Serum Response Factor Contributes Selectively to Lymphocyte Development*

Serum response factor (SRF), is a crucial transcription factor for murine embryonic development and for the function of muscle cells and neurons. Gene expression data show that SRF and its transcriptional cofactors are also expressed in lymphocyte precursors and mature lymphocytes. However, the role of SRF in lymphocyte development has not been addressed in vivo so far, attributed in part to early embryonic lethality of conventional Srf-null mice. To determine the in vivo role of SRF in developing lymphocytes, we specifically inactivated the murine Srf gene during T or B cell development using lymphocyte-specific Cre transgenic mouse lines. T cell-specific Srf deletion led to a severe block in thymocyte development at the transition from CD4/CD8 double to single positive stage. The few residual T cells detectable in the periphery retained at least one functional Srf allele, thereby demonstrating the importance of SRF in T cell development. In contrast, deletion of Srf in developing B cells did not interfere with the growth and survival of B cells in general, yet led to a complete loss of marginal zone B cells and a marked reduction of the CD5+ B cell subset. Our study also revealed a contribution of SRF to the expression of the surface molecules IgM, CD19, and the chemokine receptor 4 in B lymphocytes. We conclude that SRF fulfills essential and distinct functions in the differentiation of different types of lymphocytes.

proteins. Its activity is regulated by the interaction with transcriptional cofactors, some of which are expressed in a cell typeselective fashion (2). SRF interacts directly with at least two classes of signal-regulated cofactors, the ternary complex factor (TCF) subfamily of Ets domain proteins (SAP-1, Elk-1, and Net) which respond to mitogen-activated protein kinase (MAPK) signaling (3) and members of the myocardin-related transcription factor (MRTF) family (4 -6), which may be regulated through Rho GTPase/actin signaling. SRF activity can be triggered by a multitude of means such as serum, ionizing radiation, growth factors, and intracellular calcium-regulating agents. A steadily increasing set of SRF target genes is being identified, which includes immediate early genes (IEGs), cytoskeletal protein-encoding genes, and muscle differentiation genes (7,8). Regarding cellular function, recent in vivo and in vitro studies revealed essential contributions of SRF to murine embryogenesis (9), neuronal development (10 -12), heart development (13)(14)(15), skeletal muscle function (16,17), programmed cell death (18,19), and processes of cell morphogenesis, adhesion, and migration (20).
Cell culture analyses point to an additional requisite role for SRF in lymphocyte function and development. SRF is widely expressed in hematopoietic cell lines (21,22), activated in response to various cytokines (23), and responds to signaling via the receptors of T cells (24 -26) and B cells (27,28). Furthermore, SRF is involved in the regulation of lymphocyte-specific genes (e.g. Il2, IL2R␣, IFN␥) (24 -26). MAP kinase and Rho GTPase signaling, which have been shown to trigger SRF responses (2), are vitally important for the selection, proliferation, and maturation of thymocytes (24, 29 -32) and B cells (27,28,33).
Stimulation of lymphocyte antigen receptors (B cell receptor (BCR) and T cell receptor (TCR)) rapidly induces expression of c-fos and early growth response factor 1 (egr-1) (28,33,34), which are immediate early target genes of SRF. Several studies support the importance of these genes for lymphocyte development: Overexpression of c-fos in mice augments the differentiation and accumulation of peritoneal B1b cells (35), marginal zone B (MZB) cells (36), and terminally differentiated antigenspecific B cells (37). Activator protein 1 is composed of members of the Fos and Jun family of DNA-binding proteins. Induction of activator protein-1 is required for activation of the germline ⑀ promoter. This represents an essential step preceding immunoglobulin (Ig) isotype switching to IgE (38) and plays a central role in the regulation of promoter activity for interleu-kin-2 (IL-2) and interferon-␥ in T cells (25). The immediate early gene egr-1 has been shown to play a role in limiting the number of T cell precursors and in the efficient differentiation and survival of thymocytes during the process of positive selection (39).
