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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.

Next to a prominent nutritional role during larval growth the teleost yolk cell may function during early developmental processes as a source of inducing signals. In Xenopus mesoderm-inducing signals reside in the vegetal cells of the blastula and exert their function on the more animal located cells in the margin. Because of its position reminiscent of the vegetal cells in Xenopus, the yolk cells of teleost fishes may have a similar inductive capacity. Removal of the blastoderm from the yolk cell (YC) offers an experimental approach to study to what extent blastoderm differentiation and morphogenesis depend on inductive interaction with the YC. Expression of marker genes for dorsal-ventral and anterior/posterior determination such as no tail, goosecoid and caudal was examined in blastoderm explants, which were incubated until sibling controls reached the tail bud stage. Blastoderms removed at 3 h post fertilisation expressed rudimentary dorsal-ventral polarity. Apparently signals leading to this expression had reached the margin of 3-h blastoderms. Blastoderms removed at 4 h showed in addition to dorsal-ventral polarity notochord-like structures and also expression patterns of goosecoid and caudal suggesting that gastrulation movements had occurred.

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.

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.

A carp caudal cDNA of 1.3 kb was cloned after screening an early segmentation stage cDNA library with a probe produced by PCR using conserved homeobox sequences as primers and genomic DNA as template. The homeobox gene was called carp-cdxl. The gene appears highly similar to other vertebrate caudal homologs, especially the zebrafish gene cdx(Zf-cad). The possible relationship to homeobox genes within the Hox-C gene complexes is discussed. A weak expression of the gene, detected by in situ hybridization, was found shortly before gastrulation (at 25% epiboly) in cells likely to have a posterior fate. During gastrulation expression became stronger. At the early segmentation stage, cells of the neural keel in the area of the prospective spinal cord expressed the gene. During the progression of segmentation, expression retracted in a caudal direction. The tailbud expressed the gene throughout, but the somites lost expression shortly after their formation. Only the most lateral mesoderm cells maintained expression in the trunk area. Carp-cdxl was also expressed in the endoderm. At 24 h after fertilization the gene was only expressed in the tailbud. At 48 h, no expression could be detected. The expression pattern suggests a function for carp-cdxl in gastrulation and patterning along the anterior-posterior axis of the embryo.

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 Zfcth1 gene is, as the previously cloned carp cth1 gene, related to the mammalian TIS 11 family of primary response genes and encodes a protein with two putative CCCH zinc fingers. This report describes the RNA expression of this gene during oogenesis and early embryogenesis up to gastrulation in the zebrafish (Danio rerio). Maternal cth1 message is present in the ovary of 1-month-old fish and of adult fish in oocytes at all stages of maturation. In the youngest oocytes the message is localized in the cytoplasm all around the nucleus, in larger oocytes the message becomes restricted to the future animal pole of the embryo, and in mature oocytes the expression is sharply localized in the cortical layer under the micropyle. After ovulation the cth1 messenger spreads over the cytoplasmic cap and is distributed over the blastomeres during subsequent cleavages. In subsequent stages maternal expression of cth1 gradually disappears. From early epiboly stages onward embryonic cth1 expression is localized to the germ ring and the hypoblast cells in the central part of the embryonic shield. In the shield, cth1 expression largely overlaps with the area of gooscoid expression in the first involuting cells. In stages after 70% of epiboly cth1 expression diminishes and soon can no longer be detected in the embryo. Next to a developmental role in cell fate determination we propose a function for cth1 during oocyte maturation.

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.

Early development of cyprinid teleosts is summarized with special attention to gastrulation. Making use of comparison with Xenopus, functions are suggested for the uncleaved yolk cell, concerning induction and patterning processes before and during gastrulation. It is concluded that cyprinid development has a number of very specific aspects, involving morphogenesis and, very likely, gene functions. This should be realized when gene expression patterns during development are studied in this group of teleosts as a model of development in higher vertebrates.

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.

