Distribution of calretinin during development of the olfactory system in the brown trout, Salmo trutta fario: Comparison with other immunohistochemical markers

Immunocytochemical techniques were used to investigate the appearance and distribution of calretinin in the olfactory system of developing and adult brown trout (Salmo trutta fario L.). The earliest calretinin-immunoreactive (CR-ir) cells were detected in the olfactory placode of 5-mm embryos. In 8-mm embryos, a CR-ir olfactory nerve was observed. The number of CR-ir olfactory receptor cells increased rapidly, and in fry and adults they were characterized by light and electron microscopy as pertaining to three morphological types of receptor cell, called microvillous, ciliated and rod-like cells or crypt cells. Comparisons of the cells labeled with CR and with more general olfactory markers (acetylated tubulin and keyhole limpet haemocyanin) in alevins and fry revealed that CR-ir cells represent only a subpopulation of olfactory receptor cells. Large cells located in the primordial mitral cell layer were the first CR-ir neuronal population of the olfactory bulbs and were observed in 7-mm embryos. These cells express high HuC/D immunoreactivity and were negative for glutamic acid decarboxylase and tyrosine hydroxylase. CR immunoreactivity diminished with development and most large cells of the mitral cell layer were CR-negative in fry. In later embryos and in alevins, CR-ir granule-like cells were observed in the olfactory bulbs. Comparisons of the terminal fields of primary olfactory fibers labeled with CR and with a more general olfactory marker in the olfactory bulbs of fry and adults revealed significant differences, with most glomeruli of the dorsomedial field receiving CR-negative olfactory fibers. These results suggest new criteria for understanding the organization of the olfactory system of the trout, and hence of teleosts. Our results also suggest that CR is involved in specific functions in the olfactory system during development.     

Presence and development of thyrotropin-releasing hormone-immunoreactive amacrine cells in the retina of a teleost, the brown trout (Salmo trutta fario)

The presence of thyrotropin-releasing hormone-immunoreactive (TRHir) amacrine cells is described for the first time in the retina of a teleost. These amacrine cells were mostly located in the inner nuclear layer, with occasional perikarya in the ganglion cell layer. Their processes formed a conspicuous plexus at the level of the ganglion cell perikarya. The TRHir amacrine cells appeared in posthatching stages, with the total number in retinas of juveniles approximately four times the number of cells in adults. Two types of TRHir cells, large and small, can be distinguished in developing stages, small cells outnumbering large cells. The TRHir cells of adults appears mainly to correspond to large, multistratified amacrine cells of developing stages. The possibility of transient expression of TRH in small amacrine cells during development is discussed.     

Expression of the neurofibromatosis 2 (NF2) gene isoforms during rat embryonic development

The neurofibromatosis 2 (NF2) gene product, merlin, encodesa 595 amino acid protein with sequence similarity to a familyof proteins linking cell membrane proteins to the cytoskeleton.Two isoforms of merlin have been described which differ by thepresence (type 2 merlin) or absence (type 1 merlin) of exon16 sequences inserted into the extreme carboxyl terminus ofthe protein. To determine the role of this important negativegrowth regulator during normal embryonic development, the expressionof these two merlin isoforms was examined at representativestages of rat embryogenesis and in adult tissues. Partial sequenceanalysis of the rat merlin gene demonstrated striking aminoacid identity to the published mouse and human merlin gene sequences.In situ hybridization and RT—PCR analyses demonstratedthat rat merlin is widely expressed during embryogenesis andearly postnatal life in most tissues but becomes restrictedto the brainstem, cerebellum, dorsal root ganglia, spinal cord,adrenal gland and testis in adult animals. The elucidation ofthe pattern of merlin gene expression in adult and embryonictissues provides the foundations for future studies aimed atdetermining the function(s) of this protein during cell differentiationand embryonic development.     

