Expression of Hoxb-1 during gastrulation and segmentation stages of carp (Cyprinus carpio).

This report describes the cDNA sequence and embryonic RNA expression pattern of carp Hoxb-1. Carp Hoxb-1 is a labial-like, homeobox-containing gene of the 3' end of the Hox gene cluster. The expression pattern in carp is compared to that of homologs in other vertebrates. As holds for other Hox genes, carp Hoxb-1 is expressed with highest intensity at a sharp anterior boundary, and expression fades out towards posterior. At later stages, gaps were found in the domain. The gene is expressed from late gastrulation onwards, first mainly in the hypoblast but later in all germ layers. Its most prominent expression area is rhombomere 4 (r4) of the hindbrain. Transcripts were also found in the neural tube, mesoderm (lateral, head and presomite), epidermis and neural crest. At 30 hours post fertilization, Hoxb-1 was still expressed in r4, in the anterior trunk neural tube and in the branchial arches posterior to r4. Hox genes are thought to be involved in the specification of positional values along the embryonic anterior-posterior axis, and Hoxb-1 expression in r4 is supposed to be important for specifying the unique identity of this hindbrain segment. The conserved expression in r4 suggests that this is also true for carp Hoxb-1.

Blastomeres and cells with mesendodermal fates of carp embryos express cth1, a member of the TIS11 family of primary response genes.

The carp cth1 gene, related to the mammalian TIS11 family of primary response genes, encodes a novel fish protein with two putative CCCH zinc fingers. This report describes the RNA expression of this gene during cleavage, blastula and gastrula stages of carp embryos. Cth1 mRNA is present in all cleavage stage blastomeres as a maternal message. After the late blastula stage, the maternal expression decreases, revealing a spot of higher expression at the margin of the blastoderm of the dome stage embryo. Further decrease of the maternal message reveals a ring of cth1 expressing cells at the blastoderm margin from the stage of 40% epiboly onwards. By alpha-amanitin treatment we established that this local cth1 expression is of zygotic origin. At the onset of gastrulation the cells of the cth1 ring involute, starting with those in the shield region, and at approximately 60% epiboly the ring is fully involuted and occupies the hypoblast layer. All cth1 transcripts have disappeared at completion of epiboly. We discuss a possible role for the putative cth1 protein during cleavage and gastrulation.

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.

Blastoderm structure, cell migration and formation of the embryonic shield during gastrulation in the carp (Cyprinus Carpio); a scanning electron microscopic study.

This paper describes an ultrastructural study of the cell movements during the gastrulation of the common carp, Cyprinus carpio, using scanning electron microscopy. The morphology of the deep cells was studied in several consecutive stages ranging from 0-100% epiboly. Furthermore, the formation of the embryonic shield was followed from its earliest appearance at 50% epiboly onwards. This paper gives morphological evidence for the existence of two different pathways for involving and convergent movements. Firstly, cells may move along the inner surface of the not (yet) involuted cells. Secondly, a much smaller group may use the YSL as their substrate. These results are discussed in the light of the hypothesis that the two migrating cell populations may be differently induced, subsequently leading to the formation of mesoderm and endoderm.

Morphological indications of secretion by uterine epithelial cells and changes in the ratio between acidic and basic uterine proteins during early pregnancy in Chinese Meishan pig.

In Chinese Meishan pig embryonic mortality appears relatively low compared to European breeds. Most of embryonic loss in pig is believed to take place during early pregnancy. It is of interest to know possible specific features associated with low mortality. Therefore, the ultrastructure of the endometrial epithelium of Meishan pig was studied between Days 4 and 12 of pregnancy, and compared with earlier results on Yorkshire/Dutch Landrace interbreed (Y/DL). Furthermore, total protein and the relative amounts of acidic and basic proteins were determined in the uterine flushings, and compared with earlier results on Dutch Landrace (DL). As holds for European breeds, uterine glandular and luminal epithelium have to be considered as functionally different cell populations. Their morphology differs and suggests the synthesis of different secretory products. The periods of secretion are not the same: the luminal epithelium shows signs of product release during the whole period studied, the glands deliver their secretions from Day 12. This is correlated with a sudden increase in total protein in uterine flushings. Between Days 4 and 12, the relative amount of acidic proteins decreases from 92% to 47% in DL and from 88% to 38% in Meishan, resulting in a shift from acidic protein dominance to a mild dominance of basic proteins in both breeds, but most prominent in Meishan.

The development of the stomach in clarias lazera and the intestinal absorption of protein macromolecules.