SRF has the potential to regulate cell growth, survival or apoptosis by altering the expression of specific genes involved in these processes. Experiments in a human B cell line demonstrated that for apoptosis to proceed, the transcriptional events promoting cell survival and proliferation in which SRF is involved must first be inactivated by a caspase-mediated cleavage of the SRF protein (18). One mechanism by which SRF inhibits apoptosis is the transcriptional regulation of members of the B cell leukemia/lymphoma (Bcl) family of anti-apoptotic genes (Bcl-2, Bcl-xl, and Myeloid cell leukemia sequence 1 (Mcl-1)). Neither Bcl-xl nor Bcl-2 is absolutely required for T cell development to maturity (40 -42). Conditional Mcl-1 mutants, however, display a profound reduction in B and T lymphocytes (43).
TCFs are thought to function primarily through serum response elements via formation of ternary complexes with SRF. Recently, members of the TCF family of SRF cofactors have been characterized by knock-out studies. Elk-1 deficient mice display normal immune responses and mildly impaired neuronal gene inactivation (44) and Net mutants show defects in cell migration (45), vasculature development (46), and impaired angiogenesis during wound healing (47). The strongest phenotype with regard to immune functions is observed in SAP-1-null mice where thymocyte development is severely impaired and a decrease in the amount of CD4 ϩ and CD8 ϩ single positive (SP) cells is seen resulting from defective thymocyte positive selection (48). The relatively mild phenotypes observed upon inactivation of single TCFs suggest functional redundancy between the different TCFs. Gene targeting in mice has been particularly helpful in deciphering the genetic networks underlying lymphocyte development and function.
Classical disruption of both Srf-alleles in mice leads to early embryonic lethality associated with a gastrulation defect (9), thereby precluding the analysis of SRF function in subsequent developmental processes. To investigate potential contributions of SRF to lymphocyte differentiation and function, we conditionally ablated the Srf gene in developing lymphocytes of the mouse. Potential functional redundancies of TCF proteins are thereby also addressed since SRF mutagenesis inactivates all TCF functions that are dependent on wild-type SRF. We show that mice with ablation of the Srf gene in T cells display a substantial reduction in mature SP thymocytes and nearly complete abrogation of peripheral T cells. B cell-specific depletion of SRF, on the other hand, resulted in the loss of MZB cells and decrease of B1 cells only, whereas the majority of the conventional B2 cells was not affected. Thus our study demonstrates for the first time in vivo that SRF is an essential transcription factor for murine lymphopoiesis.

EXPERIMENTAL PROCEDURES
Lymphocyte Specific Deletion of Srf in Mice-Animals were bred at the Helmholtz Centre for Infection Research under specific pathogen-free conditions and all animal experiments were performed in accordance with institutional guidelines. Srf was deleted specifically in murine lymphocytes by using mice carrying the conditional Srf allele Srf flex1neo (abbreviated fl), which is converted into the Srf lx deletion allele (abbreviated lx) by Cre-mediated recombination (49). Conditional Srf fl mice (49) were bred to CD4-Cre (50) or CD19-Cre (51) mice on a mixed 129SvEv/C57Bl/6 background. Detection of the Srf fl allele was performed by polymerase chain reaction (PCR) using two primer combinations (SRF-E/R and SRF-L/R) as previously described (49). The Cre transgene was detected by PCR using primer 1 (5Ј-ACGACCAAGTGACAGCAATG-3Ј) and 2 (5Ј-CTCGACCAGTTTAGTTACCC-3Ј). Srf deletion was assessed by Southern blot analysis performed on BglII-digested genomic DNA isolated from FACS-sorted lymphocytes. As described previously (49), hybridization with an external 3Ј probe allowed discrimination between wt (4.6 kb), fl (3.8 kb), and lx alleles (1.4 kb).
Histology-Formalin-fixed organs were embedded in paraffin and sectioned at 4 m thickness. Immunohistochemistry for T lymphocytes was performed with a rat-anti-CD3 antibody (CD3-12, Serotec Ltd.). A biotinylated rabbit-anti-rat antibody was used for detection of the bound primary antibody. B lymphocytes were detected with a biotin-conjugated rat-antimouse-CD45R/B220 monoclonal antibody (RA3-6B2, BD Biosciences). The avidin-biotin complex (ABC) method with diaminobenzidine as chromogen was used for detection of the biotinylated antibody.
