Molecular mechanisms of cell-cell signaling by the Spemann-Mangold organizer.

We review how studies on the first Spemann-Mangold organizer marker, the homeobox gene goosecoid, led to the discovery of secreted factors that pattern the vertebrate embryo. Microinjection of goosecoid mRNA formed secondary axes and recruited neighboring cells. These non-cell autonomous effects are mediated in part by the expression of secreted factors such as chordin, cerberus and Frzb-1. Unexpectedly, many of the molecules secreted by the Spemann-Mangold organizer turned out to be antagonists that bind growth factors in the extracellular space and prevent them from binding to their receptors. The case of chordin is reviewed in detail, for this molecule has provided biochemical insights into how patterning by Spemann's organizer can be regulated by diffusion and proteolytic control. The study of the BMP-binding repeats of Chordin, which are present in many extracellular proteins, may provide a new paradigm for how cell-cell signaling is regulated in the extracellular space not only in embryos, but also in adult tissues.

Overexpression of the Xenopus tight-junction protein claudin causes randomization of the left-right body axis.

This study presents Xenopus claudin (Xcla), a tight-junction protein that is abundantly expressed in eggs and neuroectodermal precursors during early development. It was isolated via a differential screen for mRNAs enriched in microsomes in the Xenopus blastula. The Xcla protein contains four transmembrane domains and a carboxy-terminal cytoplasmic region with a putative PDZ-binding site. We show that this PDZ-binding site of Xcla is critical for its correct localization on the cell membrane and that a truncated form leads to delocalization of the tight-junction protein ZO-1. Overexpression of Xcla causes changes in the cell adhesion properties of blastomeres and leads to visceral situs randomization. The results suggest that left-right axial patterning is very sensitive to changes in regulation of cell-cell interactions and implicate a tight-junction protein in the determination of left-right asymmetry.

Regulation of the Sex combs reduced gene in Drosophila.

The Sex combs reduced gene of the Antennapedia complex specifics the identities of the anterior thoracic and posterior head segments, including the primordium of the larval salivary gland. The Sex combs reduced transcription unit spans over 30 kb of genomic DNA, with another 40 kb of upstream cis-regulatory sequences. The pattern of Sex combs reduced transcription is set in the early embryo by the segmentation genes and is then maintained by two competing sets of proteins, the Polycomb group and the trithorax group. One of the trithorax group genes required for activation, the brahma gene, encodes an evolutionarily conserved DNA-stimulated ATPase that is part of a large protein complex. This complex facilitates the action of sequence-specific, DNA-binding proteins in regulating target genes, possibly by altering chromatin structure.

The Drosophila homeotic gene moira regulates expression of engrailed and HOM genes in imaginal tissues.

moira is a member of the trithorax group of homeotic gene regulators in Drosophila melanogaster. We show that moira is required for the function of multiple homeotic genes of the Antennapedia and bithorax complexes (HOM genes) in most imaginal tissues and that the requirement for moira function is at the level of transcription. moira is also required for transcription of the engrailed segmentation gene in the imaginal wing disc. The abnormalities caused by the loss of moira function in germ cells suggests that at least one other target gene requires moira for normal oogenesis.

Genetic analysis of the brahma gene of Drosophila melanogaster and polytene chromosome subdivisions 72AB.

The brahma gene is required for activation of the homeotic genes of the Antennapedia and bithorax complexes in Drosophila. We have isolated and characterized 21 mutations in brahma. We show that both maternal and zygotic functions of brahma are required during embryogenesis. In addition, the severe abnormalities caused by loss of maternal brahma expression show that the homeotic genes are not the only targets for brahma activation. The complex pattern of interallelic complementation for the 21 brahma alleles suggests that brahama may act as a multimer. In addition to mutations in brahma, we have isolated mutations in four other essential genes within polytene chromosome subdivisions 72AB. Based on a compilation of similar studies that include about 24% of the genome, we estimate that about 3600 genes in Drosophila can mutate to cause recessive lethality, with fewer than 900 additional genes essential only for gametogenesis. We have identified three times more transcripts than lethal complementation groups in 72AB. One transcript in 72AB is the product of the essential arf-like gene and encodes a member of the ARF subfamily of small GTP-binding proteins. Two other transcripts are probably the products of a single gene whose protein products are similar to the catalytic subunits of cAMP-dependent protein kinases.

