Intestinal fatty acid binding protein gene expression reveals the cephalocaudal patterning during zebrafish gut morphogenesis.

Intracellular fatty acid-binding proteins (FABPs) are small and highly conserved cytoplasmic proteins that bind long-chain fatty acids and other hydrophobic ligands. We have examined, as a model for studying intestinal epithelial cell differentiation, the cell-specific and spatio-temporal expression of intestinal fatty acid-binding protein (i-fabp) gene during zebrafish larval development. After molecular cloning of zebrafish I-FABP cDNA, whole-mount in situ hybridization analysis revealed that i-fabp is expressed in the intestinal tube around day 3 postfertilization. By day 4, highest level of i-fabp transcript is encountered in the proximal columnar epithelium. From day 5 onwards, i-fabp is strongly expressed in the anterior intestine and its rostral expansion, slightly expressed in the esophagus mucosa and rectum, while no mRNA could be detected in the posterior intestine. Therefore, the regional differentiation of the intestine precedes first feeding and complete yolk resorption. I-fabp expression in the anterior intestine of the fed larvae is correlated with an intracellular storage of lipid droplets in the enterocytes and the massive synthesis of very low-density lipoprotein particles. In conclusion, the cephalocaudal expression pattern of i-fabp demarcates early during zebrafish gut morphogenesis the anterior fat absorbing to posterior cells of the intestine. This gene could be used as a marker for screening for mutations that affect the events of intestinal epithelial differentiation, cephalocaudal patterning, and asymmetric gut looping morphogenesis.

Apolipoprotein E gene expression correlates with endogenous lipid nutrition and yolk syncytial layer lipoprotein synthesis during fish development.

During embryogenesis of teleost fish, the formation of a yolk syncytial layer (YSL) enables the resorption of the yolk reserves and development up to the larval stage. We have examined the changes of the yolk cell structure in relation to yolk and oil-globule lipid utilization during development of the turbot (Scophthalmus maximus). After encapsulation by the YSL, resorption of the single, large oil globule occurred predominantly after yolk resorption and was slower in fasting larvae. The YSL was in contact with an enlarged perisyncytial space, but no vascular network or red blood cells were present within the walls of the yolk sac. Intrasyncytial channels infiltrated by pigmented lining cells were observed in the YSL surrounding the oil globule. Apolipoprotein E (apoE) has a prominent role in lipid metabolism because of its ability to interact with lipoprotein receptors. We performed molecular cloning of the putative low-density lipoprotein-receptor binding domain of turbot apoE. In situ hybridization analysis revealed a very high level of apoE transcripts in the YSL, while no expression could be detected in the intestine. YSL apoE expression was correlated with the synthesis of very low density lipoprotein (VLDL) particles. An extraordinarily high number of VLDL particles were poured into the perisyncytial space, and intrasyncytial channels enabled the transfer of yolk- and oil globule-derived lipids to the developing embryo or larva. The pattern of apoE mRNA distribution in relation to YSL lipoprotein synthesis indicates that apoE expression is a suitable molecular marker for monitoring endogenous lipid nutrition during the endoexotrophic period of teleost fish development.

Effects of extracellular environment on the osmotic signal transduction involved in activation of motility of carp spermatozoa.

The mechanism by which a hypo-osmotic shock activates motility of carp spermatozoa was studied. The direct role of osmolality at the axoneme was investigated after demembranation of spermatozoa with Triton X-100 and reactivation in various ionic or anionic solutions containing Mg-ATP: demembranated spermatozoa remain motile in solutions of osmolality up to 550 mOsm kg-1 while non-demembranated spermatozoa are immotile when osmolality rises above 250 mOsm kg-1 with the same salt solutions as well as in non-ionic solutions. Suspension in hypo-osmotic saline solutions triggered the swelling of native carp spermatozoa. No motility or swelling occurred above 200-300 mOsm kg-1 and this osmolality is probably that of the cytosol. The swelling of carp spermatozoa is the result of an entrance of water but this was not affected by pCMBS, an inhibitor of the aquaporin CHIP28, or by various inhibitors of the co-transport of water with ions. Various pharmacological agents that affect the motility of different sperm species had no effect on carp sperm motility when used under similar conditions. However, prolonged exposure to a solution devoid of K+ or Cl- affects the activation of motility in a reversible manner, suggesting that these ions have a role in the perception or transduction of the osmotic signal. Altering the concentration of intracellular second messengers such as Ca2+ and cAMP, and the pH did not affect the motility of carp spermatozoa. However, DMSO at 1-20% (400-3200 mOsm kg-1) affects the motility of carp spermatozoa 3-4 min after mixing. These results show that the activation signal of carp sperm motility differs from that known for spermatozoa of other species of fish such as trout. Our results indicate that the activation mechanism may involve a co-transport of ions or specific 'stretch-activated channels' that are sensitive to osmotic pressure.