The embryonic ectoderm of the pig differentiated and became part of the outer barrier of the blastocyst (earlier formed by the trophectoderm alone) before shedding of the overlying polar trophectoderm around Day 10, thus securing the integrity of the rapidly expanding blastocyst. Ferritin, added to the medium of the blastocyst, was taken up rapidly by trophectoderm cells, but did not reach the blastocoele, and consequently no tracer was found within hypoblast cells. Embryonic ectoderm cells did not absorb the macromolecule, before or after loss of the polar trophectoderm. When ferritin was injected into the blastocoele, trophectoderm, hypoblast and embryoblast cells all absorbed the tracer. At Day 11, blastocyst diameter and embryoblast cell number varied widely and were hardly correlated. We suggest that embryoblast development may be a more reliable indicator for the developmental stage of a blastocyst than its diameter, which may merely be an indication of the viability of the trophoblast.

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 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.

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.

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.

The ability of the trophoblast of the ovine preimplantation blastocyst to take up and metabolise proteins has been investigated using two experimental approaches, microscopical and radiochemical. The ultrastructure of the expanded blastocyst obtained from 14 and 17 day pregnant ewes was examined. The morphology of tissues maintained in culture for 24 h has been compared with that of fresh tissues. After culture, the cellular morphology of the explants was well preserved. Fresh and 24 h cultured tissues were incubated with horseradish peroxidase and ferritin and these proteins subsequently were found to be localized in coated pits, caveolae and secondary lysosomes of the trophoblast. Comparison of the uptake of [3H]dextran and of 125I-labelled bovine serum albumin indicated that proteins could be taken up by cultured tissue by mechanisms in addition to simple fluid phase endocytosis. During culture of explants of blastocyst with l2SI-labelled bovine serum albumin, a large fraction of the radioactivity taken up by the tissue appeared in the TCA-soluble fraction of the culture medium indicating that cultured trophoblast hydrolysed proteins. That amino acids released from captured protein could be used for protein synthesis by the trophoblast was indicated by the labelling of tissue and medium proteins after culturing explants with β-lactamase labelled with [14C]leucine. A major product (Mr approximately 17X103) present in the medium was likely to have been ovine trophoblast protein-1. It is concluded that, during the expansion of the ovine blastocyst, the trophoblast has the ability to take up proteins, transport them to lysosomes and degrade them to amino acids which are used for protein synthesis. Thus proteins, as well as free amino acids, present in the histotrophe may be an important source of nitrogen for the sheep conceptus in the critical period just prior to implantation.

