Adamts9 is widely expressed during mouse embryo development

ADAMTS metalloproteases constitute a family of 19 secreted protein or proteoglycan processing enzymes. ADAMTS9 and its closest mammalian relative, ADAMTS20, are related to gon-1, a metalloprotease required for gonadal morphogenesis in Caenorhabditis elegans. Although expressed at generally low levels in embryonic subectodermal mesenchyme, ADAMTS20 is required for melanoblast colonization of skin. Mutations in Adamts20 cause Belted, one of several white spotting alleles in the mouse. In contrast to Adamts20, we previously showed by Northern blotting that Adamts9 was expressed highly throughout mouse development. Using RNA in situ hybridization, we determined the spatial and temporal regulation of Adamts9 during mouse embryogenesis. At 7.5 dpc Adamts9 is expressed in the allantois, trophoblast, parietal endoderm and decidual tissue. At 9.5 dpc it is expressed in head mesoderm and in the developing heart. From 11.5 to 12.5 dpc, Adamts9 is strongly expressed in posterior mesoderm, in the craniofacial region, ventral body wall and diaphragm. After 14.5 dpc, Adamts9 was highly expressed in the mesenchyme of developing lung, kidney, and mesentery. It is expressed during skeletogenesis, being present from 13.5 dpc in perichondrium, in the proliferation zone of growth plates after 15.5 dpc and it is highly expressed in newly formed bone. It is expressed in vascular endothelium and during formation of the pituitary and cochlea, but expression in the central nervous system is limited to the floor plate of the diencephalon, to the ventricular zone of the cerebral cortex and to the choroid plexus.     

Cytokeratin 8 is a suitable epidermal marker during zebrafish development

We have cloned and characterized the zebrafish (Danio rerio) homologous cytokeratin 8 (zf-K8) cDNA. This cytokeratin belongs to the gene family of intermediate filaments and it is a component of the cytoskeleton of epithelial cells. Gene expression analysis during embryonic development and at adult stages presented here revealed that zf-K8 mRNA is inherited maternally and that it is present in the oocyte, the zygote and in the cleavage stage embryo. After mid blastula transition this gene is expressed in all surface cells, notably in those of the enveloping layer (EVL) and of the periderm, as well as in a subpopulation of the deep cells (DEL) presumed to be intestinal progenitors. During later embryonic stages zf-K8 mRNA is strongly expressed in the developing pectoral fin. In adult zebrafish, the zf-K8 gene is not only expressed in simple epithelia such as the colorectal intestine, but also, in contrast to other vertebrates, it is present in stratified skin and differentiated fins. These observations suggest that the zf-K8 gene is an appropriate epidermal marker during zebrafish ontogenesis.     

Acetylglutamate kinase is required for both gametophyte function and embryo development in Arabidopsis thaliana

The specific functions of the genes encoding arginine biosynthesis enzymes in plants are not well characterized. We report the isolation and characterization of Arabidopsis thaliana N-acetylglutamate kinase(NAGK), which catalyzes the second step of arginine biosynthesis. NAGK is a plastid-localized protein and is expressed during most developmental processes in Arabidopsis. Heterologous expression of the Arabidopsis NAGK gene in a NAGK-deficient Escherichia coli strain fully restores bacterial growth on arginine-deficient medium. nagk mutant pollen tubes grow more slowly than wild type pollen tubes and the phenotype is restored by either specifically through complementation by NAGK in pollen, or exogenous supplementation of arginine. nagk female gametophytes are defective in micropylar pollen tube guidance due to the fact that female gametophyte cell fate specification was specifically affected. Expression of NAGK in synergid cells rescues the defect of nagk female gametophytes. Lossof-function of NAGK results in Arabidopsis embryos not developing beyond the four-celled embryo stage. The embryo-defective phenotype in nagk/NAGK plants cannot be rescued by watering nagk/NAGK plants with arginine or ornithine supplementation. In conclusion,our results reveal a novel role of NAGK and arginine in regulating gametophyte function and embryo development, and provide valuable insights into arginine transport during embryo development.     

