SummaryWe have generated a transgenic mouse line, Tg(Stra8-cre)1Reb (Stra8-cre), which expresses improved Cre recombinase under the control of a 1.4 Kb promoter region of the germ cell-specific stimulated by retinoic acid gene 8 (Stra8). cre is expressed only in males beginning at postnatal day (P)3 in early-stage spermatogonia, and is detected through pre-leptotene-stage spermatocytes. To further define when cre becomes active, we crossed Stra8-cre males with Tg(ACTB-Bgeo/ GFP)21Lbe (Z/EG) reporter females and compared the expression of Enhanced Green Fluorescent Protein (EGFP) with the protein encoded by the zinc finger and BTB domain containing 16 (Zbtb16) gene, PLZF -a marker for undifferentiated spermatogonia. Co-expression of EGFP is observed in the majority of PLZF+ cells. We also tested recombination efficiency by mating Stra8-cre;Z/EG males and females with wild-type mice and examining EGFP expression in the offspring. Recombination is detected in >95% of Z/EG+ pups born to Stra8-cre;Z/EG fathers but in none of the offspring born to transgenic mothers, a verification that cre is not functional in females. The postnatal, premeiotic, male germ cell-specific activity of Stra8-cre makes this mouse line a unique resource to study testicular germ cell development. KeywordsCre recombinase; Stra8 promoter; spermatogonia; spermatocytes; Z/EG These experiments were initiated to produce a transgenic mouse line that expresses cre in undifferentiated spermatogonia. To date, the use of cre-mediated recombination to inactivate genes in developing germ cells at specific stages has been limited by the restricted expression patterns of available cre drivers. Recombination in primordial germ cells is possible using the alkaline phosphatase, liver/bone/kidney Alpl tm1(cre)Nagy mouse line (Lomeli et al., 2000). However, significant developmental events occur between the onset of cre at embryonic day (E)9.5 and the appearance of spermatogonia in the postnatal testis. Additional cre activity is detected in the placenta, intestine, and neural tube. Excision of floxed DNA in primary spermatocytes and elongating spermatids is achievable using synaptonemal complex protein 1 Tg(Sycp1-cre)4Min (Vidal et al., 1998) and protamine 1 Tg(Prm-cre)58Og (O'Gorman et al., 1997), respectively, but the temporal expression of these transgenes is too late to be of use in undifferentiated spermatogonia. Meanwhile, the expression of cre driven by the growth differentiation factor 9 (Gdf9) and zona pellucida 3 (Lan et al., 2004;Lewandoski et al., 1997).We therefore selected the promoter of the premeiotic male and female germ cell-specific gene Stra8 to drive expression of improved Cre recombinase. Endogenous Stra8 is first expressed in ovarian germ cells at E12.5 and continues until E16.5, while in males it is first transcribed in early-stage spermatogonia in the postnatal testis and persists in premeiotic germ cells throughout adulthood (Menke et al., 2003;Oulad-Abdelghani et al., 1996). A 1.4 Kb Stra8 promoter fragment was recently fused to EGF...

