Cre activity causes widespread apoptosis and lethal anemia during embryonic development

Cre-mediated excision of targeted loxP sites is widely used to delete or to activate gene expression in temporal or tissue-specific fashions. We examine three previously described cre alleles and find that Cre activity alone causes dramatic developmental defects, such as loss of hematopoietic activity and dramatically upregulated apoptosis in many embryonic tissues in two of these lines. These results demonstrate that cre expression generates spurious phenotypes that can confound genetics analyses. We also find that most recently published studies fail to include cre-positive controls, and thus may have attributed roles to a targeted gene, which were in reality partly or wholly due to Cre toxicity. This information will be critical in both evaluating previously published work using cre alleles and in designing future experiments.     

ADAM17 overexpression promotes angiogenesis by increasing blood vessel sprouting and pericyte number during brain microvessel development

The angiogenic process is precisely regulated by different molecular mechanisms, with a balance between stimulatory and inhibitory factors in embryonic development. Transmembrane proteins of the ADAM (a disintegrin and metalloprotease) family play a critical role in embryogenesis and are involved in protein ectodomain shedding, as well as cell-cell and cell-matrix interactions. In the present study, we found that ADAM17 is expressed spatiotemporally in the tectal layers during chicken embryonic development. To investigate the effect of ADAM17 overexpression on angiogenesis, chicken ADAM17 plasmids were transfected into the developing tectum in vivo by electroporation. Results showed that overexpression of ADAM17 induces morphological changes of brain microvessels, such as an increase in diameter, of capillary sprouting from radial microvessels and an increase in the number of pericytes, but not of endothelial cells. Our data suggest that overexpression of ADAM17 in the developing tectum promotes angiogenesis by increasing the number of pericytes and capillary sprouting in the radial vessels.     

Expression of CPI-17 in smooth muscle during embryonic development and in neointimal lesion formation

Ca²⁺ sensitivity of smooth muscle (SM) contraction is determined by CPI-17, an inhibitor protein for myosin light chain phosphatase (MLCP). CPI-17 is highly expressed in mature SM cells, but the expression level varies under pathological conditions. Here, we determined the expression of CPI-17 in embryonic SM tissues and arterial neointimal lesions using immunohistochemistry. As seen in adult animals, the predominant expression of CPI-17 was detected at SM tissues on mouse embryonic sections, whereas MLCP was ubiquitously expressed. Compared with SM α-actin, CPI-17 expression doubled in arterial SM from embryonic day E10 to E14. Like SM α-actin and other SM marker proteins, CPI-17 was expressed in embryonic heart, and the expression was down-regulated at E17. In adult rat, CPI-17 expression level was reduced to 30% in the neointima of injured rat aorta, compared with the SM layers, whereas the expression of MLCP was unchanged in both regions. Unlike other SM proteins, CPI-17 was detected at non-SM organs in the mouse embryo, such as embryonic neurons and epithelium. Thus, CPI-17 expression is reversibly controlled in response to the phenotype transition of SM cells that restricts the signal to differentiated SM cells and particular cell types. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Nicotine induces multinuclear formation and causes aberrant embryonic development in bovine

The present study was designed to investigate the effects of nicotine on development of bovine embryos derived from parthenogenetic activation (PA) and in vitro fertilization (IVF). Nicotine caused disfigured secondary meiotic spindle structures and affected embryonic development in a dose-dependent manner. Concentrations at 0.01-0.5 mM resulted in cleavage and blastocyst rates similar to the controls for both PA and IVF embryos. Nicotine at 2.0 and 4.0 mM significantly decreased the cleavage rates and none of the embryos developed beyond the 16-cell stage. Nicotine might disrupt the polymerization of microfilaments leading to impaired chromosome alignment or segregation, and induce the formation of polynuclei with a variety of abnormal nuclear structures such as 2-6 nuclei, 2-4 metaphase plates, 2-4 sets of anaphase/telophase plates, and the co-existence of polynuclei and 2-4 sets of anaphase/telophase plates. Nicotine adversely affected blastocyst chromosomal composition. Fifty-six to 70% of the IVF blastocysts and 71-88% of the PA blastocysts were polyploid and/or mixoploid after culture in 0.2-1.0 mM nicotine-containing media, which were higher (P < 0.05 or P < 0.01) than the controls. Cell numbers of the nicotine-cultured blastocysts were significantly lower than the control. In conclusion, nicotine induced disfigured spindles and irregular chromosome alignment and possibly impaired cytokinesis, which lead to decreased quality of the yielded blastocysts.     