Fibromodulin is expressed by both chondrocytes and osteoblasts during fetal bone development

Fibromodulin, a keratan-sulfate proteoglycan, was first isolated in articular cartilage and tendons. We have identified fibromodulin as a gene regulated during BMP-2-induced differentiation of a mouse prechondroblastic cell line. Because expression of fibromodulin during endochondral bone formation has not been studied, we examined whether selected cells of the chondrocytic and osteoblastic lineage expressed fibromodulin. Fibromodulin mRNA was detected in conditionally immortalized murine bone marrow stromal cells, osteoblasts, and growth plate chondrocytes, as well as in primary murine calvarial osteoblasts. We, therefore, investigated the temporo-spatial expression of fibromodulin in vivo during endochondral bone formation by in situ hybridization. Fibromodulin was first detected at 15.5 days post coitus (dpc) in the perichondrium and proliferating chondrocytes. Fibromodulin mRNA was also detected at 15.5 dpc in the bone collar and periosteum. At later time points fibromodulin was expressed in the primary spongiosa and the endosteum. To determine whether fibromodulin was expressed during intramembranous bone formation as well, in situ hybridization was performed on calvariae. Fibromodulin mRNA was present in calvarial osteoblasts from 15.5 dpc. These results demonstrate that fibromodulin is developmentally expressed in cartilage and bone cells during endochondral and intramembranous ossification. These findings suggest that this extracellular matrix protein plays a role in both endochondral and intramembranous bone formation.     

Cyclin-dependent kinase 5 influences Rohon-Beard neuron survival in zebrafish

Cyclin-dependent kinase 5 (cdk5), a member of the cyclin-dependent kinase family, is expressed predominantly in post-mitotic cell populations. Unlike the other cdks, cdk5 is abundant and most active in differentiated neurons. Here, we describe the function of a cdk5 ortholog in zebrafish. Cdk5 catalytic activity is meager but present in early stages of development. However, at 24 h post-fertilization (hpf), the activity is remarkably higher and continues to be high through 48 and 72 hpf. Knocking down cdk5 by micro-injection of a specific siRNA resulted in decreased cdk5 protein level accompanied by reduced kinase activity. In the cdk5 siRNA-injected embryos, the number of primary sensory Rohon-Beard (RB) neurons was significantly reduced and there were more apoptotic cells in the brain. These phenotypes were rescued by co-injection of cdk5 mRNA. Within the first two days of development, RB neurons undergo apoptosis in zebrafish. To examine whether cdk5 has a role in RB neuron survival, cdk5 mRNA was injected into the one- to two-cell embryos. In these embryos, RB neuron apoptosis was inhibited compared with the uninjected control embryos. These results suggest that in zebrafish, cdk5 influences RB neuron survival and potentially regulates early neuronal development.     

The anaphase-promoting complex is required in both dividing and quiescent cells during zebrafish development

The anaphase-promoting complex/cyclosome (APC/C) regulates multiple stages of the cell cycle, most prominently mitosis. We describe zebrafish with mutations in two APC/C subunits, Cdc16 and Cdc26, whose phenotypes reveal a multifaceted set of defects resulting from the gradual depletion of the APC/C. First, loss of the APC/C in dividing cells results in mitotic arrest, followed by apoptosis. This defect becomes detectable in different organs at different larval ages, because the subunits of the APC/C are maternally deposited, are unusually stable, and are depleted at uneven rates in different tissues. Second, loss of the APC/C in quiescent or differentiated cells results in improper re-entry into the cell cycle, again in an apparently tissue-specific manner. This study is the first demonstration of both functions of the APC/C in a vertebrate organism and also provides an illustration of the surprisingly complex effects that essential, maternally supplied factors can have on the growing animal over a period of 10 days or longer.     

nemo-like kinase is an essential co-activator of Wnt signaling during early zebrafish development

