Subtractive hybridization reveals tissue-specific expression of ahnak during embryonic development.

The gene product ahnak has been identified from extra-embryonic mesoderm cDNA enriched using a subtractive hybridization approach modified for using small amounts of starting material. Clones for cyclin D2 and H19 have also been isolated as being preferentially enriched in the extra-embryonic mesoderm compared with the embryo proper of embryonic day (E) 7.5 neural plate stage mouse embryos. The differential expression of these genes was confirmed at gastrulation stage using in situ hybridization. More detailed analysis of the human genomic ahnak sequence suggests that its highly repetitive structure was formed by unequal cross-over and gene conversion. During organogenesis, ahnak is expressed in a variety of tissues, including migratory mesenchyme. By E12.5, the major site of expression of ahnak is craniofacial mesenchyme. Immunohistochemical analysis has shown that ahnak protein is expressed mainly at the cell membrane of migratory mesenchymal cells, primarily in the nucleus of bone growth plate cells and mostly in the cytoplasm of differentiating nasal epithelia. The potential functions of ahnak are discussed in light of these results.

Embryonic expression and function of the chemokine SDF-1 and its receptor, CXCR4.

Directed cell movement is integral to both embryogenesis and hematopoiesis. In the adult, the chemokine family of secreted proteins signals migration of hematopoietic cells through G-coupled chemokine receptors. We detected embryonic expression of chemokine receptor messages by RT-PCR with degenerate primers at embryonic day 7.5 (E7.5) or by RNase protection analyses of E8.5 and E12.5 tissues. In all samples, the message encoding CXCR4 was the predominate chemokine receptor detected, particularly at earlier times (E7.5 and E8.5). Other chemokine receptor messages (CCR1, CCR4, CCR5, CCR2, and CXCR2) were found in E12.5 tissues concordant temporally and spatially with definitive (adult-like) hematopoiesis. Expression of CXCR4 was compared with that of its only known ligand, stromal cell-derived factor-1 (SDF-1), by in situ hybridization. During organogenesis, these genes have dynamic and complementary expression patterns particularly in the developing neuronal, cardiac, vascular, hematopoietic, and craniofacial systems. Defects in the first four of these systems have been reported in CXCR4- and SDF-1-deficient mice. Our studies suggest new potential mechanisms for some of these defects as well as additional roles beyond the scope of the reported abnormalities. Earlier in development, expression of these genes correlates with migration during gastrulation. Migrating cells (mesoderm and definitive endoderm) contain CXCR4 message while embryonic ectoderm cells express SDF-1. Functional SDF-1 signaling in midgastrula cells as well as E12.5 hematopoietic progenitors was demonstrated by migration assays. Migration occurred with an optimum dose similar to that found for adult hematopoietic cells and was dependent on the presence of SDF-1 in a gradient. This work suggests roles for chemokine signaling in multiple embryogenic events.

Reproducible proliferative responses of salamander (Ambystoma mexicanum) lymphocytes cultured with mitogens in serum-free medium.

There are several reports that proliferative responses (tritiated thymidine incorporation (3HTdR)) of salamander splenocytes cultured with phytohemagglutinin-P (PHA) or concanavalin A (Con A) in 1% fetal bovine serum (FBS)-supplemented medium are either statistically insignificant or never approach the magnitude typically observed in similarly treated cultures of frog lymphocytes. The present study confirms these findings, but also reports highly significant and reproducible PHA-induced proliferation of axolotl splenocytes and thymocytes when the medium is supplemented with 0.25% bovine serum albumin (BSA) rather than 1% FBS. In one study, splenocytes from six of six axolotls cultured in BSA-supplemented medium displayed a dose-dependent response to PHA with stimulation indices (SLs) ranging from 4.2 to 14.1. In contrast, SLs of PHA-treated cells from the same animals, cultured in parallel in FBS-supplemented medium, ranged from 0.8 to 3.0. In a kinetic study (cells harvested from days 3-7), maximal proliferation in BSA-supplemented medium was noted after 5 days; cells cultured in parallel in FBS-containing medium were not responsive to the mitogen at any time point. Although axolotl splenocytes do not exhibit PHA-stimulated growth in FBS-supplemented medium, they are reproducibly stimulated in this serum-containing medium by phorbol 12-myristate, 13-acetate (PMA). This suggests that FBS may interfere with (or does not support) some early step(s) in lectin-induced signalling, rather than with proliferation itself.

Immune system activation associated with a naturally occurring infection in Xenopus laevis.

Immune system activation correlated with a naturally occurring infection has been found in the South African clawed frog Xenopus laevis. The microorganism thought to be the cause of this infection is coccobacilloid and approximately 1 micron in diameter. Since this microorganism does not grow on conventional bacterial media and it has been observed intracellularly, it may be an obligate intracellular bacterium. It has been found in Xenopus peripheral blood and in highly vascularized organs such as the spleen and liver. Splenomegaly is the only pathology thus far described for infected frogs; infection is not associated with increased morbidity or mortality. This infection has been found in all outbred frogs examined in shipments from one South African and three separate North American vendors, and has been transmitted to animals bred and raised in our laboratory. This infection has a profound effect on the immune system of Xenopus. Significant numbers of splenocytes from infected individuals exhibit morphology commonly associated with activated T lymphocytes. There is constitutive production of T-cell growth factor (TCGF) and both IgM and IgY. Freshly harvested splenocytes from infected animals proliferate in response to a TCGF-containing supernatant, indicating that they express receptors for TCGF, a trait exclusively exhibited by activated lymphocytes. These splenocytes also show an increase in the activation marker recognized by the monoclonal antibody FJ17.