Flow Cytometry-Single cell suspensions were prepared from thymus, spleen, lymph nodes, peritoneal cavity, and bone marrow. Peripheral blood was taken from the tail vein and heparinized using 375 I.E. heparin-sodium. Erythrocytes were depleted by lysis. After washing, cells were stained with various combinations of antibodies. To exclude dead cells, propidium iodide was used. Electrophoretic Mobility Shift Assay (EMSA)-EMSA studies were performed as described previously (52). A [␣ 32 P]ATP-labeled DNA fragment containing the c-fos serum response element (SRE) was used as a DNA-binding probe either with 4 g T cell lysates from mice with a T cell-specific SRF deletion or 8 g of B or T cell lysates from mice with a B cell-specific SRF deletion. To detect specific binding of SRF to the probe, SRF antiserum (G20, Santa Cruz Biotechnology) was included for supershift analysis. Control shifts were performed with the nuclear factor of activated T cells (NFAT) tandem site of the NFATc1-P1-promoter (53).
DNA Microarray Hybridization and Analysis-Sample preparation from FACS-sorted populations of splenocytes (B cells: CD19 ϩ IgM ϩ IgD ϩ ), hybridization, washing, staining, and scanning of Affymetrix GeneChips was performed as previously described (54). Data analysis was performed using the Affymetrix Microarray Suite 5.0, Affymetrix MicroDB 3.0, and Affymetrix Data Mining Tool 3.0. All array experiments were scaled to a target intensity of 150, otherwise using the default values of the Microarray Suite. Genes were considered as regulated when their fold change was greater than or equal to an increase or decrease of 1.5, the statistical parameter for a significant change was less than 0.001 or greater than 0.999, and the signal difference of a certain gene was greater than 40.

RESULTS
T Cell Lineage-specific Deletion of Srf-To delete Srf specifically in lymphocytes we used a conditional Srf fl mouse strain where Cre-mediated recombination results in formation of the Srf lx allele deleting the complete coding region of exon 1 (49). To the best of our knowledge, there is not a protein product being derived from this allele. However, the presence of such a truncated protein cannot be ruled out with 100% certainty (13). If such a hypothetical product were derived from the Srf lx locus, it would be predicted to have lost all the important functions of nuclear localization, dimerization, and DNA binding. To restrict deletion of the Srf fl allele to developing T cells, the lymphocyte-specific CD4-Cre transgene (50) was introduced by breeding. The resulting Cre expression is directed by the murine CD4 enhancer/promoter/silencer, which leads to efficient Cre-mediated deletion in CD4 ϩ CD8 ϩ double positive (DP) and CD4 ϩ SP thymocytes, in mature CD4 ϩ T cells, and, to a lesser extent, in CD4 Ϫ CD8 Ϫ double negative (DN) thymo-cytes (55). The resulting mutant CD4-CreSrf fl/fl progeny were obtained at Mendelian frequency and exhibited no obvious phenotypic abnormalities.
T Cell-specific Srf Deletion Leads to a Block in T Cell Development-We first looked whether CD4-CreSrf fl/fl mice still possessed T lymphocytes. Immunohistochemical staining of spleen sections with a T cell-specific anti-CD3 antibody showed a striking decrease of CD3-positive cells, as compared with control specimen (Fig. 1A). This was verified by FACS analysis, which showed decreased numbers of peripheral T cells in spleen, blood, and lymph nodes of CD4-CreSrf fl/fl mice when compared with littermate control mice (i.e. Srf fl/fl , Srf fl/wt , and CD4-Cre-Srf fl/wt ) (data not shown). The reduction of peripheral T cells suggested an impaired T cell development in the thymus. The overall number of thymocytes was similar in wild-type and SRF-deficient mice and no major differences in DN and DP thymocyte populations were detected by FACS analysis. We observed, however, a dramatic reduction of SP CD4 ϩ (ϳ80%) and CD8 ϩ (ϳ50%) T cells among the thymocytes from SRF-deficient CD4-CreSrf fl/fl mice (Fig. 1B).