Wnt/beta-catenin signaling regulates many aspects of early vertebrate development, including patterning of the mesoderm and neurectoderm during gastrulation. In zebrafish, Wnt signaling overcomes basal repression in the prospective caudal neurectoderm by Tcf homologs that act as inhibitors of Wnt target genes. The vertebrate homolog of Drosophila nemo, nemo-like kinase (Nlk), can phosphorylate Tcf/Lef proteins and inhibit the DNA-binding ability of beta-catenin/Tcf complexes, thereby blocking activation of Wnt targets. By contrast, mutations in a C. elegans homolog show that Nlk is required to activate Wnt targets that are constitutively repressed by Tcf. We show that overexpressed zebrafish nlk, in concert with wnt8, can downregulate two tcf3 homologs, tcf3a and tcf3b, that repress Wnt targets during neurectodermal patterning. Inhibition of nlk using morpholino oligos reveals essential roles in regulating ventrolateral mesoderm formation in conjunction with wnt8, and in patterning of the midbrain, possibly functioning with wnt8b. In both instances, nlk appears to function as a positive regulator of Wnt signaling. Additionally, nlk strongly enhances convergent/extension phenotypes associated with wnt11/silberblick, suggesting a role in modulating cell movements as well as cell fate.     

Role of Yes kinase during early zebrafish development

We have identified the Yes kinase in zebrafish eggs and investigated its role in development of the zebrafish embryo. In situ hybridization as well as immunofluorescence techniques demonstrated that Yes kinase is maternally expressed and is localized to the cortical region of the unfertilized egg. Fertilization resulted in concentration of Yes kinase to the blastodisc where it continued to be localized to the blastoderm cells through cleavage, gastrulation, and later development. Yes kinase activity was found to decrease abruptly at fertilization, then increase progressively during epiboly, and was maintained at high levels throughout gastrulation. The role of Yes kinase in development was tested by treating embryos with chemical protein tyrosine kinase (PTK) inhibitors such as 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d] pyrimidine (PP2) and by injection of antisense morpholinos. Both treatments resulted in the arrest of development at the beginning of the epiboly. Co-immunoprecipitation studies demonstrated that Yes kinase participates in a stable complex with focal adhesion kinase (FAK), which is phosphorylated in vitro. These results demonstrate that Yes kinase plays an important role in epiboly and indicate that Yes kinase participates in signaling by focal adhesion kinase during early development.     

The heat-inducible zebrafish hsp70 gene is expressed during normal lens development under non-stress conditions

In the present study, we show that the stress-inducible hsp70 gene in zebrafish is strongly and specifically expressed during normal lens formation from 28 to 38 hours post-fertilization, and is subsequently downregulated by 2 days of age. Only weak constitutive hsp70 mRNA signal was sporadically observed in other embryonic tissues. Similarly, transgenic fish carrying a 1.5 kb fragment of the hsp70 promoter linked to eGFP exhibited fluorescence only in the lens. In contrast, both the endogenous hsp70 gene and the transgene were strongly expressed throughout the embryo following heat shock at the same developmental stages.     

The ethylene biosynthetic and perception machinery is differentially expressed during endosperm and embryo development in maize

The maize endosperm undergoes programmed cell death late in its development so that, with the exception of the aleurone layer, the tissue is dead by the time the kernel matures. Although ethylene is known to regulate the onset of endosperm cell death, the temporal and spatial control of the ethylene biosynthetic and perception machinery during maize endosperm development has not been examined. In this study, we report the isolation of the maize gene families for ACC synthase, ACC oxidase, the ethylene receptor, and EIN2 and EIL, which act downstream of the receptor. We show that ACC oxidase is expressed primarily in the endosperm, and only at low levels in the developing embryo late in its development. ACC synthase is expressed throughout endosperm development but, in contrast to ACC oxidase, it is transiently expressed to a significantly higher level in the developing embryo at a time that corresponds with the onset of endosperm cell death. Only two ethylene receptor gene families were identified in maize, in contrast to the five types previously identified in Arabidopsis. Members of both ethylene receptor families were expressed to substantially higher levels in the developing embryo than in the endosperm, as were members of the EIN2 and EIL gene families. These results suggest that the endosperm and embryo both contribute to the synthesis of ethylene, and they provide a basis for understanding why the developing endosperm is especially sensitive to ethylene-induced cell death while the embryo is protected.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by G. Jürgens     

Lost-a-fin encodes a type I BMP receptor, Alk8, acting maternally and zygotically in dorsoventral pattern formation

