Deciphering the heterogeneity of the Lyve1+ perivascular macrophages in the mouse brain.

Perivascular macrophages (pvMs) are associated with cerebral vasculature and mediate brain drainage and immune regulation. Here, using reporter mouse models, whole brain and section immunofluorescence, flow cytometry, and single cell RNA sequencing, besides the Lyve1+F4/80+CD206+CX3CR1+ pvMs, we identify a CX3CR1- pvM population that shares phagocytic functions and location. Furthermore, the brain parenchyma vasculature mostly hosts Lyve1+MHCII- pvMs with low to intermediate CD45 expression. Using the double Cx3cr1GFP x Cx3cr1-Cre;RosatdT reporter mice for finer mapping of the lineages, we establish that CD45lowCX3CR1- pvMs are derived from CX3CR1+ precursors and require PU.1 during their ontogeny. In parallel, results from the Cxcr4-CreErt2;Rosa26tdT lineage tracing model support a bone marrow-independent replenishment of all Lyve1+ pvMs in the adult mouse brain. Lastly, flow cytometry and 3D immunofluorescence analysis uncover increased percentage of pvMs following photothrombotic induced stroke. Our results thus show that the parenchymal pvM population is more heterogenous than previously described, and includes a CD45low and CX3CR1- pvM population.

Crumbs homologue 1 in polarity and blindness.

Several retinal dystrophies, including retinitis pigmentosa type 12 and Leber congenital amaurosis, are caused by a large variety of mutations in the CRB1 (Crumbs homologue 1) gene. This discovery led to an increased focus on the function of CRB1 and the Drosophila homologue Crumbs. In the present study, we review the current knowledge on Crumbs and its vertebrate homologues, their function in cell polarity and their pathogenicity in retinal degeneration.

Localization of an activin/activin receptor system in the porcine ovary.

The aim of this study was to locate a possible activin/activin receptor system within porcine ovaries containing functional corpora lutea. In situ hybridization was used to assess the gene expression of beta(A)- and beta(B)-activin subunits, and immunohistochemical studies were done to detect activin-A protein and activin receptor type II. mRNA expression of the beta(A)- and beta(B)-activin subunits was found in the granulosa from the unilaminar follicle stage onward, in the developing thecal layer of multilaminar and small antral follicles, in the theca interna of mid-sized antral follicles, in corpora lutea, and in the ovarian surface epithelium. Immunoreactive activin A protein could be detected at the same ovarian sites, but in thecal tissue of small antral follicles only. This protein was also demonstrated at the peripheral zone of oocytes from multilaminar and antral follicles. A positive immunoreaction for activin receptor was found in granulosa cells from multilaminar and older follicles and in oocytes from the earliest stages of follicular development onward. In late multilaminar follicles and in antral follicles, the oolemma was stained. Except for small antral follicles, a positive activin receptor immunoreaction was absent in the follicular theca. Activin receptor immunoreaction was furthermore present in corpora lutea and in the ovarian surface epithelium. It is concluded that, within porcine ovaries containing functional corpora lutea, an activin/activin receptor system is present in all intact follicles, the corpora lutea and the surface epithelium. Within follicles, granulosa and theca cells are the main sites of activin synthesis, while oocytes and granulosa cells are the main activin binding sites.

Comparison of anterior-posterior development in the porcine versus chicken embryo, using goosecoid expression as a marker.

During early embryonic development, pig and chicken embryos share striking morphological similarities. In the present study, the timing and location of expression of mRNA for goosecoid (gsc), a gene classically expressed in the nodal region of developing embryos, was examined and compared in preprimitive streak and gastrulating pig and chicken embryos. The expression of gsc appeared first in the hypoblast and second in the hypoblast of pig and chicken embryos. Because gsc expression in these tissues was not symmetrical, gsc appears to be a useful marker for the onset of embryonic polarity. During gastrulation in both species, gsc expression became confined to cells in and around the node, in the epiblast and mesoderm layers. The only significant species-related difference in the distribution of gsc expression at these stages of development was the presence of gsc expression in the gut endoderm of chicken but not pig embryos. Certainly, our results suggest that the molecular mechanisms that control anterior-posterior development in different classes of vertebrates are remarkably similar. In addition, we were able to demonstrate that the pattern of gsc expression appears to provide a more sensitive and accurate means of determining the developmental stage of early porcine embryos than the more commonly used trophoblast or embryoblast size. Using gsc expression and accompanying embryo morphometric changes, we were able to develop a four-point scale that may offer a more accurate means of quantifying early embryo development in pigs.

Uterine-embryonic interaction in pig: activin, follistatin, and activin receptor II in uterus and embryo during early gestation.

