Differential gene expression during terminal erythroid differentiation

Abstract

Terminal erythroid differentiation in mammals is the process whereby nucleated precursor cells accumulate erythroid-specific proteins such as hemoglobin, undergo extensive cellular and nuclear remodeling, and ultimately shed their nuclei to form reticulocytes, which then become mature erythrocytes in the circulation. Little is known about the mechanisms that enable erythroblasts to undergo such a transformation. We hypothesized that genes involved in these mechanisms were likely expressed at restricted times during the differentiation process and used differential display reverse transcriptase polymerase chain reaction as a first step in identifying such genes. We identified three differentially expressed cDNAs that we termed late erythroblast (LEB) 1-3. None of these cDNAs were previously identified as being expressed in erythroblasts and their patterns of expression indicated they are likely to be involved in the differentiation process. LEB-1 cDNA was derived from the gene A330102K04Rik (approved gene symbol Apoll1), and shares homology with members of the apolipoprotein L family in humans. LEB-3 cDNA was derived from the novel gene D930015E06Rik, that has no known function. LEB-2 cDNA was derived from the gene ranBP16 (approved gene symbol Xpo7), a nuclear exportin. D930015E06Rik mRNA is also strongly expressed in the testis and was localized to a region of the seminiferous tubule where secondary spermatocytes and early spermatids are found, suggesting a role for D930015E06Rik in spermatogenesis as well as terminal erythroid differentiation. We have thus identified three genes not previously described as being expressed in erythroblasts that could be relevant in elucidating mechanisms involved in terminal erythroid differentiation.

Figure 1

Detection of LEB gene expression in mouse tissues using 5′-RACE generated probes. A. Top, northern blot probed with the LEB-1 5′RACE probe. Bottom, RNA samples on blot prior to transfer. U, uterus; SM, skeletal muscle; Sp, spleen; K, kidney; L, liver; B, cerebrum; LI, large intestine; T, testis; O, ovary; Lu, lung; H, heart; SI, small intestine; 0, FVA cells at the intiation of culture; 24, FVA cells after 24 hours of culture with EPO; 48, FVA cells after 48 hours of culture with EPO. B, Top, northern blot probed with the LEB-2 5′RACE probe. Bottom, RNA samples on blot prior to transfer. Labels for gel lanes are as above. C. Top, northern blot probed with the LEB-3 5′RACE probe. Bottom, RNA samples on blot prior to transfer. Labels for gel lanes are as above.

Figure 2

Detection of LEB gene expression in mouse tissues using RT-PCR generated probes. A. Expression of LEB-1/A330102K04 during the terminal differentiation of FVA cells. A. Top, northern blot probed with the LEB-1/A330102K04 RT-PCR probe. Bottom, same blot probed with the mouse 18S ribosomal RNA probe. B. Expression of LEB-2/Xpo7 during the terminal differentiation of FVA cells. Top, northern blot probed with the LEB-2 /Xpo7 RT-PCR probe. Bottom, same blot probed with the mouse 18S ribosomal RNA probe. C. Expression of LEB-3/D930015E06Rik during the terminal differentiation of FVA cells. Top, northern blot probed with the LEB-3/D930015E06Rik RT-PCR probe. Bottom, same blot probed with the mouse 18S ribosomal RNA probe. Labels for lanes: 2, FVA cells after 2 hours of culture with EPO; 24, FVA cells after 24 hours of culture with EPO; 48, FVA cells after 48 hours of culture with EPO; T, testis.

Figure 3

Detection of LEB gene expression in normal mouse tissues by RT-PCR. Top. cDNA amplified using LEB-1/A330102K04 primers. Middle. cDNA amplified using LEB-3/D930015E06Rik primers. Bottom. cDNA amplified using primers specific for GAPDH. K, kidney. B, bone marrow. T, testis. FVA, FVA cells after 48 hours of culture in the presence of EPO. Numbers indicate whether kidney or bone marrow was from mouse 1, 2 or 3. RT reactions were prepared as described in Methods and Materials and diluted as follows for the PCR reactions: mouse 1, 1:100; mouse 2, 1:200; mouse 3, 1:100; testis, 1:200; FVA, 1:400.

Figure 4

Analysis of LEB-3/D930015E06Rik expression in mouse testis by in situ hybridization. A. Low magnification photomicrograph of a mouse seminiferous tubule hybridized with the LEB-3/D930015E06Rik antisense riboprobe. Aggregates of silver grains form a band between the basal and apical regions of the tubule. B. Low magnification photomicrograph of a mouse seminiferous tubule hybridized with the LEB-3/D930015E06Rik sense riboprobe. Only background silver grains are evident and no aggregation of silver grains is seen between the basal and apical regions of the tubule C. Higher magnification photomicrograph of an area contained in A. Silver grains can be seen in greatest density over areas of mouse seminiferous tubules corresponding to where secondary spermatocytes and early spermatids are located. sg, are of tubule where spermatogonia and primary spermatoctes are found; sc, are of tubule where secondary spermatocytes and early spermatids are found; st, area near the lumen of the tubule where late spermatids are found. D. Higher magnification of an area in B. No specific hybridization is present over the seminiferous tubule after hybridization with the sense riboprobe.