Peripheral T Cells Lacking SRF Are Non-Viable-The remaining T cells may have survived without functional SRF or may represent a minority population that failed to delete the Srf gene. To address this question we sorted T and B cells from thymus and spleen and performed Southern blot analysis to distinguish the active Srf fl allele from the inactive Srf lx allele ( Fig. 2A). Consistent with the absence of CD4 promoter activity in B cells, only the Srf fl , but not the deleted Srf lx allele could be detected in B cells from spleens of CD4-CreSrf fl/fl mice ( Fig. 2A, right panel). In contrast, the Srf fl allele was completely recombined to Srf lx in CD4 ϩ CD8 ϩ DP cells from thymi of CD4-CreSrf fl/fl mice ( Fig. 2A, left panel). Thus, CD4-Cre-mediated excision of Srf fl resulted in selective perturbations of T cell populations. The Srf lx allele was also detectable in peripheral T cells isolated from the spleens of mutant animals ( Fig. 2A, center). However, in these splenic T cells we detected an additional band, which was specific for the intact Srf fl allele. We then decided to investigate SRF protein content of lymphocyte cells directly by EMSA. Protein extracts prepared from CD4 Ϫ CD8 Ϫ DN cells (in which Cre is not yet expressed) from thymi of mutant mice contain SRF as shown by the protein-DNA complex in Fig. 2B (left  panel). In contrast, no such SRF-DNA complex was visible when we used CD4 ϩ CD8 ϩ DP cell extracts from CD4-CreSrf fl/fl mice. The presence of SRF protein in these shifted complexes was verified by supershift experiments with SRF antiserum. To prove the general capability of our protein extracts for specific DNA binding, we performed EMSA control experiments employing a tandem DNA binding site for the NFAT transcription factor (data not shown). Furthermore, when lymphocyte extracts from spleen were used for gel shift assays we detected SRF protein not only in B cells of CD4-CreSrf fl/fl mice, but also in the remaining peripheral T cells (Fig.  2B, right panel). The latter result is in agreement with the Southern blot analysis, indicating the presence of the non-recombined Srf fl allele in some splenic T cells. Taken together, our data suggest that peripheral T cells lacking both copies of Srf are non-viable and that the residual T cells found in CD4-CreSrf fl/fl mice are derived from cells that have escaped Cremediated deletion.
B Lineage-specific Srf Deletion-The CD19-Cre mouse strain used here for B cell-specific recombination of the Srf fl allele has been generated by a knockin of the Cre recombinase into the CD19 locus. CD19-Cre hemizygous mice are phenotypically normal and can be used for B lineage-specific deletion of a floxed target gene (56). To disrupt Srf specifically in B cells we crossed CD19-Cre mice with Srf fl/fl animals. Mutant CD19-CreSrf fl/fl mice were born with Mendelian distribution and appeared phenotypically normal. Southern blot analysis showed a complete B cell-specific deletion of the Srf fl allele in FACS-sorted splenocytes of CD19-Cre-Srf fl/fl mice (Fig. 3A). Presence of SRF in the protein-DNA complexes was monitored by EMSA, including supershift assays with SRF antiserum (Fig. 3B). Again, NFAT DNA-binding was used in control EMSAs to check for the integrity of cell extracts (data not shown). The remaining SRF protein in splenic B cells from CD19-Cre-Srf fl/fl mice is in contrast to the complete recombination of Srf seen in the Southern blot analysis and can probably be attributed to the long half-life of the SRF protein (15,57). This has also been noticed in embryonic myocytes (15) and in fibroblasts where a half-life of at least 12 h was estimated for SRF (57).
