Distinct developmental expression of two elongase family members in zebrafish

Despite the known importance of long-chained polyunsaturated fatty acids (LC-PUFA) during development, very little is known about their utilization and biosynthesis during embryogenesis. Combining the advantages of the existence of a complete range of enzymes required for LC-PUFA biosynthesis and the well established developmental biology tools in zebrafish, we examined the expression patterns of three LC-PUFA biosynthesis genes, Elovl2-like elongase (elovl2), Elovl5-like elongase (elovl5) and fatty acyl desaturase (fad) in different zebrafish developmental stages. The presence of all three genes in the brain as early as 24 hours post fertilization (hpf) implies LC-PUFA synthesis activity in the embryonic brain. This expression eventually subsides from 72 hpf onwards, coinciding with the initiation of elovl2 and fad expression in the liver and intestine, 2 organs known to be involved in adult fish LC-PUFA biosynthesis. Collectively, these patterns strongly suggest the necessity for localized production of LC-PUFA in the brain during in early stage embryos prior to the maturation of the liver and intestine. Interestingly, we also showed a specific expression of elovl5 in the proximal convoluted tubule (PCT) of the zebrafish pronephros, suggesting a possible new role for LC-PUFA in kidney development and function.     

Developmentally regulated expression of the LRRTM gene family during mid-gestation mouse embryogenesis

We have analysed the expression during mid-gestation mouse development of the four member LRRTM gene family which encodes type 1 transmembrane proteins containing 10 extracellular leucine rich repeats and a short intracellular tail. Each family member has a developmentally regulated pattern of expression distinct from all other members. LRRTM1 is expressed in the neural tube, otic vesicle, apical ectodermal ridge, forebrain and midbrain up to a sharp central boundary. LRRTM2 is expressed in a subset of progenitors in the neural tube. LRRTM3 is expressed in a half somite wide stripe in the presomitic mesoderm adjacent to the boundary with the most recently formed somite. Additional expression is seen in the neural tube, forebrain and hindbrain. LRRTM4 is expressed in the limb mesenchyme, neural tube, caudal mesoderm and in three distinct regions of the head. Later expression occurs in a subset of the developing sclerotome. Each family member has a unique expression domain within the neural tube.     

Developmentally regulated expression of specific tau sequences

Tau protein undergoes a shift in its molecular mass and its electrophoretic complexity during early postnatal development. We have sequenced a tau cDNA from an adult rat brain expression library and have found two inserted sequences. One of these inserts predicts a fourth repeated sequence homologous to the other three in the carboxyl end of tau that have the property of microtubule binding. Oligonucleotide probes directed against the insert hybridized only to tau mRNA at postnatal time points, even though tau is first expressed as early as embryonic day 13. A probe directed against the junction revealed expression of non-insert-containing tau mRNA from embryonic day 14 until postnatal day 8, after which time there was an abrupt decline in the expression of this immature form. Comparison of the developmentally expressed tau sequences with those sequences obtained directly from Alzheimer paired helical filaments revealed the presence of both the mature and the immature tau mRNA sequences.     

Developmentally regulated expression of hemoglobin subunits in avascular tissues

We investigated the spatio-temporal profile of hemoglobin subunit expression in developing avascular tissues. Significant up-regulation of hemoglobin subunits was identified in microarray experiments comparing blastocyst inner cell masses with undifferentiated embryonic stem (ES) cells. Hemoglobin expression changes were confirmed using embryoid bodies (derived from in vitro differentiation of ES cells) to model very early development at pre-vascular stages of embryogenesis; i.e. prior to hematopoiesis. We also demonstrate, using RT-PCR, Western blotting and immunocytochemistry, expression of adult and fetal mouse hemoglobin subunits in the avascular ocular lens at various stages of development and maturation. Hemoglobin proteins were expressed in lens epithelial cells (cytoplasmic) and cortical lens fiber cells (nuclear and cell-surface-associated); however, a sensitive heme assay demonstrated negligible levels of heme in the developing lens postnatally. Hemoglobin expression was also observed in the developing eye in corneal endothelium and retinal ganglion cells. Gut sections showed, in addition to erythrocytes, hemoglobin protein staining in rare, individual villus epithelial cells. These results suggest a paradigm shift: hemoglobin subunits are expressed in the avascular lens and cornea and in pre-hematopoietic embryos. It is likely, therefore, that hemoglobin subunits have novel developmental roles; the absence of the heme group from the lens would indicate that at least some of these functions may be independent of oxygen metabolism. The pattern of expression of hemoglobin subunits in the perinuclear region during lens fiber cell differentiation, when denucleation is taking place, may indicate involvement in the apoptosis-like signaling processes occurring in differentiating lens fiber cells.     