Recent studies by Dorshkind, Yoder, and colleagues show that embryonic (E9) B-cell progenitors located in the yolk sac and intraembryonic hemogenic endothelium before the initiation of circulation give rise to B-1 and marginal zone B cells but do not give rise to B-2 cells. In studies here, we confirm and extend these findings by showing that distinct progenitors for B-1 and B-2 cells are present in the adult spleen. Furthermore, we show that the splenic B-cell progenitor population (lin -CD19 + /B220 lo/− /CD43 -) that gives rise to B-1 cells is likely to be heterogeneous because, in some recipients, it also gives rise to B cells expressing the marginal zone phenotype (B220 hi IgM hi IgD lo CD21 hi ) and to some (CD19 -CD5 hi ) T cells. In addition to the well-known function differences between B-1 and B-2, our studies demonstrate that substantial developmental differences separate these B-cell lineages. Thus, consistent with the known dependence of B-2 development on IL-7, all B-2 progenitors express IL-7R. However, >30% of the B-1 progenitors do not express this marker, enabling the known IL-7 independent development of B-1 cells in IL-7 −/− mice. In addition, marker expression on cells in the early stages of the B-2 development pathway (CD19 -/c-Kit lo/− / Sca-1 lo/− ) in adult bone marrow distinguish it from the early stages of B-1 development (CD19 hi /c-Kit + /Sca-1 + ), which occur constitutively in neonates. In adults, in vivo inflammatory stimulation (LPS) triggers B-1 progenitors in spleen to expand and initiate development along this B-1 developmental pathway. A fter many years of smoke but no clear fire, Dorshkind, Yoder, and colleagues (1) finally identified embryonic (E9) B-cell progenitors that give rise to B-1 and marginal zone (MZ) B cells in adoptive recipients, but do not give rise to B-2 cells. Located in the yolk sac and intraembryonic hemogenic endothelium before the initiation of circulation, these B-1 and MZ B progenitors predate the emergence of the well-known B-cell progenitors in the fetal liver, which collectively give rise to B-1, B-2, and MZ B cells (1-3). The fetal liver B-cell progenitors are detectable in the fetus from day 13 onwards. These progenitors start to give rise to mature IgM-bearing B cells shortly before birth and collectively continue to populate the B-cell compartments during neonatal life and beyond.

In the companion article by Yang and colleagues [Yang Y, et al. (2012) Proc Natl Acad Sci USA , 109, 10.1073/pnas.1121631109], we have shown that priming with glycolipid (FtL) from Francisella tularensis live-vaccine strain ( i ) induces FtL-specific B-1a to produce robust primary responses (IgM >>IgG); ( ii ) establishes persistent long-term production of serum IgM and IgG anti-FtL at natural antibody levels; and ( iii ) elicits FtL-specific B-1a memory cells that arise in spleen but rapidly migrate to the peritoneal cavity, where they persist indefinitely but divide only rarely. Here, we show that FtL rechallenge alone induces these PerC B-1a memory cells to divide extensively and to express a unique activation signature. However, FtL rechallenge in the context of a Toll-like receptor 4 agonist-stimulated inflammatory response readily induces these memory cells to migrate to spleen and initiate production of dominant IgM anti-FtL secondary responses. Thus, studies here reveal unique mechanisms that govern B-1a memory development and expression, and introduce B-1a memory as an active participant in immune defenses. In addition, at a practical level, these studies suggest previously unexplored vaccination strategies for pathogen-associated antigens that target the B-1a repertoire.

B-1a cells are primarily thought of as natural antibody-producing cells. However, we now show that appropriate antigenic stimulation induces IgM and IgG B-1a antibody responses and long-lived T-independent antigen-specific B-1a memory that differs markedly from canonical B-2 humoral immunity. Thus, we show here that in the absence of inflammation, priming with glycolipid (FtL) from Francisella tularensis live vaccine strain induces splenic FtL-specific B-1a to mount dominant IgM and activation-induced cytidine deaminase-dependent IgG anti-FtL responses that occur within 3-5 d of FtL priming and fade within 1 wk to natural antibody levels that persist indefinitely in the absence of secondary FtL immunization. Equally surprising, FtL priming elicits long-term FtL-specific B-1a memory cells (IgM>>IgG) that migrate rapidly to the peritoneal cavity and persist there indefinitely, ready to respond to appropriately administrated secondary antigenic stimulation. Unlike B-2 responses, primary FtL-specific B-1a responses and establishment of persistent FtL-specific B-1a memory occur readily in the absence of adjuvants, IL-7, T cells, or germinal center support. However, in another marked departure from the mechanisms controlling B-2 memory responses, rechallenge with FtL in an inflammatory context is required to induce B-1a secondary antibody responses. These findings introduce previously unexplored vaccination strategies for pathogens that target the B-1a repertoire.B-1 | memory B cells | vaccine