Maternal control of vertebrate development before the midblastula transition: mutants from the zebrafish I

Maternal factors control development prior to the activation of the embryonic genome. In vertebrates, little is known about the molecular mechanisms by which maternal factors regulate embryonic development. To understand the processes controlled by maternal factors and identify key genes involved, we embarked on a maternal-effect mutant screen in the zebrafish. We identified 68 maternal-effect mutants. Here we describe 15 mutations in genes controlling processes prior to the midblastula transition, including egg development, blastodisc formation, embryonic polarity, initiation of cell cleavage, and cell division. These mutants exhibit phenotypes not previously observed in zygotic mutant screens. This collection of maternal-effect mutants provides the basis for a molecular genetic analysis of the maternal control of embryogenesis in vertebrates.     

Enhanced detection of human immunodeficiency virus type 1-specific T-cell responses to highly variable regions by using peptides based on autologous virus sequences

The antigenic diversity of human immunodeficiency virus type 1 (HIV-1) represents a significant challenge for vaccine design as well as the comprehensive assessment of HIV-1-specific immune responses in infected persons. In this study we assessed the impact of antigen variability on the characterization of HIV-1-specific T-cell responses by using an HIV-1 database to determine the sequence variability at each position in all expressed HIV-1 proteins and a comprehensive data set of CD8 T-cell responses to a reference strain of HIV-1 in infected persons. Gamma interferon Elispot analysis of HIV-1 clade B-specific T-cell responses to 504 overlapping peptides spanning the entire expressed HIV-1 genome derived from 57 infected subjects demonstrated that the average amino acid variability within a peptide (entropy) was inversely correlated to the measured frequency at which the peptide was recognized (P = 6 x 10(-7)). Subsequent studies in six persons to assess T-cell responses against p24 Gag, Tat, and Vpr peptides based on autologous virus sequences demonstrated that 29% (12 of 42) of targeted peptides were only detected with peptides representing the autologous virus strain compared to the HIV-1 clade B consensus sequence. The use of autologous peptides also allowed the detection of significantly stronger HIV-1-specific T-cell responses in the more variable regulatory and accessory HIV-1 proteins Tat and Vpr (P = 0.007). Taken together, these data indicate that accurate assessment of T-cell responses directed against the more variable regulatory and accessory HIV-1 proteins requires reagents based on autologous virus sequences. They also demonstrate that CD8 T-cell responses to the variable HIV-1 proteins are more common than previously reported.     

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.     

Modulation of BMP activity in dorsal-ventral pattern formation by the chordin and ogon antagonists

We analyzed the interactions between mutations in antagonistic BMP pathway signaling components to examine the roles that the antagonists play in regulating BMP signaling activity. The dorsalized mutants swirl/bmp2b, snailhouse/bmp7, lost-a-fin/alk8, and mini fin/tolloid were each analyzed in double mutant combinations with the ventralized mutants chordino/chordin and ogon, whose molecular nature is not known. Similar to the BMP antagonist chordino, we found that the BMP ligand mutants swirl/bmp2b and snailhouse/bmp7 are also epistatic to the putative BMP pathway antagonist, ogon, excluding a class of intracellular antagonists as candidates for ogon. In ogon;mini fin double mutants, we observed a mutual suppression of the ogon and mini fin mutant phenotypes, frequently to a wild type phenotype. Thus, the Tolloid/Mini fin metalloprotease that normally cleaves and inhibits Chordin activity is dispensable, when Ogon antagonism is reduced. These results suggest that Ogon encodes a Tolloid and Chordin-independent antagonistic function. By analyzing genes whose expression is very sensitive to BMP signaling levels, we found that the absence of Ogon or Chordin antagonism did not increase the BMP activity remaining in swirl/bmp2b or hypomorphic snailhouse/bmp7 mutants. These results, together with other studies, suggest that additional molecules or mechanisms are essential in generating the presumptive gastrula BMP activity gradient that patterns the dorsal-ventral axis. Lastly we observed a striking increased penetrance of the swirl/bmp2b dominant dorsalized phenotype, when Chordin function is also absent. Loss of the BMP antagonist Chordin is expected to increase BMP signaling levels in a swirl heterozygote, but instead we observed an apparent decrease in BMP signaling levels and a loss of ventral tail tissue. As has been proposed for the fly orthologue of chordin, short gastrulation, our paradoxical results can be explained by a model whereby Chordin both antagonizes and promotes BMP activity.     

Hyphal tip growth in Achlya bisexualis. I. Distribution of 1,3-{beta}-glucans in elongating and non-elongating regions of the wall

We have approached the problem of hyphal tip growth by comparing the cell wall composition of elongating and non-elongating regions of the hyphae of Achlya bisexualis. To ensure that we could distinguish between elongating and non-elongating hyphae, light microscopic observations were used to determine the rates of elongation under growing and non-growing conditions. When elongation was measured in 10 min intervals it was found to consist of fluctuating periods of fast and slow growth rates, in the form of cycles. Even under our growing conditions, however, a very small number of hyphae in a colony are not elongating. SEM analysis revealed that elongating hyphae have tapered apices, whereas non-elongating hyphae have a rounded apex. The major matrix wall components, 1,3-β-glucans, were localized with an indirect immunogold technique specific for these polymers. This method resulted in their localization to all regions of both elongating and non-elongating hyphae, including the apex.     