The mRNA expression patterns of activin beta(A) and follistatin in the uterus and embryo, the mRNA expression of the activin receptor II in the embryo, and the localization in the uterus of the immunoreactive activin beta(A) and the receptor II proteins in the uterus were examined at gestation days 7-12 after ovulation in pig. Activin was located predominantly at the mesometrial side of the uterus during all stages of pregnancy studied. Follistatin mRNA was absent in the uterus during these stages, suggesting that activin of uterine origin is not inhibited by intra-uterine follistatin. The receptor was localized throughout the glandular and luminal epithelium of the uterus. In the embryo, activin was expressed predominantly in the epiblast before unfolding, but after unfolding of the epiblast activin expression shifted to the trophoblast. The expression pattern of follistatin mRNA was contrarily to that of activin, i.e., before unfolding predominantly in the trophoblast (days 8-9), and shifted to the epiblast at day 10. During streak stages, follistatin was detected in the node and primitive streak. Activin receptor II mRNA was first detected at day 8 in the embryoblast. At day 11, it was expressed in trophoblast cells near the epiblast, and in the first ingressing mesoderm cells. During the streak stages, it was expressed predominantly in the trophoblast. The presence of activin and its receptor in uterine epithelium and early embryonic tissues indicate that both embryonic and uterine activin are involved in intra-uterine processes, such as attachment and early embryonic development. Mol. Reprod. Dev. 59: 390-399, 2001.

Expression of the organizer specific homeobox gene goosecoid (gsc) in porcine embryos.

The homeobox gene goosecoid is one of the first genes expressed in the organizer region of vertebrates and specifies future dorsal regions along the anterior/posterior axis of the embryo. Goosecoid (gsc) expression marks the posterior end of the anterior/posterior axis and might be a good marker to visualise early events in embryonic axis formation and differentiation processes in the epiblast at the onset of gastrulation. The aim of the present study was to evaluate gsc expression in porcine embryos. For this the homeobox containing region of the porcine gsc was isolated using RT-PCR. The sequence of the PCR product appeared to be highly homologous to the sequence in the mouse, human, and chicken. We concluded that the isolated region represents part of the porcine gsc messenger. Relative levels of gsc expression were estimated in porcine embryos from day 9 to day 12 of pregnancy. Gsc was expressed in embryos of all ages and localisation on one side of the embryoblast was demonstrated with in situ hybridisation on whole- mount embryos at day 10 of pregnancy. In embryos collected at day 13 of pregnancy gsc expression was localised anterior to the primitive streak. The correlation between embryo size and level of gsc expression was low. Levels and pattern of expression varied within and between litters collected at similar days of pregnancy. It is concluded that gsc expression can be used as an early marker of differentiation and to describe embryo diversity in the pig.

Effects of vasopressin and elimination of corticotropin-releasing hormone-target cells on pro-opiomelanocortin mRNA levels and adrenocorticotropin secretion in ovine anterior pituitary cells.

Although arginine-vasopressin (AVP) is reported to produce greater ACTH biosynthetic and secretary responses than does corticotropin-releasing hormone (CRH) in sheep anterior pituitary cells, neither factor appears to increase pro-opiomelanocortin (POMC) mRNA levels, as does CRH in the cells of some other species. Since only a fraction of cells that express POMC mRNA may be able to respond to AVP, the aim of this study was to further delineate the regulation of POMC mRNA in ovine anterior pituitary corticotrophs, as a whole and in functional subpopulations of corticotrophs. We measured the effects of AVP, CRH or activation of protein kinase C by phorbol myristate acetate (PMA) in cultured cells. We compared responses in intact populations with those of cultures from which CRH-target cells were pharmacologically eliminated. Dissociated adult ovine anterior pituitary cells were cultured overnight, treated with either vehicle (intact) or a CRH-toxin conjugate that specifically eliminates CRH-target cells (CRH-target-depleted), washed, returned to culture and subsequently challenged with vehicle, AVP (100 nM), CRH (10 nM) or PMA (1 microM) for 5 h. The media were assayed for ACTH by RIA and the cells for POMC mRNA by Northern blot analysis. In intact populations, AVP and CRH increased ACTH secretion from 6.5 +/- 1.2 to 216 +/- 22 and 81 +/- 14 ng/well respectively, but only AVP caused an increase in steady-state POMC mRNA levels (+48 +/- 10%). Direct activation of protein kinase C with PMA mimicked the effect of AVP on ACTH secretion (318 +/- 16 ng/well), but did not alter POMC mRNA levels. In CRH-target-depleted populations, control ACTH secretion (11 +/- 3 ng/well) and POMC mRNA (+69 +/- 7%) were elevated, compared with intact populations. AVP (55 +/- 8 ng/well) and PMA (120 +/- 17 ng/ well), but not CRH, increased ACTH secretion; POMC mRNA was not significantly elevated by any of the treatments. Taken together, these data provide further support for the notion of dissociation between secretion of ACTH and expression of POMC mRNA, and demonstrate that AVP increases steady-state POMC mRNA levels in ovine anterior pituitary cells. The data are also consistent with the concept that complex interactions, possibly including those between cells, influence ACTH secretion and steady-state POMC mRNA levels.