The development of the stomach of the teleost, Clarias lazera, during the early posthatching period, is described, and the developing stomach is compared with that of adult Clarias. The stomach develops in two distinct parts: the corpus, which differentiates first, and the pylorus. The corpus contains a mucous surface epithelium, arranged in folds, and a tubular gland system containing only one type of gland cell, to which the secretion of pepsinogen and HCl is attributed. The pyloric region does not contain tubular glands. From the ultrastructure of the gland cells, the 3H-thymidine labeling index, and the onset of acid production (as determined with pH indicators) it is concluded that a functional stomach is present in juveniles with a standard length of +/- 11 mm (approximately 12 days after fertilization at 23-24 degrees C). The ultrastructure of the intestinal epithelium has also been studied. The intestine consists of three segments, similar to those described for stomachless teleosts and a number of fish larvae. In larvae as well as in juveniles, the enterocytes of the second segment show pinocytosis of horseradish peroxidase, although in the juveniles the stomach has already developed. This second segment has the same relative length in all studied larvae and juveniles and is also present in adult Clarias. It is therefore concluded that the capacity to absorb protein macromolecules is not specifically related to the absence of a functional stomach in this teleost species.

The ultrastructure and renewal of the intestinal epithelium of the juvenile grasscarp, Ctenopharyngodon idella (Val.).

The intestinal absorptive epithelium of starved and fed fish has been studied electron microscopically. After feeding, cells of the proximal segment of the intestine show morphological characteristics of lipid absorption. Absorptive cells in the middle segment contain many pinocytotic vesicles in both fasted and fed specimens. Absorption of protein macromolecules is supposed to be one of the main functions of this part of the gut. In the most caudal part of the intestine, absorptive cells carry relatively few and short microvilli. The proximal and distal segments show structural indications of a function in osmoregulation. The renewal of the epithelium has been studied with light microscopic autoradiography, using tritiated thymidine. The intestinal mucosal fold epithelium represents a cell renewal system. The cells proliferate at the base of the fold and migrate towards the apex in 10--15 days at 20 degrees C. The functional absorptive cells proved to be generally present in the intestinal epithelium, including the proliferative area. Undifferentiated cells have not been identified. The results will be compared with data on absorption of lipid and protein macromolecules in teleostean and mammalian intestines and with descriptions of the cell renewal system in the mammalian intestine.

Embryonic and uterine development during early pregnancy in pigs.

Comparison of the timing of pig preimplantation development, alterations in the ultrastructure of embryonic germ layers, and cytological changes of the uterine epithelial cells leads to the supposition that a close relationship exists between embryonic and uterine development during early pregnancy. The results of in-vitro studies of embryonic development and of experiments concerning asynchrony between embryos and uterine environment confirm this supposition, especially as far as the post-hatching period is concerned. It is suggested that successive steps in embryonic germ layer differentiation may induce specific developmental events and secretory activity of the embryos. A mutual influence of maternal and embryonic tissues appears to exist, but we can only speculate about the causes of many of the described phenomena.

The pig blastocyst: its ultrastructure and the uptake of protein macromolecules.

Between days 8 and 11 of pregnancy spherical blastocysts from 0.3 to 10 mm in diameter were flushed from the uterine horns of Dutch Landrace pigs. A description of their ultrastructure is given, and the uptake of horseradish peroxidase and ferritin is demonstrated. The ultrastructure of the trophoblast was similar at all ages studied. The trophoblast which has many apical microvilli is able to take up and digest the macromolecules which were offered in the in vitro incubation medium. The hypoblast consists of flattened cells. In blastocysts 2 mm and larger, compact cells bearing microvilli are found below the embryoblast. Cell organelles indicating protein synthesis are found within hypoblast cells of such blastocysts. In the embryoblast, local concentrations of cell organelles are visible, indicating that differentiation has started. After the disappearance of Rauber's layer, which takes place when the blastocyst reaches a diameter of about 2 mm, superficial embryoblast cells develop short microvilli. The cells do not absorb ferritin or peroxidase but are dependent on the trophoblast for their food requirements. All cell layers in the blastocyst contain mitochondria that have characteristics of those found in steroid-producing cells. The significance of the uptake and digestion of macromolecules by trophoblast cells, the synthesis of protein by hypoblast cells and the possible synthesis of steroids is discussed with respect to the relationship between the cell layers of the blastocyst and in the context of concepto-maternal relationships.

REgional functional differentiation in the gut of the grasscarp, Ctenopharyngodon idella (Val.).

A regional differentiation--reflecting structural differences--of the intestine of larval and juvenile grasscarps can be illustrated by studying the activity of alkaline phosphatase and the uptake of orally administered horseradish peroxidase. Pinocytosis takes place in a welldefined area of about 23% of the length of the gut (segment II). Neither the rostral +/- 68% (segment I) nor the caudal +/- 9% (segment III) shows absorption of the enzyme. Alkaline phosphatase activity, mainly localized at the microvilli of the enterocytes is high in the first segment of the gut and low in the second segment. In larvae, the activity decreases sharply at the transition from segment I to segment II. The activity is weak or absent in the caudal third segment. Quantitative histochemical data are confirmed by biochemical analyses. Alkaline phosphatase activity is found all over the mucosal folds of the first segment, with relatively weak activity at the base and at the tip of the folds. This may be related to a renewal of the epithelium. Our results suggest that active absorption of digested food takes place mainly in the rostral first segment, while the uptake of macromolecules by pinocytosis is a function of the second segment. Comparison of the results with information available in literature leads to a rejection of the hypothesis that the uptake of protein macromolecules in Cyprinids is to be attributed to the absence of a stomach and therefore to an inefficient digestion of proteins.