B Cell-specific Srf Deletion Leads to a Decrease in B Cell Numbers, CD19, and IgM Expression-We next analyzed the overall distribution of B cells in spleen, bone marrow, peripheral blood, and lymph nodes and found that the respective cell populations of pre-B cells (IgM Ϫ IgD Ϫ ), newly generated B cells (IgM ϩ IgD Ϫ ), and mature recirculating B cells (IgM ϩ IgD ϩ ) exhibited comparable percentile representations in both SRF-deficient (CD19-CreSrf fl/fl ) and control animals (Srf fl/fl and CD19-CreSrf fl/wt ) (data not shown). Quantification of the number of lymphocytes and CD19 ϩ cells from bone marrow, however, showed a decrease in the overall number of CD19 ϩ lymphocytes (Fig. 4A, left panel), which could not be attributed to the reduction of an individual B cell population (Fig. 4A, right panel). To our surprise, SRF-deficient mature B cells (CD19-CreSrf fl/fl ) from spleen, lymph nodes, bone marrow, and blood displayed a lower expression of the surface molecule IgM in comparison to controls (Fig. 4B, left; shown for blood only), whereas the expression of IgD, which is cotranscribed, remained constant (Fig. 4B, right). We also observed down-regulation in the expression of CD19 in SRF-deficient B cells from peripheral blood. This is only partly caused by the hemizygosity of the CD19 allele, as shown by FACS analysis comparing B cells from CD19-CreSrf fl/fl mice with B cells from control animals expressing Cre recombinase (CD19-CreSrf wt/wt and CD19-CreSrf fl/wt ) (Fig. 4C). Next, we tested the ability of SRF-deficient splenic B cells to elicit a calcium response to the F(abЈ)2 fragment of anti-IgM antibodies. No differences in [Ca 2ϩ ] i between wild-type and mutant cells were observed when the splenic B cells were stimulated by suboptimal or optimal concentrations of anti-IgM antibodies (data not shown). This analysis confirms that the BCR complex on SRFdeficient B cells is capable of receiving and delivering signals. In   (58). This analysis with SRF-deficient splenocytes showed that MZB cells were specifically absent (Fig. 5A). MZB cell populations were also examined by microscopy of splenic cryosections stained with anti-IgM and anti-IgD (Fig. 5B). Normal microanatomic structures containing T cell areas, B cell follicular structures, and marginal zone and metallophilic macrophages were present and correctly localized. However, in agreement with the cytometric results, the characteristic ring of IgM ϩ IgD Ϫ MZB cells (arrows in Fig. 5B) peripheral to follicles was missing in the absence of SRF. This result confirms the absence of MZB cells in CD19-CreSrf fl/fl mice despite normal follicular architecture. Next, we examined a possible influence of SRF on the self-renewing mature CD5 ϩ B1 B cells. Analysis of peritoneal cells from CD19-CreSrf fl/fl mice revealed a substantial decrease of CD19 ϩ IgM ϩ CD5 ϩ B cells in comparison to littermate controls (Srf fl/fl and CD19-CreSrf fl/wt ) (Fig. 6).
Down-regulation of SRF Target Genes in the B Cell-specific Srf Mutant-To identify SRF target genes that could account for the observed phenotypic differences, we compared the gene expression profiles of FACS-sorted splenic B cells from mutant (CD19-CreSrf fl/fl ) and control mice (Srf fl/fl ) using cDNA expression arrays (Affymetrix MOE430A GeneChip, 22690 transcripts) in two independent experiments. The microarray data were deposited in the NCBI gene expression and hybridization array data repository with assigned accession number (GEO Series Acc GSE7412). We filtered for genes whose expression was at least 1.5-fold differentially regulated. In addition to the expected down-regulation of known SRF target genes, such as the immediate early genes egr-1, junB, and c-fos and the structural genes encoding ␥-actin, ␤-actin, and vimentin, a contribution of SRF was observed to the regulation of the accessory immunoglobulins Ig␣ and Ig␤, and CD19, which we already knew from our FACS analysis (see Fig. 4C), and the chemokine receptor CXCR4. Also, in the case of CXCR4 we were able to confirm its down-regulation on protein level by FACS analysis (Fig. 7).

DISCUSSION
Our data show substantial evidence that SRF is involved in controlling lymphocyte maturation. We deleted the conditional Srf fl allele (49) using mice expressing the Cre recombinase in developing T cells (CD4-Cre) (50) or developing B cells (CD19-Cre) (51). The phenotypes of these mutant mice reveal distinct requirements for SRF in the developmental control of different lymphocytes.
T cell-specific Srf knock-out mice showed a substantial reduction in mature SP thymocytes and nearly complete absence of peripheral T cells, prohibiting a further functional analysis of this specific T cell subset. The few remaining T cells in the periphery still contained the non-recombined Srf fl allele and SRF DNA-binding activity, suggesting that the residual peripheral T cells had undergone population expansion from the few T cells that had escaped Cre-mediated deletion. These data provide evidence of the requirement of SRF for T cell maturation.