TGFbeta signaling pathways of the bone morphogenetic protein (BMP) subclass are essential for dorsoventral pattern formation of both vertebrate and invertebrate embryos. Here we determine by chromosomal mapping, linkage analysis, cDNA sequencing and mRNA rescue that the dorsalized zebrafish mutant lost-a-fin (laf) is defective in the gene activin receptor-like kinase 8 (alk8), which encodes a novel type I TGFbeta receptor. The alk8 mRNA is expressed both maternally and zygotically. Embyros that lack zygotic, but retain maternal Laf/Alk8 activity, display a weak dorsalization restricted to the tail and die by 3 days postfertilization. We rescued the laf dorsalized mutant phenotype by alk8 mRNA injection and generated homozygous laf/alk8 mothers to investigate the maternal role of Laf/Alk8 activity. Adult fish lacking Laf/Alk8 activity are fertile, exhibit a growth defect and are significantly smaller than their siblings. Embryos derived from homozygous females, which lack both maternal and zygotic Laf/Alk8 activity, display a strongly dorsalized mutant phenotype, no longer limited to the tail. These mutant embryos lack almost all gastrula ventral cell fates, with a concomitant expansion of dorsal cell types. During later stages, most of the somitic mesoderm and neural tissue circumscribe the dorsoventral axis of the embryo. Zygotic laf/alk8 mutants can be rescued by overexpression of the BMP signal transducer Smad5, but not the Bmp2b or Bmp7 ligands, consistent with the Laf/Alk8 receptor acting within a BMP signaling pathway, downstream of a Bmp2b/Bmp7 signal. Antibodies specific for the phosphorylated, activated form of Smad1/5, show that BMP signaling is nearly absent in gastrula lacking both maternal and zygotic Laf/Alk8 activity, providing further evidence that Laf/Alk8 transduces a BMP signal. In total, our work strongly supports the role of Laf/Alk8 as a type I BMP receptor required for the specification of ventral cell fates.     

The cytoskeletal effector xPAK1 is expressed during both ear and lateral line development in Xenopus

xPAK1, a probable effector of stress activated MAP-kinase SAPK1/JNK activation and cytoskeletal dynamics, was found to be ubiquitously expressed within the Xenopus laevis ear and lateral line system during the development and differentiation of these organs. xPAK1 expression was very strong in the otic placode from its condensation, and expression continued in the otic vesicle up until stage 35/36, after which it abruptly ceased. At stage 29/30 expression occurred specifically in the epithelium of the otic vesicle, which includes the prospective sensorial epithelium. Expression of xPAK1 was also observed in the lateral line system from stage 35/36, at which stage the lateral line primordia have begun to migrate from the region of the otic vesicle. Lateral line expression continued at least until stage 37/38, at which time xPAK1 was noted in association with the differentiating lateral line organs. To our knowledge, xPAK1 is the first ubiquitous lateral line marker that is also expressed in the ear. In the context of previous studies, our data suggest that xPAK1 either plays a role in the differentiation of the mechano-sensors of the auditory system or in the formation of the otic vesicle epithelium and the lateral line primordia.     

The keratocan gene is expressed in both ocular and non-ocular tissues during early chick development.

Extracellular matrix (ECM) keratan sulfate proteoglycans (KSPGs) are core proteins with sulfated polylactosamine side chains (KS). The KSPG core protein keratocan gene (Kera) is expressed almost exclusively in adult vertebrate cornea, but its embryonic expression is little known. Embryonic chick in situ hybridization reveals Kera mRNA expression in corneal endothelium from embryonic day (E) 4.5, Hamburger-Hamilton (HH) 25, in stromal keratocytes from E6.5, HH30, and in iris distal surface cells from E8, HH34. As highly sulfated, antibody I22-positive KS increases extracellularly from posterior to anterior across the stroma, nerves enter and populate only anterior stroma and epithelium. RT-PCR and in situ hybridization demonstrate that developmentally regulated Kera mRNA expression initiates in midbrain and dorsolateral mesenchyme at E1, HH7, then spreads caudally in hindbrain and cranial and trunk mesenchyme flanking the neural tube through E2, HH20. Cranial expression extends ventrally through the developing head, and concentrates in mesenchyme surrounding eye anterior regions and cranial ganglia, and in subepidermal pharyngeal arch mesenchyme by E3.5, HH22. Kera expression in the trunk at E3.5, HH22 and E4.5, HH25, is strong in dorsolateral subepidermal, sclerotomal and nephrogenic mesenchymes, but absent in neural tube, dorsal root ganglia, nerve outgrowths, notochord, heart and gut. Early limb buds express Kera mRNA throughout their mesenchyme, then in restricted proximal and distal mesenchymes. I22-positive KS appears only in notochord in E3.5, HH22 and E4.5, HH25, embryos. Results suggest the hypothesis that keratocan, or keratocan with minimally sulfated KS chains, may play a role in structuring ECM for early embryonic cell and neuronal migrations.     