The expression of two kallikrein gene family members in the rat kidney

The mRNAs for two kallikrein gene family members expressed in the rat kidney have been characterized. One mRNA (PS) has previously been found in the pancreas and submaxillary gland and encodes true kallikrein. The second mRNA (K1) encodes a novel kallikrein-like enzyme expressed in the kidney and submaxillary gland that retains many of the key amino acid residues for the characteristic enzymatic cleavage specificity of kallikrein. Two oligonucleotide hybridization probes specific for the K1 mRNA demonstrate that the K1 mRNA is expressed in the kidney and submaxillary gland, but in none of the other eight tissues known to express one or more members of the rat kallikrein gene family. The K1 mRNA is the dominant kallikrein-related mRNA of the kidney, expressed at roughly 10 times the level of the true kallikrein (PS) mRNA. In the submaxillary gland the K1 mRNA is expressed at roughly one-fourth the level of true kallikrein mRNA.     

Characterization of zebrafish PSD-95 gene family members

The PSD-95 family of membrane- associated guanylate kinases (MAGUKs) are thought to act as molecular scaffolds that regulate the assembly and function of the multiprotein signaling complex found at the postsynaptic density of excitatory synapses. Genetic analysis of PSD-95 family members in the mammalian nervous system has so far been difficult, but the zebrafish is emerging as an ideal vertebrate system for studying the role of particular genes in the developing and mature nervous system. Here we describe the cloning of the zebrafish orthologs of PSD-95, PSD-93, and two isoforms of SAP-97. Using in situ hybridization analysis we show that these zebrafish MAGUKs have overlapping but distinct patterns of expression in the developing nervous system and craniofacial skeleton. Using a pan-MAGUK antibody we show that MAGUK proteins localize to neurons within the developing hindbrain, cerebellum, visual and olfactory systems, and to skin epithelial cells. In the olfactory and visual systems MAGUK proteins are expressed strongly in synaptic regions, and the onset of expression in these areas coincides with periods of synapse formation. These data are consistent with the idea that PSD-95 family members are involved in synapse assembly and function, and provide a platform for future functional studies in vivo in a highly tractable model organism.     

Developmentally regulated expression of alpha 6 integrin in avian embryos.

The distribution pattern of the avian alpha 6 integrin subunit was examined during early stages of development. The results show that this subunit is prevalent in cells of the developing nervous system and muscle. alpha 6 is first observed on neuroepithelial cells of the cranial neural plate and trunk neural tube. With time, immunoreactivity becomes prominent near the lumen and ventrolateral portions of the neural tube, co-distributing with neurons and axons, particularly notable on commissural neurons. The alpha 6 expression pattern is dynamic in the neural tube, with immunoreactivity peaking by embryonic day 6 (stage 30) and decreasing thereafter. The ventral roots and retina exhibit high levels of immunoreactivity throughout development. In the peripheral nervous system, alpha 6 immunoreactivity first appears on a subpopulation of sympathoadrenal cells around the dorsal aorta and later in the dorsal root ganglia shortly after gangliogenesis. Immunoreactivity appears on prospective myotomal cells as the somites delaminate into the dermomyotome and sclerotome, remaining prominent on myoblasts and differentiated muscle at all stages. The mesonephros also has intense immunoreactivity. In the periphery, alpha 6 immunoreactive regions often in proximity to laminin, which is thought to be the ligand of alpha 6 beta 1 integrin.     

Developmentally regulated expression of temporally distinct beta-glucosidase isozymes in Dictyostelium discoideum

During development of Dictyostelium discoideum, the cellular specific activity of beta-glucosidase increases before aggregation, declines to low levels during pseudoplasmodium formation, and increases rapidly during culmination. In addition, two electrophoretically distinct isozymes of beta-glucosidase are present at different times of development. Using enzyme-specific monoclonal antibodies, we have shown that changes in the level of enzyme specific activity are closely paralleled by changes in the relative rate of beta-glucosidase synthesis in vivo and by corresponding changes in the relative cellular concentration of functional beta-glucosidase mRNA. Thus, the developmental synthesis of beta-glucosidase is controlled at a pretranslational level. Furthermore, our experiments have demonstrated that both beta-glucosidase isozymes consist of a single subunit of identical molecular weight. This result is consistent with the previous finding that both isozymes are encoded by the same gene and suggests that the isozymes differ solely with respect to post-translational modification.     