T cell development is controlled at multiple stages by signals from cell surface receptors. The reduced percentage of mature cells in the T cell specific Srf mutant mice suggests that SRF is required for positive selection, an advanced stage of T cell differentiation. Here, CD4 ϩ CD8 ϩ DP T cells interact with peptides associated with major histocompatibility complex (MHC) proteins to either differentiate or undergo apoptosis. If positively selected, immature DP thymocytes down-regulate either CD4 or CD8 to become SP T cells (59). A contribution of SRF to the process of positive selection is consistent with data showing the necessity of the Ras/Erk (MAPK) pathway for this process. MAPK signaling is known to trigger SRF via its TCF cofactors. Dominant interfering forms of Ras, MEK, and ERK are able to block positive selection (31,60). This was confirmed by the phenotype of ERK deficient mice (60). The transcription factor egr-1 is targeted by activated ERK in thymocytes (61) and egr-1-deficient mice exhibit impaired positive selection (39), whereas enforced egr-1 expression promotes positive selection (62). The TCFs are known to regulate a MAPK-responsive subset of SRF target genes, including egr-1, c-fos, and Mcl-1. Recent results from Costello et al. (48) prove the TCF SAP-1 to be a direct link between ERK signaling and the transcriptional effectors of positive selection for T cells. SAP-1 is rapidly phosphorylated upon TCR activation via the ERK pathway, and SAP-1 deficiency caused impairment of egr-1 and Id-3 gene activation and decreased the amount of positively selected thymocytes. T lineage-specific deletion of Srf increased the severity of the SAP-1 phenotype leading to stronger reductions in the proportion of CD4 ϩ SP and CD8 ϩ SP thymocytes and peripheral T cells. This probably reflects the functional redundancy between the TCFs. Although SAP-1 was demonstrated to be the main constituent of ternary complexes in thymocytes, Elk-1 was also shown to be present (48). We demonstrated that SRF is required for T cell development following the DP stage, coinciding temporally with CD4-Cre expression and Srf deletion. However, it is likely that SRF has important functions also in the early, TCR-independent phase of T cell development, during e.g. T-lineage commitment, the transition from the CD4 Ϫ CD8 Ϫ DN to the CD4 ϩ CD8 ϩ DP stage, or for the survival of mature peripheral T cells. These assumptions are supported by the finding that SRF binding activity is already abundant early in thymocyte development at the DN stage (26). Moreover, studies with egr-1 knock-out mice demonstrated defective positive selection and an increased amount of DN thymocyte precursor cells (39). Also, conditional deletion of the SRF target gene Mcl-1 arrested T cell development at the DN   stage, whereas deletion in peripheral T cell populations resulted in their rapid loss (43). The effects of a SRF deficiency on the very early stages of T cell development may be addressable using lck-Cre mice (63), permitting Cre-mediated deletion at the most immature CD44 ϩ DN stage. On the other hand, an influence of SRF on late stages of T cell development may be detectable using inducible CD4-Cre or inducible mx-Cre mouse strains (64).
B cell development in adult mice occurs in the bone marrow. Progenitor and precursor cells differentiate into immature B cells after expressing a surface immunoglobulin receptor (IgM). Newly generated B cells leave the bone marrow and some of these cells, after further positive and negative selection steps, give rise to mature B cells. Based on phenotypic, topographic, and functional characteristics, B cells are classified into at least three subsets. The heterogeneous recirculating B2 cells localize to the B-lymphoid follicles of lymph nodes and spleen, the selfrenewing nonrecirculating B1 B cells are enriched in the pleural and peritoneal cavities, and the mostly nonrecirculating MZB cells are enriched in the marginal zone of the spleen (65).
