subsets of these minor transcripts lie close to sequences known to be involved in globin gene regulation (i.e. the TATA, CAAT and the CACCCT boxes) or other conserved sequences; others lie close to developmentally regulated DNase I hypersensitive sites around the globin and GSHPx genes.

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. In the present paper, the same dual approach is applied to a different experimental system in which hemoglobin synthesis in Friend virus-transformed mouse cells is induced by treatment with dimethylsulfoxide (DMSO) (2). The results obtained by in situ and conventional hybridization agree in showing that there is a minimum period of treatment with DMSO required in order that globin mRNA and hemoglobin may accumulate. M A T E R I A L S AND METHODS Cell CultureFriend cells (clone 707) and lymphoma cells (L 5178Y) were grown in Ham's FI2 medium (Flow Laboratories, Irvine, Scotland), supplemented with minimal essential medium amino acids, penicillin (60 U/ml), and 10-15% horse serum and buffered with HEPES or bicarbonate. LS cells were grown in Eagle's minimal essential medium plus 5% calf serum.All other methods are fully described in the accompanying paper (1). RESULTS