The formation of germ layers during gastrulation and the specification and patterning of the body axes are important events in the development of the embryo. The investigations described in this thesis aimed to isolate and characterize the distribution of transcripts of genes, in particular novel genes, that are expressed during the formation of mesoderm and the patterning of the anteroposterior axis in the carp (Cyprinus carpio), a cyprinid teleost fish. Such studies may contribute to a better insight in the molecular mechanisms underlying the above processes, in two respects. Firstly, the characterization of a gene's expression pattern is one of the first steps towards the elucidation of its function. Especially the characterization of novel genes may provide a key to new insights in development. Furthermore, in studies of development, it is of importance to have markers that identify specific cell types, for example early mesodermal precursor cells. The isolation of genes that are specifically expressed in certain cell types provides such markers.Two molecular approaches were chosen for gene isolation. Firstly, we specifically searched for homeobox genes, which encode transcription factors with important regulatory functions during development. A particular class of homeobox genes, the Hox genes, provides cells along the anteroposterior axis with positional information and the expression patterns of members of this class are excellent markers of position on this axis. Our second approach was a subtractive hybridization strategy. It was applied to isolate genes that are differentially expressed between the oocyte and the early segmentation stage, a period during which mesoderm is induced.For the identification of homeobox-containing sequences in a carp early segmentation stage cDNA library, we used a probe that was composed of a mixture of homeobox fragments, produced by PCR. The PCR primers were designed against the most conserved regions of the homeobox. This approach yielded a number of different genes of which two are described in this thesis. The gene cdx1(Chapter 2) is a member of the caudal class of homeobox genes and is expressed in ventrolateral cells of the embryo prior to gastrulation. During gastrulation, transcripts of this gene accumulate in the posterior half of the embryo. The functions of caudal class genes of Drosophila, mouse and Xenopus indicate that genes of this class mediate the specification of posterior positional values in the embryo. Because of their characteristic distribution, cdx1 transcripts are useful markers of (ventro) posterior position in the embryo and have been used as such in the studies of mesoderm formation and anteroposterior patterning in blastoderm explants, performed in our laboratory.A second gene isolated in the search for homeobox genes was the Hoxb-1 gene, described in Chapter 3. This gene belongs to the class of Hox genes, whose members are organized into clusters in the genome of many species. With expression reaching into the hindbrain, the Hoxb-1 gene is one of the most anteriorly expressed Hox genes. Its most prominent expression, especially during segmentation, is found in rhombomere 4. In the late segmentation stage embryo, Hoxb-1 expression is a valuable marker of this rhombomere and the neural crest cells at that level of the hindbrain.Chapter 4 describes the cloning of genes on the basis of their differential gene expression between the oocyte and the early segmentation stage, using a subtractive hybridization strategy. Fifteen genes, identified from the oocyte stage cDNA library, are expressed in early development, when mesoderm induction occurs, and their expression disappears before the beginning of segmentation. From the early segmentation stage cDNA library, 26 genes were selected whose expression was activated during segmentation but not yet in early development, coinciding with the differentiation of the mesoderm and the patterning of the anteroposterior axis. In total 27 genes appeared to code for novel proteins and are therefore candidates for further studies and may provide a better insight into molecular mechanisms underlying developmental processes. Also in light of the large scale mutagenesis screens of zebrafish that have recently been undertaken in a number of laboratories and for which the affected genes yet need to be molecularly identified, it is important that the search for novel genes continues for only few candidate sequences are available so far. The subtractive hybridization strategy described in this thesis appears a worthwhile technique to obtain such candidate sequences.Further investigations of these novel genes were restricted to a detailed characterization of the expression of one gene: cth1.Chapter 5 gives a description of the distribution of the mRNA transcripts of this gene during cleavage, blastula and gastrula stages. Whereas maternal cth1 mRNA is ubiquitously distributed in the blastomeres, the embryonically transcribed cth1 mRNA is expressed after the late blastula stage, in cells at the blastoderm margin which have mesodermal and endodermal fates. The cth1 transcripts disappear around midgastrulation, coinciding with the commitment of cells to the mesodermal (or endodermal) fate. In the cth1 protein, motifs containing three cysteines and one histidine (C3H) are present that are most likely zinc fingers, structures involved in the regulation of expression of target genes. The cth1 mRNA expression pattern and the gene's homology to the TIS11 family, a family of primary response genes whose expression is activated after treatment with for example growth factors, suggest a function for the cth1 gene of maintenance of the cellular potential in cells with mesodermal (and endodermal) fates, and in cells of cleavage stages. By inhibiting the expression of certain target genes, cth1 could prevent or delay the selection of certain differentiation pathways, such as for example the commitment to a mesodermal fate before midgastrulation.Proteins containing C3H motifs are expressed in a number of species.For example in C.elegans, the PIE-1 protein is required to keep germlineblastomeres totipotent during early development, most likely by suppressing the transcription in these blastomeres. In Chapter 6 the literature on the C3H class of proteins is shortly reviewed and the hypothesis is proposed that they may be widely involved in preserving cellular potency in specification events during development.In Chapter 7 the results presented in previous chapters are discussed, with emphasis on the proposed role for cth1 and how its activity could affect the fate of the cells expressing this gene.