The arflike gene encodes an essential GTP-binding protein in Drosophila.

We have identified a Drosophila gene (arflike, arl) encoding a protein that is structurally related (approximately 55% identity) to the ADP-ribosylation factors (ARFs) of yeast and mammals. Biochemical analyses of purified recombinant arl-encoded protein revealed properties similar to the ARF proteins, including the ability to bind and hydrolyze GTP. Clear functional differences between arl and ARF proteins, including a complete lack of ARF activity, suggest that arl is not a functional homolog of ARF. A recessive lethal arl mutation was recovered, demonstrating that the arl locus is an essential gene. We conclude that the arl locus encodes an essential member of the ARF subfamily of small GTP-binding proteins in Drosophila.

Fibrinogenolytic afibrinogenemia after envenomation by western diamondback rattlesnake (Crotalus atrox).

The absence of fibrinogen and the presence of plasmic fragments X, Y, D, and E were demonstrated in a patient bitten by a western diamondback rattlesnake, Crotalus atrox. The factor VIII level and the platelet count were within normal limits. There were distinct changes of protease inhibitors in the patient's plasma. Alpha-1-protease inhibitor was elevated. Antithrombin-III was only slightly decreased after the envenomation, but alpha 2-antiplasmin and alpha 2-macroglobulin were initially significantly lowered, returning to normal values in 38 and 3 days, respectively. Plasmin-alpha 2-antiplasmin complex was present until day 10 after the envenomation. However, purified plasminogen was not activated in vitro by the venom. Cultured endothelial and smooth muscle cells from human blood vessels released an increased amount of plasminogen activator upon incubation with the venom. The release did not result from cell lysis. Platelets in normal human platelet-rich plasma were aggregated by 10 micrograms/ml of the venom, without serotonin secretion. The aggregation kinetics and serotonin secretion induced by adenosine diphosphate (ADP) or arachidonate were not significantly affected by the venom at 1-10 micrograms/ml. It is concluded that the predominant mechanism of afibrinogenemia in the patient after Crotalus atrox bite resulted from primary fibrinogenolysis and not from a consumptive coagulopathy. The lytic state seemed to be induced through an indirect activation of plasminogen by vascular plasminogen activator, which was probably released from endothelial cells and smooth muscle cells by the snake venom.

The interference of plasmic degradation products of human crosslinked fibrin with clot formation.

The role of plasmic degradation products of human crosslinked fibrin on polymerization of fibrin monomer and clot formation was studied. Both reactions were inhibited by Fragment DD, which formed a complex with fibrin monomer in a molar ratio 1 : 1. The rate of polymerization was slightly increased by Fragment E but it was not affected by (DD)E complex and Fragment A. Approximately the same amount of fibrin was formed in the presence and absence of Fragments A, E and the complex. It was concluded that of the degradation products of crosslinked fibrin, only Fragment DD is a potent anticoagulant at physiologic pH. The (DD)E complex is inert and Fragments A and E have only marginal effects.

The differential effects of chemical carcinogens on vitamin A status and on microsomal drug metabolism in normal and vitamin A-deficient rats.

Male Sprague-Dawley rats were maintained on a vitamin A-deficient diet for 5 weeks. Although serum and hepatic levels of vitamin A were significantly lower at this time, no outward signs of vitamin A deficiency were present. Hepatic microsomal levels of cytochrome P-450 in the vitamin A-deficient animals were 70% that of the control animals. Of the three microsomal enzymes studied, ethylmorphine N-demethylase, aniline hydroxylase, and aminopyrine N-demethylase, only the last one was adversely affected by vitamin A deficiency. 3-Methylcholanthrene, phenobarbital, and 2-acetylaminofluorene had a greater inductive effect and cytochrome P-450 in vitamin A-deficient rats. 4-Dimethylaminoazobenzene treatment decreased in the level of cytochrome P-450 in control rats more than in deficieny rats. The hepatic concentration of vitamin A was significantly reduced in control rats that were given injections of 3-methylcholanthrene, 2-acetylaminofluorene, or phenobarbital. Benzo(a)pyrene and 4-dimethylaminoazobenzene had less effect.