Expression patterns of the Wtx/Amer gene family during mouse embryonic development

WTX/AMER1 is a novel negative regulator of the WNT/β‐catenin pathway with mutations detected in Wilms' tumors and an X‐linked sclerosing bone dysplasia. WTX/AMER1 (Fam123b) shares several domains of homology with two other recently identified proteins: AMER2 (Fam123a) and AMER3 (Fam123c). Here, we describe an in‐depth expression analysis of all three members of this gene family during mouse embryonic development. All genes were strongly expressed in the central as well as the peripheral nervous system, thus suggesting important roles of this gene family during neurogenesis. Specific expression was found in the retina, inner ear, and nasal epithelium. Outside of the nervous system Wtx/Amer1 showed the broadest expression domains including cephalic and limb mesenchyme, skeletal muscle, bladder, gonads, lung bud, salivary glands, and the kidneys. The widespread expression pattern of Wtx/Amer1, together with its role as a modulator of the Wnt signaling pathway, suggest that Wtx/Amer1 serves pleiotropic roles during mammalian organogenesis. Developmental Dynamics 239:1867–1878, 2010. © 2010 Wiley‐Liss, Inc.     

Cytogenetic studies of PCB77 on brown trout (Salmo trutta fario) using the micronucleus test and the alkaline comet assay

Polychlorinated biphenyls (PCBs) are stable pollutants, whichcan be found in almost every compartment of terrestrial andaquatic ecosystems. They are very lipophilic and therefore havethe potency of accumulating in the fat stores of animals. Themechanisms by which PCBs exert their adverse effects are stillunclear. It is known that PCBs induce some important biotransformationenzymes, but their mutagenic properties are still controversial.The DNA breakage and clastogenic potency of a planar PCB77 (3,3', 4, 4'-tetrachlorobiphenyl) was determined in vivo in fish,using the single cell gel electrophoresis or comet assay andthe micronucleus test, on erythrocytes of the brown trout exposedfor 3, 9 and 14 days to initial PCB concentrations of 780 and918 pg/ml, dissolved in the water. Blood was taken by a caudalpuncture and the erythrocytes were either deposited in an agarosegel (0.6%) for the comet assay or smeared directly on slidesfor the micronucleus test. Five fish were studied per treatmentand 50 and 2000 erythrocytes per concentration and per animalwere analysed for the comet assay and the micronucleus testrespectively. Ethyl methanesulphonate (EMS) at a concentrationof 25 mg/I water was used as a positive control. Although EMSinduced a statistically significant increase of single strandbreaks in the comet assay, in neither of the two tests used,were mutagenic effects due to PCB exposure observed. ³To whom correspondence should be addressed     

Promoter of the heat shock testis-specific Hsp70.2/Hst70 gene is active in nervous system during embryonic development of mice

The Hsp70.2/Hst70 gene is a unique member of the 70 kDa heat shock proteins multigene family whose activity is regulated developmentally; in adult mice and rats its expression is restricted mostly to meiotic and postmeiotic male germ cells. In aim to analyze activity of the Hsp70.2/Hst70 promoter in developing embryos we have constructed transgenic mice expressing EGFP reporter gene under control of the rat Hst70 promoter. The appearance of EGFP fluorescence coincides with series of major developmental events, such as extra-embryonic membranes formation, axial rotation, formation of neural tube and the primordium of central nervous system, formation of differentiated somites, extensive remodeling of the heart, development of fingers and toes, and sensory organs formation. Activity of the Hst70 promoter localizes mostly inside nervous system indicating the role of Hsp70.2/Hst70 gene in development of this system.     

Lineage and development of the parasympathetic nervous system of the embryonic chick heart

 We were interested in the contribution of the cardiac neural crest to the complete anterior and posterior nerve plexus of the chick heart. This includes the pathways by which these cardiac neural crest-derived neuronal precursors enter the heart. As lineage techniques we used the traditional quail-chick chimera in combination with the newly introduced technique of retroviral reporter gene transfer to premigratory cardiac neural crest cells. Retrovirally infected embryos (n=23) and quail-chick chimeras (n=19) between stages HH27 and 40, were immunohistochemically evaluated, using the lineage markers LacZ (retroviral reporter) and QCPN (anti-quail nuclear marker), respectively and the neuronal differentiation markers HNK-1, RMO-270 and DO-170. Between stages HH27 and 33, quail-derived and LacZ positive cells were situated around the arterial cardiac vagal branches at the arterial pole, and vagal branches along the anterior cardinal veins and the sinal vagal branch at the venous pole. From stage HH35 onward, QCPN/LacZ-positive cardiac ganglia were observed throughout the anterior and posterior plexus and were mainly concentrated in the subepicardium near the distal ends of the arterial cardiac vagal branches and the sinal cardiac vagal branch respectively. From stage HH36 both the anterior and posterior plexus contained a population of large cardiac ganglion cells and a population of smaller cells along nerve branches as well as in the cardiac ganglia, which means that differentiation starts in both plexus at the same time. Furthermore only nerve fiber connections between the anterior and posterior plexus were observed. These results show that the cardiac neural crest contributes to the cardiac ganglion cells from both the entire anterior and posterior plexus. Furthermore these results suggest that these precursor cells enter the arterial pole via the arterial cardiac vagal branches and the venous pole via the sinal cardiac vagal branch without intermixing. Finally we show that in addition to the cardiac ganglia, the cardiac neural crest contributes to small myocardial glia or undifferentiated cells along nerve fibers, and some myocardial nerve fibers as well as nerve tissue in the adventitia of the large veins at the venous pole and in the adventitia of the coronary arteries. Accepted: 30 March 1998     