Cyclin-dependent kinase 5 is an upstream regulator of mitochondrial fission during neuronal apoptosis

Under physiological conditions, mitochondrial morphology dynamically shifts between a punctuate appearance and tubular networks. However, little is known about upstream signal transduction pathways that regulate mitochondrial morphology. We show that mitochondrial fission is a very early and kinetically invariant event during neuronal cell death, which causally contributes to cytochrome c release and neuronal apoptosis. Using a small molecule CDK5 inhibitor, as well as a dominant-negative CDK5 mutant and RNAi knockdown experiments, we identified CDK5 as an upstream signalling kinase that regulates mitochondrial fission during apoptosis of neurons. Vice versa, our study shows that mitochondrial fission is a modulator contributing to CDK5-mediated neurotoxicity. Thereby, we provide a link that allows integration of CDK5 into established neuronal apoptosis pathways.     

Mepe is expressed during skeletal development and regeneration

Matrix extracellular phosphoglycoprotein (Mepe) is a bone metabolism regulator that is expressed by osteocytes in normal adult bone. Here, we used an immunohistochemical approach to study whether Mepe has a role in murine long bone development and regeneration. Our data showed that Mepe protein was produced by osteoblasts and osteocytes during skeletogenesis, as early as 2 days postnatal. During the healing of non-stabilized tibial fractures, which occurs through endochondral ossification, Mepe expression was first detected in fibroblast-like cells within the callus by 6 days postfracture. By 10 and 14 days postfracture (the hard callus phase of repair), Mepe was expressed within late hypertrophic chondrocytes and osteocytes in the regenerating tissues. Mepe became externalized in osteocyte lacunae during this period. By 28 days postfracture (the remodeling phase of repair), Mepe continued to be robustly expressed in osteocytes of the regenerating bone. We compared the Mepe expression profile with that of alkaline phosphatase, a marker of bone mineralization. We found that both Mepe and alkaline phosphatase increased during the hard callus phase of repair. In the remodeling phase of repair, Mepe expression levels remained high while alkaline phosphatase activity decreased. We also examined Mepe expression during cortical bone defect healing, which occurs through intramembranous ossification. Mepe immunostaining was found within fibroblast-like cells, osteoblasts, and osteocytes in the regenerating bone, through 5 to 21 days postsurgery. Thus, Mepe appears to play a role in both long bone regeneration and the latter stages of skeletogenesis.     

Elfin is expressed during early heart development

Elfin (previously named CLIM1) is a protein that possesses an N-terminal PDZ domain and a C-terminal LIM domain. It belongs to the family of Enigma proteins. Enigma proteins are a family of cytoplasmic proteins that contain an N-terminal PDZ domain and a series of C-terminal LIM domains. By virtue of these two protein interacting domains, Enigma proteins are capable of protein-protein interactions. It has been proposed that Enigma proteins may act as adapters between kinases and the cytoskeleton. We have previously shown that Elfin is most abundantly expressed in the heart and it colocalizes with alpha-actinin 2 at the Z-disks of the myocardium. In this report, Elfin was shown to localize at the actin stress fibers of myoblasts, as revealed by green fluorescent protein (GFP) tagging. In situ hybridization and immunostaining showed that Elfin expression begins at an early stage in mouse development and is present throughout the developing heart. Taken together, our experimental results suggest that Elfin may play an important role in myofibrillogenesis and heart development.