Our findings imply a specialized role for SRF in CD5 ϩ B1 cells and MZB cells. No obvious B cell function was described for the SRF cofactor SAP-1 (48), possibly indicating functional redundancy of different TCFs in B1 cells. These populations are substantially decreased or even lost, respectively, in the B cellspecific Srf mutant, which precludes a further functional analysis of these cell types. Surprisingly, we found SRF to be largely dispensable for the development of conventional B2 lymphocytes. These display no obvious change in BCR function, and there is no difference in the basal serum immunoglobulin concentrations in mutant animals as compared with wild types.
FACS analysis of the remaining B2 cells revealed a downregulation of the surface molecules CD19, IgM, and CXCR4. Using gene arrays we detected decreased expression of c-fos, egr-1, junB, Ig␣, Ig␤ and several genes coding for structural proteins. As IgM and IgD are transcribed as a common pre-RNA, a transcriptional change should lead to altered expression of both genes, but the level of IgD in our mutants remained unchanged. We interpret this to be due to the reduced Ig␣ message observed in the gene array. Ig␣ is necessary for the display of IgM and IgD on the cell surface, but IgD in contrast to IgM, can be alternatively anchored via a lipid tail in the cell membrane (66).
An interesting yet difficult to address question concerns the mechanism by which SRF promotes the development of MZB and B1-type B cells. There is distinct uncertainty in the field regarding the identity of immediate precursors to these B cell subsets and signals leading to their development. The fact that numerous mutations that modulate BCR signaling (67,68) result in altered development of MZB or B1 cells led to the conclusion that increased BCR signaling is involved in the development of these B cell subtypes. If this were correct one would imagine an interplay between SRF signaling and BCR signaling. Hints for this were found in the observed reductions in the amount of the B cell coreceptor CD19, the surface molecule IgM, and the associated signal transducing elements Ig␣ and Ig␤ in the remaining B cells from our mutants. In that respect, it is worth mentioning that CD19-deficient mice pres-ent multiple B cell defects, including a severe reduction in MZB and B1 cells (56,69). However, we did not detect any obvious change in BCR function as determined by measuring calcium flux activation.
Our array data proved c-fos to be an SRF target gene in B cells. As overexpression of c-fos in mice elicited an increase in peritoneal B 1 cells and MZB cells (35,36), up-regulation of c-fos by SRF could be one possible mechanism to influence the size of these specific B cell subsets. Defects in cell migration might similarly account for the observed phenotypes in our B cell specific SRF mutants. Indeed, ES cells lacking SRF display impaired cell-cell interactions, disorganization of the cytoskeleton, and down-regulation of surface proteins (20). Furthermore, an influence of the actin cytoskeleton and integrin-mediated processes in lymphocyte retention and localization to the marginal zone has been shown (70,71). Altered cytoskeletal activity in SRF deficient B cells may be indicated by the downregulation of the actin, vimentin and vinculin genes, as detected by our array analysis. In addition, we noticed a slight decrease in the amount of beta1 and beta7 integrin (data not shown). Decreased expression of the chemokine receptor CXCR4 could likewise contribute to the phenotypic differences seen in our SRF mutant. B cell-specific inactivation of CXCR4 affects B cell migration leading to reductions in the B1 and MZB cell compartments (72). However, a general migration defect in B cells from our mutants can be excluded, as they can be detected in the peripheral lymph nodes.
In summary, we showed that SRF is required for the development of T cells from the DP stage onward and for the development of the B1 and MZB subsets of B cells. Surprisingly, SRF deficiency does not perturb the development or survival of conventional B cells, which suggests that SRF and its cofactors mediate distinct physiological functions in different types of lymphocytes. Thus, SRF ablation does not have pleiotropic effects on the general transcription machinery in all lymphocytes, suggesting that alternative transcriptional programs exist to regulate the survival and maintenance of B2 cells. In addition, there has to be a redundancy in factors regulating known SRFtarget genes like Mcl-1, which was shown to be required for the survival of all sets of B lymphocytes. However, as the CD19-Cre regulatory element used to direct Cre recombinase is not active from the beginning of B lymphocyte development one could imagine that deletion at an earlier time might result in a more severe phenotype. This hypothesis is supported by the general decrease in the number of conventional B cells seen in our mutants. Using a Cre transgenic mouse line deleting earlier in B cell development, i.e. MB1-Cre (73) should reveal additional roles of SRF in the early processes of B cell differentiation.