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. 1998 Apr 14;95(8):4481-6.
doi: 10.1073/pnas.95.8.4481.

Impaired fetal T cell development and perinatal lethality in mice lacking the cAMP response element binding protein

D Rudolph  1 , A Tafuri, P Gass, G J Hämmerling, B Arnold, G Schütz
Affiliations

Impaired fetal T cell development and perinatal lethality in mice lacking the cAMP response element binding protein

D Rudolph et al. Proc Natl Acad Sci U S A. .

Abstract

CREB, the cAMP response element binding protein, is a key transcriptional regulator of a large number of genes containing a CRE consensus sequence in their upstream regulatory regions. Mice with a hypomorphic allele of CREB that leads to a loss of the CREBalpha and delta isoforms and to an overexpression of the CREBbeta isoform are viable. Herein we report the generation of CREB null mice, which have all functional isoforms (CREBalpha, beta, and delta) inactivated. In contrast to the CREBalpha delta mice, CREB null mice are smaller than their littermates and die immediately after birth from respiratory distress. In brain, a strong reduction in the corpus callosum and the anterior commissures is observed. Furthermore, CREB null mice have an impaired fetal T cell development of the alpha beta lineage, which is not affected in CREBalpha delta mice on embryonic day 18.5. Overall thymic cellularity in CREB null mice is severely reduced affecting all developmental stages of the alpha beta T cell lineage. In contrast gamma delta T cell differentiation is normal in CREB mutant mice.

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Figures

Figure 1
Figure 1
Targeting strategy for the generation of a CREB null allele. (A) (Top) Gene structure of the 3′ region of the CREB locus. Exons are shown as boxes. Solid boxes, coding regions; shaded boxes, nontranslated regions. (Middle) Targeting vector. (Bottom) Gene structure of the targeted allele. E, EcoRI; R, EcoRV; LacZ-Neo, fusion gene encoding β-galactosidase and neomycin phosphotransferase; P, probes used for detection of homologous recombination events. Arrows show the expected size of the restriction fragments detected with these probes. (B) Southern blot analysis of EcoRI-digested genomic DNA from E18.5 newborns. The 3′ probe was used, the size of the restriction fragments is indicated. (C) RT–PCR analysis using a cDNA derived from total brain RNA of a CREB −/− mouse and wild-type littermate, respectively, demonstrating the absence of the 3′ part of the CREB transcript in mutant mice. Primers for the CREB gene were derived from the 3′ end of the gene; primers for Hprt (hypoxanthine guanine phosphoribosyltransferase) were used as control for RNA integrity. The CREB PCR product is indicated by an arrow, and the Hprt PCR product is indicated by an asterisk. −RT, −reverse transcriptase.
Figure 2
Figure 2
Severe atelectasis of the lung and reduced expression of SP-D in CREB null mice. Histological analysis of lungs from a CREB −/− animal (B) and a control wild-type littermate (A), stained with hematoxylin/eosin and showing a severe atelectasis of the lung in CREB mutant animals. (C) Northern blot analyses of total lung RNA (5 μg) using probes specific for SP-A, SP-B, SP-C, and SP-D. Filters were rehybridized with β-actin as a loading control.
Figure 3
Figure 3
Histological analysis of the CNS of CREB −/− mice on E18.5. (A and B) A severe reduction in the corpus callosum (arrowheads and lower right of micrographs) and the anterior commissures (arrows and lower left of micrographs) was observed in CREB −/− mice. LV, lateral ventricle. (C and D) A marked up-regulation of CREM was observed in the developing hippocampus as well as other forebrain areas (data not shown) of CREB −/− animals. Wild-type littermates only showed faint CREM immunoreactivity. Arrowheads indicate immunostained nuclei of the pyramidal neurons of the hippocampus.
Figure 4
Figure 4
Fetal T cell development in CREB null mice and CREBαΔ mice on E18.5. (Upper) Thymocytes from E18.5 CREB null mice, wild-type and heterozygous littermates, and CREBαΔ mice were analyzed by flow cytometry using antibodies against CD4 and CD8. The thymocyte subsets from one representative animal of each genotype are shown. (Lower) Forward side scatter (FSC) versus side scatter (SSC) plots are shown for thymocytes from the indicated genotypes. CREB null mice had a larger percentage of thymocytes with a higher FSC. CREBαΔ mice did not differ from the control animals.
Figure 5
Figure 5
Normal fetal γδ T cell differentiation but severely reduced numbers of mature CD3high TCRαβhigh CD4+ CD8− SP thymocytes in CREB null mice on E18.5. (A) CD4+ CD8− thymocytes from CREB null mice and control littermates were stained with antibodies against the TCRαβ complex, the TCRVβ8.1,2,3 chain, and CD3ɛ as indicated and were analyzed by flow cytometry. CREB null mice had a severely reduced percentage of mature CD4+ SP cells characterized by high TCRαβ, TCRVβ8.1,2,3, and CD3ɛ surface expression. CD4− CD8− (B) and CD4−CD8+ (C) thymocytes from CREB null mice and control littermates were stained with an antibody against TCRγδ and analyzed by flow cytometry. No difference in γδ T cell differentiation was observed between CREB −/− mice and their control littermates. (D) Northern blot analysis of RNA (500 ng) from sorted DN thymocytes derived from five animals of each genotype. A TCRβ-chain-specific probe containing exon cβ2 was used (Upper). The filter was rehybridized with β-actin as a loading control (Lower). (E) Quantitative RT–PCR using a cDNA derived from sorted DP cells of mutant or wild-type animals, respectively. One representative experiment of five is shown. Gene-specific primers were used for the TCRα chain. Primers for β-actin were used as a standard. Aliquots of the PCR products were taken after 19, 21, and 23 cycles as indicated, separated on an agarose gel, and blotted. Filters were hybridized with internal oligonucleotides to detect the two PCR products: the TCRα product is indicated by an arrow and the β-actin product is indicated by an asterisk.
Figure 6
Figure 6
Altered thymocyte subsets defined by CD4/CD8 surface staining in CREB null mice on E18.5. (Upper) The absolute thymocyte numbers (×106) of each of the thymocyte populations indicated above for one representative animal of each genotype. Open bars, +/+ animal; hatched bars, +/− animal; solid bars, the CREB null mouse. The absolute number of the thymocytes in each of the subsets was decreased in CREB null mice. (Lower) Relative percentage of the thymocyte subsets given as the mean from n = 8 +/+ (open bars), n = 13 +/− (hatched bars), and n = 15 CREB −/− mice (solid bars). The standard deviation is indicated. The relative percentage of CD4− CD8− cells (P < 0.00005, Wilcoxon rank sum test) and immature CD4− CD8+ TCRαβ− SP cells (P < 0.00005, Wilcoxon rank sum test) was increased in CREB null mice, whereas the relative percentage of the more mature CD4+ CD8+ (P < 0.00005, Wilcoxon rank sum test) and the CD4+ CD8− TCRαβ+ SP cells (P < 0.0159, Wilcoxon rank sum test, n = 5) was reduced compared with the littermate controls. We did not observe statistically significant differences between wild-type and heterozygous littermates.
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