Cell fates can be established either by preformation or by epigenesis. With respect to primordial germ cells (PGCs) it has been shown that the Amphibia exhibit both types of cell fate establishment. Therefore, it is important to study the germ cell origin of the evolutionary lower class of fishes.In order to study the origin and cell fate establishment of a specific cell type, two approaches are possible. Firstly, its morphological characteristics may be studied, and traced back to the earliest possible developmental stage. Using this approach, we studied in Barbus conchonius, a cyprinid fish, the presence, location and morphology of cells, containing nuage, an ultrastructural electron dense perinuclear material, generally accepted to be characteristic for germ cells (Chapter 2). The results show that nuage containing cells, PGCs, could be found from 10 h after fertilization (a.f.) onwards (around 100% epiboly). They translocate between 10 h and 12 h a.f. from a position within the mesoderm towards a position between mesoderm and yolk syncytial layer (YSL). However, PGCs remain separated from the YSL by extensions of endodermal cells. The location of PGCs at the stage of first identification may indicate that they originate within the mesoderm. Contacts with endodermal cells may be involved in the formation of their characteristics.A second approach for studying the origin of PGCs concerns the labeling of blastomeres during cleavage stages and following their progeny up to the stage of morphological recognition of PGCs (Chapter 3). Using this method, a cell lineage tracer, Lucifer Yellow - Dextran (LY-D), was injected into individual blastomeres of the 64 cell stage of Barbus conchonius embryos. Study of the fate of their progeny revealed that a lower layer cell (LLC) could give rise to both somatic cells and a number of PGCs (in about 25% of the cases), while injections into upper layer cells (ULC) only resulted in labeled somatic cells. The distribution of somatic progeny after injection of a certain blastomere was unpredictable with respect to both the later location within the embryo and the tissue type of its descendants. Unpredictable cell lineages were probably due to extensive intermingling of cells during epiboly. The results suggest that the formation of PGCs as well as that of somatic cells is an epigenetic process. As mentioned above, the PGCs may originate within the mesoderm (Chapter 2).Since in Barbus conchonius cells apparently are not yet committed to their fate during cleavage and start extensive intermingling during epiboly, the question arose when cell fate restrictions occur (Chapter 4). These restrictions may also be important for the determination of a diversity of cell types, including PGCs. Changes in communication properties between cells or cell groups may be correlated with differences of their developmental pathways. Since it was known that during early epiboly certain deep cells are dye-coupled to the yolk syncytial layer (YSL), injections of Lucifer Yellow (LY) into the YSL at several stages of epiboly were used in order to study changes in communication properties. The formation of a group of dye-coupled cells, indicating a group of gap junctionally communicating cells, was described. At the onset of epiboly LY appeared to be transferred from the YSL to all blastodermal cells. Between 40% and 60% epiboly we observed a ringshaped group of labeled cells, which probably had involuted during early gastrulation. This cell group correlated with the leading edge of the hypoblast, and was dye-uncoupled from the uninvoluted epiblast. From 60% epiboly onwards the blastodermal cells were dye-uncoupled from the YSL. Between 50% and 100% epiboly the ring-shaped labeled hypoblast was, due to convergent cell movements, gradually transferred towards a dorsally located bar-like structure. Gap junctions appear to connect cells with the same fate. Consequently, the appearance of part of the hypoblast as a dye-coupled cell layer, which is dye-uncoupled from the epiblast, may correlate with early restriction in cell fate. On the other hand, gap junctions between YSL and DC may be involved in the transfer of an inducing signal at the onset of gastrulation.Apparently, it is only from gastrulation onwards that developmental pathways of cell groups gradually disperse. Since cell determination appeared to be highly regulative, knowledge of the control of the directional mass cell migration during gastrula stages may be important for understanding cell fate establishment. Because fibronectin (FN) appeared to be involved in the guidance of migrating cells in amphibian embryos, the presence of FN during epiboly and gastrulation was studied in embryos of the common carp ( Cyprinus carpio ; Chapter 5). However, in order to establish that FN is also involved in early fish development, we first performed a pilot study in which the functional role of FN was blocked by treatment with GRGDS, interfering with interactions between FN and its receptor. Since this treatment resulted both in retardation of epiboly, and a lack of involution, it became interesting to study the localization of FN during respective stages. Using immunocytological methods, FN first appeared present on a number of cell membranes during early epiboly. During the progress of gastrulation (from 50% epiboly onwards) we observed a gradually increasing number of epiblastcells, which were labeled on their YSL facing surface. They were first present in front of the migratory hypoblast cells and later partly separated the hypoblast cells from the epiblast. The results suggest that also in cyprinid fishes FN may be involved in the guidance of gastrulating cells.In conclusion, PGCs in embryos of cyprinid fishes can be recognized from late gastrula stages onwards by the presence of nuage. They are supposed to arise epigenetically from the mesoderm, possibly by inducing influence of endodermal cells. Determination processes of both PGCs and somatic cells probably do not start until early gastrulation (50% epiboly). FN may be involved directly (induction processes) or indirectly (morphogenetic movements resulting in induction) in epigenetic processes.