Dynamical modelling of pattern formation during embryonic development

The combination of genetic and molecular biology techniques has uncovered the intricacies of several gene networks controlling developmental processes. In the face of such complex regulatory networks, developmental geneticists cannot rely on reasoning alone; a thorough understanding of the spatio-temporal properties of these networks clearly requires the use of proper computational tools and methods.     

The shedding activity of ADAM17 is sequestered in lipid rafts

The tumor necrosis factor-alpha (TNF) converting enzyme (ADAM17) is a metalloprotease-disintegrin responsible for the cleavage of several biologically active transmembrane proteins. However, the substrate specificity of ADAM17 and the regulation of its shedding activity are still poorly understood. Here, we report that during its transport through the Golgi apparatus, ADAM17 is included in cholesterol-rich membrane microdomains (lipid rafts) where its prodomain is cleaved by furin. Consequently, ADAM17 shedding activity is sequestered in lipid rafts, which is confirmed by the fact that metalloproteinase inhibition increases the proportion of ADAM17 substrates (TNF and its receptors TNFR1 and TNFR2) in lipid rafts. Membrane cholesterol depletion increases the ADAM17-dependent shedding of these substrates demonstrating the importance of lipid rafts in the control of this process. Furthermore, ADAM17 substrates are present in different proportions in lipid rafts, suggesting that the entry of each of these substrates in these particular membrane microdomains is specifically regulated. Our data support the idea that one of the mechanisms regulating ADAM17 substrate cleavage involves protein partitioning in lipid rafts.     

Peptide hydrolase activity during embryonic carp development]

It was established that in the carp the process of embryonic and early postembryonic development was accompanied by the increase in the activity of both the trypsin- and chemotrypsin-like peptide hydrolases. This increase is most evident in larvae with resorbing yolk. The total and protein nitrogen content suffers no changes during embryonic and early postembryonic development. The content of non-protein nitrogen increases from the stage of the middle of segmentation on attaining the maximum level in larvae with resorbing yolk.     

Analysis of pattern formation during embryonic development of Hydractinia echinata

Summary Patterning processes during embryonic development of Hydractinia echinata were analysed for alterations in morphology and physiology as well as for changes at the cellular level by means of treatment with proportioning altering factor (PAF). PAF is an endogenous factor known to change body proportions and to stimulate nerve cell differentiation in hydroids (Plickert 1987, 1989). Applied during early embryogenesis, this factor interferes with the proper establishment of polarity in the embryo. Instead of normal shaped planulae with one single anterior and one single posterior end, larvae with multiple termini develop. Preferentially, supernumerary posterior ends, which give rise to polyp head structures during metamorphosis, form while anterior ends are reduced. The formation of such polycaudal larvae coincide with an increase in the number of interstitial cells and their derivatives at the expense of epithelial cells. Treatment of further advanced embryonic stages causes an increase in length, presumably due to the general stimulation of cell proliferation observed in such embryos. Also, the spatial arrangement of cells (i.e. cells in proliferation and RFamide (Arg-Phe-amide immunopositive nerve cells) is altered by PAF. Larvae that develop from treated embryos display altered physiological properties and are remarkably different from normal planulae with respect to their morphogenetic potential: (1) Larvae lose their capacity to regenerate missing anterior parts; isolated posterior larva fragments form regenerates of a bicaudal phenotype. (2) In accordance with the frequently observed reduction of anterior structures, the capacity to respond to metamorphosis-inducing stimuli decreases. (3) The morphogenetic potential to form basal polyp parts is found to be reduced. In contrast, the potential to form head structures during metamorphosis increases, since primary polyps with supernumerary hypostomes and tentacles metamorphose from treated animals.     

Gene expression and pattern formation during early embryonic development in amphibians