The ultrastructure of the uterine epithelium of the pig during the estrous cycle and early pregnancy.

The ultrastructure of the endometrial epithelium of the pig was studied during the estrous cycle and early pregnancy up to implantation. Special attention was given to the luminal epithelium and morphological indications of protein synthesis. Although the general morphology of the luminal and glandular epithelia is similar (both tissues consist of secretory cells and ciliated cells at all the stages studied), it appears that the two epithelia should be considered as two functionally different units in the pre-implantation period. Morphological evidence suggests the presence of at least three different secretory products within luminal epithelial cells; they are released at different times, i.e. at estrus, between day 8 and 10 and after day 11. The glandular epithelium shows release of secretory products from day 10-11. Increasing amounts of glycogen were found within epithelial cells, especially in pregnant gilts from day 12. The possible significance of secretory activity of the epithelium is discussed in relation to the development of the embryos.

Differential susceptibility of early steps in carp (Cyrinus carpio) development to α-amanitin.

Carp embryos were dechorionated and their early development was studied in the presence or absence of a-amanitin. Cleavage and the formation of the enveloping layer and yolk syncytial layer were not influenced by the drug. However, a-amanitin largely blocked epiboly which started 6 h after fertilization in controls. Involution of deep cells, taking place during gastrulation movements, appeared to be blocked to a lesser degree. This might reflect differences in the degree to which maternal transcripts influence these developmental steps.

TTP, a C3H zinc finger protein gene, is expressed in mouse ovarian oocytes.

The gene TTP, encoding a C3H zinc finger protein of the TIS11 family, is expressed in growing mouse oocytes. The gene is downregulated in Graafian follicles shortly before ovulation. This corresponds to a possible function in regulation of maternal mRNA translation, a function attributed to related C3H class genes in Caenorhabditis elegans, zebrafish, and Xenopus.

The carp homeobox gene Ovx1 shows early expression during gastrulation and subsequently in the vagal lobe, the facial lobe and the ventral telencephalon.

The homeobox gene Carp-Ovx1 shows similarity to vertebrate and invertebrate Ovx genes and to Drosophila unplugged. Its expression pattern was studied by in situ hybridization in carp embryos and juveniles. During segmentation, expression becomes gradually limited to the neural tube. In juveniles up to 9 weeks old, cells in the ventral telencephalon, the facial lobe and the vagal lobe show Ovx1 expression, confining expression to parts with chemosensory projections.

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.

Proliferation and differentiation of intestinal epithelial cells during development of Barbus conchonius (Teleostei, Cyprinidae).

The processes of proliferation, cell division and differentiation of intestinal epithelial cells have been studied during development of the fish, Barbus conchonius. On the 3rd day, nearly all cells of the presumptive gut proliferate. Once the intestinal epithelium begins to differentiate, a decreasing percentage of proliferative cells can be found. On the 7th day, when intestinal folds start to develop, the proliferative cells become restricted to the future basal parts of the folds. Ultrastructural examination of 3H-thymidine-labeled cells and mitotic cells of 6-day-old larvae shows that functional enterocytes are proliferative. The same feature is suggested for older fish. Proliferating undifferentiated "dark" cells, characterized by many free ribosomes and a few organelles, are also present in the intestinal epithelium of larval fish; they are considered to be stem cells, mainly for goblet cells. Proliferating goblet cells and enteroendocrine cells were not observed. The latter cell type is scarce and has a long turnover time. A common feature of all these dividing cells is the presence of isolated spherical to cylindrical lamellar structures which may have lost contact with the cell membrane during prophase; they probably regain this contact by fusion with the cell membrane at the end of mitosis.

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.

Isolation of carp cDNA clones, representing developmentally-regulated genes, using a subtractive-hybridization strategy.

A subtractive-hybridization technique, combined with differential screenings and subsequent whole mount in situ hybridization (ISH) reactions, was used to isolate novel cDNA clones representing developmentally-regulated genes of carp. Small-scale differential screenings of an oocyte and a segmentation-stage cDNA library using oocyte-specific and segmentation stage-specific enriched probes, yielded 75 positive clones. ISH screening showed that 65% (15) of the oocyte-stage clones and 50% (26) of the segmentation-stage clones were indeed stage-specific. Partial sequence analysis suggests that approximately 65% of the 41 stage-specific clones represent novel genes. In addition, an Otxl clone was isolated. Two novel clones and the Otxl clone are of special interest for developmental studies. The clones represent genes that are locally expressed during embryonic development. The expression patterns of Otxl and one of the novel clones suggest functions in specification of the anterior-posterior axis. The three clones provide molecular markers for the study of gastrulation and the patterning of the a-p axis in teleosts.