Reprogramming of early embryonic blastomeres into endodermal progenitors by a Caenorhabditis elegans GATA factor

The END-1 GATA factor has been implicated in specifying endoderm in Caenorhabditis elegans and is the earliest known zygotic protein expressed in the lineage of E, the clonal endoderm progenitor. We report that ubiquitous end-1 expression during a critical period in embryogenesis causes all non-endodermal lineages to produce endoderm instead of ectoderm and/or mesoderm. END-1 expression bypasses the requirement for maternal SKN-1 and the maternal Wnt signaling pathway in endoderm formation. This suggests that a primary function of these maternal factors is to regulate zygotic end-1 expression, which is then sufficient to initiate the entire program for endoderm development.     

Conservation of BMP signaling in zebrafish mesoderm patterning

To investigate the conservation of mechanisms for mesodermal patterning between zebrafish and Xenopus, we isolated two cDNA clones encoding bone morphogenetic protein (BMP)-related proteins from a zebrafish cDNA library. Based on their predicted amino acid sequences, these two clones were designated as zbmp-2 and zbmp-4. Whole-mount in situ hybridization analysis revealed that in gastrula embryo, both genes were localized in the ventral part of the embryo, consistent with the proposed function of Xenopus BMP-4 in ventral mesoderm specification. zbmp-4 expression, however, was also seen in the embryonic shield, the most dorsal mesodermal structure. To examine the ability of zbmp-2 to ventralize mesoderm, we injected synthetic mRNA into zebrafish embryos and found that overexpression of this gene eliminated dorsal structures including notochord at both morphological and molecular level. In contrast, expression of ventral marker gene eve1 was expanded to the dorsal side. These effects are analogous to the ventralization of embryos caused by ectopic xBMP-4 expression. Taken together, one may conclude that the developmental mechanisms for mesodermal patterning regulated by BMPs are evolutionarily conserved between amphibians and teleosts.     

Regional cell movement and tissue patterning in the zebrafish embryo revealed by fate mapping with caged fluorescein

Determination of fate maps and cell lineage tracing have previously been carried out in the zebrafish embryo by following the progeny of individual cells injected with fluorescent dyes. We review the information obtained from these experiments and then present an approach to fate mapping and cell movement tracing, utilizing the activation of caged fluorescein-dextran. This method has several advantages over single-cell injections in that it is rapid, allows cells at all depths in the embryo to be marked, can be used to follow cells starting at any time during development, and allows an appreciation of the movements of cells located in a coherent group at the time of uncaging. We demonstrate that the approach is effective in providing additional and complementary information on prospective mesoderm and brain tissues studied previously. We also present, for the first time, a fate map of placodal tissues including the otic vesicle, lateral line, cranial ganglia, lens, and olfactory epithelium. The prospective placodal cells are oriented at the 50% epiboly stage on the ventral side of the embryo with anterior structures close to the animal pole, and posterior structures nearer to the germ ring.     

Conversion of ectoderm into a neural fate by ATH-3, a vertebrate basic helix-loop-helix gene homologous to Drosophila proneural gene atonal

We have isolated a novel basic helix-loop-helix (bHLH) gene homologous to the Drosophila proneural gene atonal, termed ATH-3, from Xenopus and mouse. ATH-3 is expressed in the developing nervous system, with high levels of expression in the brain, retina and cranial ganglions. Injection of ATH-3 RNA into Xenopus embryos dramatically expands the neural tube and induces ectopic neural tissues in the epidermis but inhibits non-neural development. This ATH-3-induced neural hyperplasia does not require cell division, indicating that surrounding cells which are normally non-neural types adopt a neural fate. In a Xenopus animal cap assay, ATH-3 is able to convert ectodermal cells into neurons expressing anterior markers without inducing mesoderm. Interestingly, a single amino acid change from Ser to Asp in the basic region, which mimics phosphorylation of Ser, severely impairs the anterior marker-inducing ability without affecting general neurogenic activities. These results provide evidence that ATH-3 can directly convert non-neural or undetermined cells into a neural fate, and suggest that the Ser residue in the basic region may be critical for the regulation of ATH-3 activity by phosphorylation.     