Temporal and spatial gene expression and inductive interactions control the establishment of the body plan during embryogenesis in invertebrates and vertebrates. The best-studied vertebrate model system is the amphibian embryo. Seventy-five years after the famous organizer experiment of Hans Spemann and Hilde Mangold in 1924 our knowledge of the molecular mechanisms of the multi-step formation of embryonic axis has substantially improved. Although in the 30s and 40s the interest of many laboratories was focussed on neural induction (determination of the central nervous system), only crude factors from so-called heterogeneous inducers (liver, bone marrow, etc.,) could be isolated by the traditional biochemical techniques available at this time. An important breakthrough was the characterization and purification of a mesoderm inducing factor, the so-called vegetalizing factor (homologous to Activin) in highly purified from chicken embryos. Much later after the introduction of molecular techniques Vgl and Activin (both belonging to the TGF-β family) and FGFs could be identified as important factors for mesoderm formation. It was in the 90s that secreted neuralizing factors (chordin, noggin, follistatin and cerberus) could be detected, which are expressed at the dorsal side of the early embryo including the Spemann organizer. In contrast to the classical view, these proteins act as antagonists to factors like BMP-4 localized on the ventral side. Of special interest was the fact that inDrosophila sog, homologous to chordin, determines the ventral side, whiledpp, homologous toBMP-4, participates in the formation of the dorsal side. These data of evolutionary conserved genes in both invertebrates and vertebrates support the view that they are descendents of common ancestors, the urbilateralia, living around 300 million years ago. The expression of those genes coding for secreted proteins is closely related to inductive interactions between cells and germ layers. Recently it was shown that planar signals are not sufficient to generate a specific anterior/posterior pattern during the primary steps of neural induction, i.e., formation of the central nervous system in amphibians.     

Signals regulating muscle formation in the limb during embryonic development

Classically, somites have been the preparation of choice for the study of muscle development, while the limb bud is the preferred model of axis formation. Nevertheless, the limb bud offers some experimental advantages for muscle studies. This review describes the successive events involved in limb muscle formation during embryonic development, the properties of the key marker molecules and resumes our current knowledge of the signalling pathways involved.     

Subcellular translocation signals regulate Geminin activity during embryonic development

BACKGROUND INFORMATION: Geminin (Gem) is a protein with roles in regulating both the fidelity of DNA replication and cell fate during embryonic development. The distribution of Gem is predominantly nuclear in cells undergoing the cell cycle. Previous studies have demonstrated that Gem performs multiple activities in the nucleus and that regulation of Gem activation requires nuclear import in at least one context. In the present study, we defined structural and mechanistic features underlying subcellular localization of Gem and tested whether regulation of the subcellular localization of Gem has an impact on its activity in cell fate specification during embryonic development. RESULTS: We determined that nuclear localization of Gem is dependent on a bipartite NLS (nuclear localization signal) in the N-terminus of Xenopus Gem protein. This bipartite motif mapped to a Gem N-terminal region previously shown to regulate neural cell fate acquisition. Microinjection into Xenopus embryos demonstrated that import-deficient Gem was incapable of modulating ectodermal cell fate, but that this activity was rescued by fusion to a heterologous NLS. Cross-species comparison of Gem protein sequences revealed that the Xenopus bipartite signal is conserved in many non-mammalian vertebrates, but not in mammalian species assessed. Instead, we found that human Gem employs an alternative N-terminal motif to regulate the protein's nuclear localization. Finally, we found that additional mechanisms contributed to regulating the subcellular localization of Gem. These included a link to Crm1-dependent nuclear export and the observation that Cdt1, a protein in the pre-replication complex, could also mediate nuclear import of Gem. CONCLUSIONS: We have defined new structural and regulatory features of Gem, and showed that the activity of Gem in regulating cell fate, in addition to its cell-cycle-regulatory activity, requires control of its subcellular localization. Our data suggest that rather than being constitutively nuclear, Gem may undergo nucleocytoplasmic shuttling through several mechanisms involving distinct protein motifs. The use of multiple mechanisms for modulating Gem subcellular localization is congruent with observations that Gem levels and activity must be stringently controlled during cell-cycle progression and embryonic development.     

Genetic control of cuticle formation during embryonic development of Drosophila melanogaster

The embryonic cuticle of Drosophila melanogaster is deposited by the epidermal epithelium during stage 16 of development. This tough, waterproof layer is essential for maintaining the structural integrity of the larval body. We have characterized mutations in a set of genes required for proper deposition and/or morphogenesis of the cuticle. Zygotic disruption of any one of these genes results in embryonic lethality. Mutant embryos are hyperactive within the eggshell, resulting in a high proportion reversed within the eggshell (the "retroactive" phenotype), and all show poor cuticle integrity when embryos are mechanically devitellinized. This last property results in embryonic cuticle preparations that appear grossly inflated compared to wild-type cuticles (the "blimp" phenotype). We find that one of these genes, krotzkopf verkehrt (kkv), encodes the Drosophila chitin synthase enzyme and that a closely linked gene, knickkopf (knk), encodes a novel protein that shows genetic interaction with the Drosophila E-cadherin, shotgun. We also demonstrate that two other known mutants, grainy head (grh) and retroactive (rtv), show the blimp phenotype when devitellinized, and we describe a new mutation, called zeppelin (zep), that shows the blimp phenotype but does not produce defects in the head cuticle as the other mutations do.     

