In many organisms the allocation of primordial germ cells (PGCs) is determined by the inheritance of maternal factors deposited in the egg. However, in mammals, inductive cell interactions are required around gastrulation to establish the germ line. Here, we show that Bmp4 homozygous null embryos contain no PGCs. They also lack an allantois, an extraembryonic mesodermal tissue derived, like the PGCs, from precursors in the proximal epiblast. Heterozygotes have fewer PGCs than normal, due to a reduction in the size of the founding population and not to an effect on its subsequent expansion. Analysis of ␤-galactosidase activity in Bmp4 lacZneo embryos reveals that prior to gastrulation, Bmp4 is expressed in the extraembryonic ectoderm. Later, Bmp4 is expressed in the extraembryonic mesoderm, but not in PGCs. Chimera analysis indicates that it is the Bmp4 expression in the extraembryonic ectoderm that regulates the formation of allantois and primordial germ cell precursors, and the size of the founding population of PGCs. The initiation of the germ line in the mouse therefore depends on a secreted signal from the previously segregated, extraembryonic, trophectoderm lineage.

Lgr5 stem cells reside at small intestinal crypt bottoms, generating both the enterocyte and secretory lineage. Entry into the latter epithelial lineage requires silencing of Notch signaling. The Notch ligand Dll1 is strongly up-regulated in a small subset of immediate stem cell daughters. Lineage tracing utilizing a novel Dll1GFP-ires-CreERT2 knock-in mouse reveals that single Dll1high cells generate small, short-lived clones of all four secretory cell types. In culture, sorted Dll1high cells can form long-lived organoids when briefly exposed to Wnt3A. When Dll1 cells are genetically marked prior to tissue damage, significant numbers of stem cell tracing events occur. Lineage specification therefore occurs already in the earliest stem cell daughters through Notch lateral inhibition. Yet, specified secretory progenitors display plasticity and can regain stemness upon tissue damage.

Defined growth conditions are essential for many applications of human embryonic stem cells (hESC). Most defined media are presently used in combination with Matrigel, a partially defined extracellular matrix (ECM) extract from mouse sarcoma. Here, we defined ECM requirements of hESC by analyzing integrin expression and ECM production and determined integrin function using blocking antibodies. hESC expressed all major ECM proteins and corresponding integrins. We then systematically replaced Matrigel with defined medium supplements and ECM proteins. Cells attached efficiently to natural human vitronectin, fibronectin, and Matrigel but poorly to laminin ؉ entactin and collagen IV. Integrin-blocking antibodies demonstrated that ␣V␤5 integrins mediated adhesion to vitronectin, ␣5␤1 mediated adhesion to fibronectin, and ␣6␤1 mediated adhesion to laminin ؉ entactin. Fibronectin in feeder cell-conditioned medium partially supported growth on all natural matrices, but in defined, nonconditioned medium only Matrigel or (natural and recombinant) vitronectin was effective. Recombinant vitronectin was the only defined functional alternative to Matrigel, supporting sustained self-renewal and pluripotency in three independent hESC lines. STEM

CALUX assay, only o,p′-DDT demonstrated weak dose-related estrogenic activity in vivo. To determine if differences in reporter gene activity may be explained by differential affinity of (xeno)estrogens to human and zebrafish ERs, fulllength sequences of the zebrafish ER subtypes R, , and γ were cloned, and transactivation by (xeno)estrogens was compared to human ERR and ER . Using transiently transfected recombinant ER and reporter gene constructs, EE2 also showed relatively potent activation of zebrafish ERR and ER compared to human ERR and ER . Zebrafish ER and ERγ showed higher transactivation by (xeno)-estrogens relative to E2 than human ER .

Cycling intestinal Lgr5+stem cells are intermingled with their terminally differentiated Paneth cell daughters at crypt bottoms. Paneth cells provide multiple secreted (e.g., Wnt, EGF) as well as surface-bound (Notch ligand) niche signals. Here we show that ablation of Paneth cells in mice, using a diphtheria toxin receptor gene inserted into the P-lysozyme locus, does not affect the maintenance of Lgr5+stem cells. Flow cytometry, single-cell sequencing, and histological analysis showed that the ablated Paneth cells are replaced by enteroendocrine and tuft cells. As these cells physically occupy Paneth cell positions between Lgr5 stem cells, they serve as an alternative source of Notch signals, which are essential for Lgr5+stem cell maintenance. Our combined in vivo results underscore the adaptive flexibility of the intestine in maintaining normal tissue homeostasis.

We describe the cloning and initial characterization of a novel cDNA from human embryonal carcinoma (EC) cells. This cDNA, which we named human growth di erentiation factor 3 (hGDF3), encodes the homologue of mouse GDF3, a TGFb superfamily member belonging to the Growth/Di erentiation Factors. We have analysed the expression of hGDF3 in human embryonal carcinoma cell lines and in primary testicular germ cell tumours of adolescents and adults (TGCTs). Expression of hGDF3 in human EC cell lines is stem cell-speci®c, is downregulated upon RA-mediated di erentiation and is increased upon culture of the cells in the presence of activin A. In TGCTs, hGDF3 expression is low in seminomas, while expression in non-seminomas is readily detectable and appears to be associated with the EC and yolk sac components in the tumours. We have also mapped the hGDF3 locus to the short arm of human chromosome 12, a region consistently overrepresented in human testicular germ cell tumours. Thus, hGDF3 represents an embryonal carcinoma stem cell-associated marker both in vitro and in vivo.

The Bm86 antigen, as originally identified in Boophilus microplus, is the basis of commercial tick vaccines against this tick species. The potential for using this antigen or homologues of the antigen in vaccination against other tick species has been assessed. We have conducted vaccine trials in cattle using the B. microplus-derived recombinant Bm86 vaccine (TickGARD) using pairs of vaccinated calves and control calves. These were infested with B. microplus and Boophilus decoloratus larvae simultaneously. For both species, the numbers of engorged female adult ticks, their weight and egg-laying capacity were all reduced, leading to a reduction in reproductive capacity of 74% for B. microplus and 70% for B. decoloratus. Hyalomma anatolicum anatolicum ticks were fed both as immatures as well as adults on vaccinated calves and non-vaccinated controls. There was an overall 50% reduction in the total weight of nymphs engorging on vaccinated calves, and a suggestion of a subsequent effect on feeding adults. For Hyalomma dromedarii there was a 95% reduction in the number of nymphs engorging and a further 55% reduction in weight of those ticks surviving. Rhipicephalus appendiculatus and Amblyomma variegatum ticks were fed simultaneously both as immatures and subsequently as adults. There was no evidence for a significant vaccination effect. Finally, the amino acid sequence of a Bm86 homologue found in H. a. anatolicum unequivocally demonstrated the conservation of this molecule in this species. Our strategy for the development of multivalent anti-tick vaccines is discussed in relation to these findings.

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