Conversion of a radioresistant phenotype to a more sensitive one by disabling erbB receptor signaling in human cancer cells

Inhibition of cell growth and transformation can be achieved in transformed glial cells by disabling erbB receptor signaling. However, recent evidence indicates that the induction of apoptosis may underlie successful therapy of human cancers. In these studies, we examined whether disabling oncoproteins of the erbB receptor family would sensitize transformed human glial cells to the induction of genomic damage by gamma-irradiation. Radioresistant human glioblastoma cells in which erbB receptor signaling was inhibited exhibited increased growth arrest and apoptosis in response to DNA damage. Apoptosis was observed after radiation in human glioma cells containing either a wild-type or mutated p53 gene product and suggested that both p53-dependent and -independent mechanisms may be responsible for the more radiosensitive phenotype. Because cells exhibiting increased radiation-induced apoptosis were also capable of growth arrest in serum-deprived conditions and in response to DNA damage, apoptotic cell death was not induced simply as a result of impaired growth arrest pathways. Notably, inhibition of erbB signaling was a more potent stimulus for the induction of apoptosis than prolonged serum deprivation. Proximal receptor interactions between erbB receptor members thus influence cell cycle checkpoint pathways activated in response to DNA damage. Disabling erbB receptors may improve the response to gamma-irradiation and other cytotoxic therapies, and this approach suggests that present anticancer strategies could be optimized.     

Conversion of anterior limb bud cells to ZPA signaling cells in vitro and in vivo

Following a graft of posterior (zone of polarizing activity or ZPA) cells into the anterior margin of the developing chick wing bud, anterior cells are induced to alter their developmental fate and form structures that are normally composed of posterior cells. When anterior cells are cultured under microdissociation conditions they develop ZPA signaling ability within 24 hr. ZPA signaling in these cultures is transient and once established the level of ZPA signaling declines with time in culture. ZPA signaling in anterior cells is sensitive to treatment with fibroblast growth factor-2 (FGF-2); the development of ZPA signaling is inhibited when nonsignaling anterior cells are cultured in the presence of FGF-2. Conversely, when anterior cells that have developed ZPA signaling are treated with FGF-2, ZPA signaling levels are maintained. Thus, our results suggest that FGF-2 maintains or stabilizes the positional character of anterior (nonsignaling) cells, as well as anterior ZPA signaling converted cells, and posterior (ZPA signaling) limb bud cells in vitro (R. Anderson, M. Landry, and K. Muneoka (1993) Development 117, 1421-1433). In addition, anterior cells will convert to ZPA signaling cells in vivo following apical ectodermal ridge (AER) removal, suggesting that a factor(s) localized to the AER prevents anterior cells from developing ZPA signaling capability during limb outgrowth. These findings indicate that nonsignaling anterior limb bud cells have the potential to become ZPA signaling cells and that FGF-2, or a related factor, functions in the maintenance of positional states in the developing limb.     

The role of ErbB receptor signaling in cell fate decisions by cortical progenitors: evidence for a biased, lineage-based responsiveness to different ligands

We recently identified the required collaborative signaling of TGFalpha and collagen type IV to regulate cell fate choice in the cerebral cortex, measured by the expression of the limbic system associated membrane protein (LAMP) by nonlimbic, sensorimotor progenitors. We show that activation of different members of the erbB receptor family can similarly modulate the specification of cortical area fate. The region of the cerebral wall from which progenitor cells arise does not influence the response to the neuregulin-1 or TGFalpha, but a subpopulation of progenitors is not competent to express LAMP in response to neuregulin-1. The heterogeneity in the responsiveness by progenitors to the two growth factors is reflected in the expression of different repertoires of erbB receptors. Using clonal analysis, we demonstrate that there may be a lineage-dependent mechanism regulating the ability of neuronal progenitors to respond to specific inductive cues that control cell fate.     