The angiogenesis inhibitor endostatin impairs blood vessel maturation during wound healing.

Endostatin is a cleavage product of collagen XVIII that strongly inhibits tumor angiogenesis. To determine if endostatin affects other angiogenic processes, we generated full-thickness excisional wounds on the back of mice that were systemically treated with recombinant murine endostatin. No macroscopic abnormalities of the wound healing process were observed. Histological analysis revealed normal wound contraction and re-epithelialization, but a slight reduction in granulation tissue formation and reduced matrix deposition at the wound edge. The blood vessel density in the wounds of endostatin-treated mice was not affected. However, ultrastructural analysis demonstrated severe abnormalities in blood vessel maturation. The wound vessels in the endostatin-treated mice were narrowed or closed with an irregular luminal surface, resulting in a severe reduction in the number of functional vessels and extravasation of erythrocytes. Endostatin treatment did not affect the expression level and localization of collagen XVIII mRNA and protein. Furthermore, the angiogenesis regulators vascular endothelial growth factor, angiopoietin-1, and angiopoietin-2 were normally expressed in the wounds of endostatin-treated mice. However, expression of the major wound matrix proteins fibronectin and collagens I and III was significantly reduced. This reduction is likely to explain the reduced density of the wound matrix. Our results demonstrate that endostatin treatment reduces the number of functional blood vessels and the matrix density in the granulation tissue, but does not significantly affect the overall wound healing process.     

Tissue-dependent regulation of protein tyrosine kinase activity during embryonic development

Protein tyrosine kinase activity was assayed in a variety of chicken tissues during embryonic development and in the adult. In some tissues protein tyrosine kinase activity decreased during embryonic development; however, in other tissues it remained high throughout development, it contrast to the level of protein tyrosine phosphorylation, which decreased during development. The highest levels of tyrosine kinase activity were detected in 17-d embryonic brain although only low levels of protein tyrosine phosphorylation were observed in this tissue. Several alternatives were examined in an effort to determine the mechanism responsible for the low levels of tyrosine phosphorylated proteins in most older embryonic and adult chicken tissues despite the presence of highly active tyrosine kinases. The results show that the regulation of protein tyrosine phosphorylation during embryonic development is complex and varies from tissue to tissue. Furthermore, the results suggest that protein tyrosine phosphatases play an important role in regulating the level of phosphotyrosine in proteins of many older embryonic and adult tissues.     

Thymidine kinase activity in cardiac muscle during embryonic and postnatal development

Cytoplasmic thymidine kinase from cardiac muscle of the rat has been characterized. It has a pH optimum of 9.0 and a K(m) value for thymidine of 1.6mum. The sedimentation coefficient of this enzyme in sucrose gradients is 4.5S, which represents a molecular weight of approx. 69000. Thymidine kinase prepared from cardiac muscle of foetal, neonatal and adult rats is inhibited by dTTP and dTDP; there is neither inhibition nor stimulation by dTMP, dCTP, dATP, dGTP or cyclic AMP. The activity of thymidine kinase in differentiating cardiac muscle of foetal and neonatal rats declines progressively with development, reaching adult values of almost zero by the fifteenth to seventeenth day of postnatal development. This represents a 70-fold decrease in enzyme activity from 3 days before birth to 17 days after birth. The loss of thymidine kinase activity in differentiating cardiac muscle correlates temporally with the cessation of DNA biosynthesis and the loss of cytoplasmic DNA polymerase activity in this tissue.     

Acid phosphatase activity in the chick ovary during embryonic development

Acid phosphatase activity was studied in the left and right ovaries of the chick during embryonic development. The cytochemical study indicated that local enzymatic activity is localized mainly in germ and somatic cells. From the results obtained in the study on the specific activity of this acid hydrolase, it can be inferred that the higher concentration of this enzyme in the right ovary would be determined by a decrease of total proteins in the total homogenate and in the cellular fractions of this organ. Besides, a decrease in the enzymatic activity of this ovary is not observed, but it occurs in the left ovary. This finding would indicate a lack of enzymatic segregation that could be related to the phenomenon of ovarian atrophy. Finally, the electrophoretic study indicated that it would apparently exist only one molecular form of the enzyme. Our results support the hypothesis that acid phosphatase would be involved in the atrophic processes of the right ovary during embryonary differentiation.