Derivatives of chitin oligosaccharides have been shown to play a role in plant organogenesis at nanomolar concentrations. Here we present data which indicate that chitin oligosaccharides are important for embryogenesis in vertebrates. We characterize chitin oligosaccharides synthesized in vitro by zebrafish and carp embryos in the late gastrulation stage by incorporation of radiolabeled N-acetyl-D-[U 14 C]glucosamine and by HPLC in combination with enzymatic conversion using the Bradyrhizobium NodZ ␣-1,6-fucosyltransferase and chitinases. A rapid and sensitive bioassay for chitin oligosaccharides was also used employing suspension-cultured plant cells of Catharanthus roseus. We show that chitin oligosaccharide synthase activity is apparent only during late gastrulation and can be inhibited by antiserum raised against the Xenopus DG42 protein. The DG42 protein, a glycosyltransferase, is transiently expressed between midblastula and neurulation in Xenopus and zebrafish embryogenesis. Microinjection of the DG42 antiserum or the Bradyrhizobium NodZ enzyme in fertilized eggs of zebrafish led to severe defects in trunk and tail development.Lipo-chitin oligosaccharides (LCOs) are signal molecules that were discovered during study of the root nodulation process in leguminous plants. Nitrogen-fixing root nodules are the result of an association of plants with bacteria belonging to the genera Rhizobium, Bradyrhizobium, and Azorhizobium, commonly called rhizobia. LCOs produced by rhizobia are key factors in the specific recognition processes that underlie the formation of root nodules (1-3). The basic structure of these LCOs is a ␤-1,4-linked N-acetylglucosamine (GlcNAc) tetraor pentasaccharide, which is N-acylated at the nonreducing glucosamine moiety (1, 2). The LCOs are synthesized and secreted by the Rhizobium nodulation (Nod) genes whose expression is induced by plant flavonoids. The NodC, NodB, and NodA proteins are involved in the synthesis of the core LCO structure and function as chitin oligosaccharide synthase, chitin oligosaccharide deacetylase, and acyl transferase, respectively. Other rhizobial enzymes function in the modification of the LCO core structure and are important for determining the host range of rhizobia. An example is the NodZ protein which transfers an ␣-1,6-linked fucose group to the C6 position of the reducing end glucosamine moiety (4; reviewed in ref. 5). The effects of LCOs are not restricted to leguminous plants since it was shown that they stimulate cell division in tobacco protoplasts (6) and that they can rescue a temperature-sensitive somatic embryogenic mutant of carrot (Daucus) (7).It was suggested that chitin oligosaccharides might also play a role in animal embryogenesis since Rhizobium NodC is homologous to the developmentally regulated DG42 protein of Xenopus laevis (8). The DG42 gene also shows homology with hyaluronan synthases and fungal chitin synthases. Homologues of the Xenopus DG42 were identified in zebrafish and mouse (9). In Xenopus, DG42 is only expressed during...

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 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.

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