Conversion of tyrosine to phenolic derivatives by Taiwan cobra venom

We have examined the ability of Taiwan cobra (Naja naja atra) venom to transform in vitro the amino acid tyrosine to phenolic oxidation products via 4-hydroxyphenylpyruvate. This amino acid can be released from neuropeptide substrates by oligopeptidases present in the venom. Using a variety of analytical techniques to probe a complicated series of reactions, we confirm that the L-amino acid oxidase present in the venom initially releases the keto form of 4-hydroxyphenylpyruvic acid and hydrogen peroxide after reacting with the tyrosine. Thereafter, there is evidence that a tautomerase in the venom promotes a partial conversion of the keto-form 4-hydroxyphenylpyruvic acid into an enol form. The enol is oxidised primarily to 4-hydroxybenzaldehyde and 4-hydroxyphenol (hydroquinone). The keto form is oxidised through to 4-hydroxyphenylacetic acid by the hydrogen peroxide co-released by the L-amino acid oxidase. The venom promotes both these spontaneous oxidation routes and also generates traces of other phenolics, some of which are as yet unidentified. We propose that reactions between the precursors of the major oxidation products may be responsible for generating unusual short-lived phenolics, possibly giving rise to special bioactivities that are relevant to venom action.     

Conversion of Thebaine to Codeine

An improved conversion of thebaine to codeine has been developed. Oxymercuration of thebaine with mercuric acetate in refluxing methanol, followed by hydrolysis of the intermediate 7-acetomercurineopinone dimethyl ketal with 3 N acetic acid, or, alternatively, reduction of the organomercury compound with sodium borohydride and mild acid hydrolysis of the resulting neopinone dimethyl ketal, gives neopinone in 95-100% yields. Either acid- or alkali-catalyzed isomerization to codeinone leads to the equilibrium mixture consisting of codeinone-neopinone, 3:1. Complete conversion to codeinone in 85-90% yield results from treatment of neopinone with anhydrous hydrogen chloride or hydrogen bromide in ether-methylene chloride, followed by elimination of hydrogen halide from the intermediate 8-halodihydrocodeinone. The known borohydride reduction of codeinone then gives codeine in 85% overall yield from thebaine.     

Determination of cell fate by c-Abl activation in the response to DNA damage

The cellular response to DNA damage includes growth arrest and activation of DNA repair. Certain insights into how DNA damage is converted into intracellular signals that control the genotoxic stress response have been derived from the finding that the c-Abl protein tyrosine kinase is activated by ionizing radiation and other DNA-damaging agents. c-Abl associates with the DNA-dependent protein kinase (DNA-PK) and is activated by DNA-PK-dependent phosphorylation. The ataxia telangiectasia mutated (ATM) gene product also contributes to c-Abl activation. The demonstration that c-Abl binds to p53, induces the transactivation function of p53 and activates p21 expression has supported involvement of c-Abl in regulation of the p53-dependent G1 arrest response. Interaction between c-Abl and the Rad51 protein has also provided support for involvement of c-Abl in recombinational repair of DNA strand breaks. Defects in G1 arrest and repair predispose to replication of damaged templates and, in the event of irreparable DNA lesions, induction of apoptosis. The available evidence indicates that c-Abl effects a proapoptotic function by a mechanism largely independent of p53. c-Abl also functions as an upstream effector of the proapoptotic JNK/SAPK and p38 MAPK pathways. In addition, c-Abl-dependent inhibition of PI 3-kinase contributes to the induction of apoptosis. The findings thus suggest that, in response to genotoxic stress, c-Abl functions in determining cell fate, that is growth arrest and repair or induction of apoptosis. The physiologic function of c-Abl may reside in control of the cellular response to DNA strand breaks that occur during DNA replication, genetic recombination and gene rearrangements.     