polyhomeotic: a gene required for the embryonic development of axon pathways in the central nervous system of Drosophila

Hypomorphic alleles of the locus polyhomeotic (ph) produce multiple, homeotic-like transformations in adult flies that mimic dominant mutations in the Antennapedia and Bithorax complexes. Analysis of null alleles of ph has revealed a complex, embryonically lethal phenotype that includes cell death of the ventral epidermis and abnormalities in the patterns of expression of homeotic and segmentation genes. There is also a dramatic alteration in the pattern of axon pathways in the central nervous system, such that the wild-type array of segmentally repeated commissures and connectives is replaced by bundles of axons confined to the hemiganglia of origin. It is possible that this axonal phenotype is the result of loss of neuronal identity caused by abnormal homeotic and segmentation gene expression.     

The timing and sequence of events in the development of the human nervous system during the embryonic period proper

Summary A documented scheme of the early development of the human nervous system is presented. It is based on (I) reports of workers who personally studied staged embryos, and (2) personal observations and confirmations. The necessity of staged embryos in determining the precise sequence of developmental events is stressed.Supported in part by National Institutes of Health (U.S.A.) General Research Support Grant, School of Medicine, Wayne State University.     

Expression of cVg1 mRNA during chicken embryonic development

Using degenerated PCR-primers to identify known and novel BMPs that are expressed in the developing chicken heart, we identified not only BMP2, -4, and -7 mRNA, but also the TGFbeta superfamily member cVg1. The expression pattern of cVg1 mRNA was determined during chicken development from HH4 to HH44. In early developmental stages, cVg1 mRNA is expressed in the primitive streak, paraxial mesoderm, developing somites, and developing neural tube. Subsequently, cVg1 mRNA is expressed in the developing central and peripheral nervous system, retina, auditory vesicle, notochord, lung alveoli, and olfactory mucosa. In the heart, cVg1 is initially expressed through the linear heart tube, but becomes restricted to the forming chamber myocardium, in an expression domain similar to that of atrial natriuretic factor (ANF) mRNA.     

Expression of protease-activated receptor-2 during embryonic development.

Protease-activated receptor-2 (PAR-2) is the second member of a novel family of G-protein-coupled receptors, activated through proteolytic cleavage within the extracellular domain to reveal a newly formed amino terminus that acts as a tethered ligand causing receptor activation. PAR-2 is expressed in a number of adult tissues, but its distribution during development has not been characterized. Knowledge of the tissue distribution of PAR-2 during development will provide clues as to its function(s) in vivo. In the current immunohistochemical study, a polyclonal antibody raised against a peptide corresponding to the post-cleavage amino terminal sequence of PAR-2 was used to localize PAR-2 expression in developing mouse tissues. In the developing central nervous system and cardiac muscle, PAR-2 expression was detectable at embryonic day 12 and persisted throughout embryogenesis. At embryonic day 14, PAR-2 expression was strong in peripheral nerves, but either weak or absent in skin, bone, skeletal muscle, and blood vessels. In embryonic day 17 and postnatal day 1 hindlimbs, however, PAR-2 staining was observed throughout the layers of the epidermis, in osteoblasts, muscle fibers, and in vascular smooth muscle and endothelium. The pattern of PAR-2 expression observed during embryonic development and the association of expression with differentiation in certain tissues suggest compelling physiological roles for this novel receptor.