Developmentally regulated expression of cell surface carbohydrates during mouse embryogenesis

Cell surface carbohydrates undergo marked alterations during mouse embryogenesis. In preimplantation embryos, many carbohydrate markers show stage-specific expression in diverse ways. In early postimplantation embryos, certain carbohydrate markers are localized in defined regions in the embryo. Important carriers of stage-specific carbohydrates are the lactoseries structure (Gal beta 1----4GlcNAc) and the globoseries structure (Gal alpha 1----4Gal). Notably, the glycoprotein-bound large carbohydrate of poly-N-acetyllactosamine-type ([Gal beta 1----4GlcNAc beta 1----3]n) carries a number of markers preferentially expressed in early embryonic cells. These markers are of practical value in analyzing embryogenesis and cell differentiation. For example, in order to monitor in vitro differentiation of multipotential embryonal carcinoma cells, stage-specific embryonic antigen-1 (SSEA-1) and the Lotus agglutinin receptor have been used as markers of the undifferentiated cells, and the Dolichos agglutinin receptor has been used as a marker of extraembryonic endoderm cells. Developmental control of cell surface carbohydrates is attained by controlled expression of activities of key glycosyltransferases; for example, the activity of N-acetylglucosaminide alpha 1----3 fucosyltransferase is lost during in vitro differentiation of embryonal carcinoma cells to parietal endoderm cells, in parallel to the disappearance of SSEA-1. Accumulating evidence suggests that poly-N-acetyllactosamine-type glycans that are abundant in early embryonic cells are involved in cell surface recognition of these cells.     

Mutants of Dictyostelium discoideum blocked in expression of all members of the developmentally regulated discoidin multigene family

Mutant strains of D. discoideum are described that can complete morphogenesis and cytodifferentiation but which express vastly reduced levels of the galactose-binding lectins discoidin I and II (less than 1% and 1%-2% respectively) compared to the wild-type control. Mutant cells proceeding through development lack lectin activity, lectin protein, and specific lectin mRNA. In contrast, the genes encoding these proteins are present in their wild-type configurations in the genome. Since these proteins are encoded by four to five discrete genes, the mutations in these strains are most likely in genes involved in the regulation of the expression of members of this multigene family. The results also indicate that the discoidin lectins may not be required for fruiting body construction in this organism. Finally, coupled with the recent ability to transform D. discoideum, these mutants open the way to identification and isolation of regulatory genes and their products.     

Developmentally regulated gene expression in Artemia: Histone gene expression in newly hatched larvae

The synthesis of histones and presence of histone mRNA sequences in embryos and larvae of the brine shrimp, Artemia, were investigated. Radiolabeling of proteins synthesized in vivo followed by electrophoretic and fluorographic analysis confirmed the prediction that histone synthesis is coordinated with the wave of DNA replication in newly hatched larvae. No histone synthesis occurs during development of encysted embryos. Hybridization of cloned Artemia histone gene DNA to total cell RNA indicated that dormant encysted embryos do not contain “masked” messenger RNA.     

Developmentally regulated expression of the P2X3 receptor in the mouse cochlea

ATP-gated non-selective cation channels assembled from P2X₃ receptor subunits contribute to transduction and neurotransmitter signaling in peripheral sensory systems and also feature prominently in the development of the central nervous system. In this study, P2X₃ receptor expression was characterized in the mouse cochlea from embryonic day 18 (E18) using confocal immunofluorescence. From E18 to P6, spiral ganglion neuron cell bodies and peripheral neurites projecting to the inner and outer hair cells were labeled. The inner spiral plexus associated with the inner hair cell synapses had a stronger fluorescence signal than outer spiral bundle fibers which provide the afferent innervation to the outer hair cells. Labeling in the cell bodies and peripheral neurites diminished around P6, and was no longer detected after the onset of hearing (P11, P17, adult). In opposition to the axiom that P2X₃ expression is neuron-specific, inner and outer sensory hair cells were labeled in the base and mid turn region at E18, but at P3 only the outer hair cells in the most apical region of the cochlea continued to express the protein. These data suggest a role for P2X₃ receptor-mediated purinergic signaling in cochlear synaptic reorganization, and establishment of neurotransmission, which occurs just prior to the onset of hearing function.