Promotion of dendritic growth by CPG15, an activity-induced signaling molecule

Activity-independent and activity-dependent mechanisms work in concert to regulate neuronal growth, ensuring the formation of accurate synaptic connections. CPG15, a protein regulated by synaptic activity, functions as a cell-surface growth-promoting molecule in vivo. In Xenopus laevis, CPG15 enhanced dendritic arbor growth in projection neurons, with no effect on interneurons. CPG15 controlled growth of neighboring neurons through an intercellular signaling mechanism that requires its glycosylphosphatidylinositol link. CPG15 may represent a new class of activity-regulated, membrane-bound, growth-promoting proteins that permit exquisite spatial and temporal control of neuronal structure.     

The fate of competing beneficial mutations in an asexual population

In sexual populations, beneficial mutations that occur in different lineages may be recombined into a single lineage. In asexual populations, however, clones that carry such alternative beneficial mutations compete with one another and, thereby, interfere with the expected progression of a given mutation to fixation. From theoretical exploration of such 'clonal interference', we have derived (1) a fixation probability for beneficial mutations, (2) an expected substitution rate, (3) an expected coefficient of selection for realized substitutions, (4) an expected rate of fitness increase, (5) the probability that a beneficial mutation transiently achieves polymorphic frequency (> or = 1%), and (6) the probability that a beneficial mutation transiently achieves majority status. Based on (2) and (3), we were able to estimate the beneficial mutation rate and the distribution of mutational effects from changes in mean fitness in an evolving E. coli population.     

Preimplantation embryo sexing by polymerase chain reaction amplification of the sry gene on single mouse blastomeres

Accurate and rapid sex determination of preimplantation embryos has great potential both in animal breeding and in human pathology. In the past, sex determination has been accomplished by cytogenetic or immunologic means and by polymerase chain reaction amplification of Y-chromosome-specific repetitive sequences. More recently, amplification of the Y-specific single-copy ZFY gene has been used in humans for sex determination of preimplantation embryos. The experiments reported here indicate that another Y-chromosome-specific single-copy gene, the sex-determining region gene (sry) can be successfully amplified from single mouse blastomeres. Blastocysts positive for sry amplification were reimplanted to foster mothers, and six of six newborns were male. We conclude that sry gene amplification can represent a good marker for embryo sex determination.     

Specification of the zebrafish nervous system by nonaxial signals

The organizer of the amphibian gastrula provides the neurectoderm with both neuralizing and posteriorizing (transforming) signals. In zebrafish, transplantations show that a spatially distinct transformer signal emanates from tissues other than the organizer. Cells of the germring (nonaxial mesendoderm) posteriorized forebrain progenitors when grafted nearby, resulting in an ectopic hindbrain-like structure; in contrast, cells of the organizer (axial mesendoderm) caused no posterior transformation. Local application of basic fibroblast growth factor, a candidate transformer in Xenopus, caused malformation but not hindbrain transformation in the forebrain. Thus, the zebrafish gastrula may integrate spatially distinct signals from the organizer and the germring to pattern the neural axis.     

Conversion of neuronal growth cone responses from repulsion to attraction by cyclic nucleotides

Nerve growth is regulated by attractive and repulsive factors in the nervous system. Microscopic gradients of Collapsin-1/Semaphorin III/D (Sema III) and myelin-associated glycoprotein trigger repulsive turning responses by growth cones of cultured Xenopus spinal neurons; the repulsion can be converted to attraction by pharmacological activation of the guanosine 3',5'-monophosphate (cGMP) and adenosine 3',5'-monophosphate signaling pathways, respectively. Sema III also causes the collapse of cultured rat sensory growth cones, which can be inhibited by activation of the cGMP pathway. Thus cyclic nucleotides can regulate growth cone behaviors and may be targets for designing treatments to alleviate the inhibition of nerve regeneration by repulsive factors.