Differential recognition of tyrosine-based basolateral signals by AP-1B subunit mu1B in polarized epithelial cells

To investigate the importance of tyrosine recognition by the AP-1B clathrin adaptor subunit mu1B for basolateral sorting of integral membrane proteins in polarized epithelial cells, we have produced and characterized a mutant form of mu1B. The mutant (M-mu1B) contains alanine substitutions of each of the four conserved residues, which in the AP-2 adaptor subunit micro2 are critical for interacting with tyrosine-based endocytosis signals. We show M-mu1B is defective for tyrosine binding in vitro, but is nevertheless incorporated into AP-1 complexes in transfected cells. Using LLC-PK1 cells expressing either wild type or M-mu1B, we find that there is inefficient basolateral expression of membrane proteins whose basolateral targeting signals share critical tyrosines with signals for endocytosis. In contrast, membrane proteins whose basolateral targeting signals are distinct from their endocytosis signals (transferrin and low-density lipoprotein receptors) accumulate at the basolateral domain normally, although in a manner that is strictly dependent on mu1B or M-mu1B expression. Our results suggest that mu1B interacts with different classes of basolateral targeting signals in distinct ways.     

Predicting demographic group structures based on DNA sequence data

The ability to infer relationships between groups of sequences, either by searching for their evolutionary history or by comparing their sequence similarity, can be a crucial step in hypothesis testing. Interpreting relationships of human immunodeficiency virus type 1 (HIV-1) sequences can be challenging because of their rapidly evolving genomes, but it may also lead to a better understanding of the underlying biology. Several studies have focused on the evolution of HIV-1, but there is little information to link sequence similarities and evolutionary histories of HIV-1 to the epidemiological information of the infected individual. Our goal was to correlate patterns of HIV-1 genetic diversity with epidemiological information, including risk and demographic factors. These correlations were then used to predict epidemiological information through analyzing short stretches of HIV-1 sequence. Using standard phylogenetic and phenetic techniques on 100 HIV-1 subtype B sequences, we were able to show some correlation between the viral sequences and the geographic area of infection and the risk of men who engage in sex with men. To help identify more subtle relationships between the viral sequences, the method of multidimensional scaling (MDS) was performed. That method identified statistically significant correlations between the viral sequences and the risk factors of men who engage in sex with men and individuals who engage in sex with injection drug users or use injection drugs themselves. Using tree construction, MDS, and newly developed likelihood assignment methods on the original 100 samples we sequenced, and also on a set of blinded samples, we were able to predict demographic/risk group membership at a rate statistically better than by chance alone. Such methods may make it possible to identify viral variants belonging to specific demographic groups by examining only a small portion of the HIV-1 genome. Such predictions of demographic epidemiology based on sequence information may become valuable in assigning different treatment regimens to infected individuals.     

Effect of linker sequence on the stability of circularly permuted variants of ribonuclease T1

Circularly permuted variants of ribonuclease T1 were constructed with a library of residues covalently linking the original amino and carboxyl terminal ends of the wild-type protein. The library of linking peptides consisted of three amino acids containing any combination of proline, aspartate, asparagine, serine, threonine, tyrosine, alanine, and histidine. Forty two unique linker sequences were isolated and 10 of these mutants were further characterized with regard to catalytic activity and overall thermodynamic stability. The 10 mutants with the different linking sequences (HPD, TPH, DTD, TPD, PYH, PAT, PHP, DSS, SPP, and TPS), in addition to GGG and GPG, were 4.0-6.2 kcal/mol less stable than the wild-type ribonuclease T1. However, these circular permuted variants were only 0.4-2.6 kcal/mol less stable than the direct parent protein that is missing the disulfide bond connecting residues 2 and 10. The most stable linking peptide was HPD.     

Tumor-derived cytokines dysregulate macrophage interferon-gamma responsiveness and interferon regulatory factor-8 expression

Tumors can evade immune responses through suppressor signals that dysregulate host effector cell function. In this study we demonstrate that tumor-derived suppressor molecules impede host antitumor immune activity through dysregulation of multiple macrophage (Mphi) pathways, including suppressed production of cytotoxic and immunostimulatory agents and impaired expression of the interferon regulatory factor-8 (IRF-8) protein, a critical transducer of interferon-gamma-mediated activation pathways. The tumor-derived immunosuppressive cytokines interleukin-10 and transforming growth factor-beta(1) constrain IRF-8 production by normal Mphis, regardless of priming, and IRF-8 is also dysregulated in primary Mphis from tumor-burdened hosts. Collectively, these data describe a new mechanism by which tumors disrupt immune function and suggest that abrogation of tumor-derived immunoregulatory factors in situ can restore immune function and enhance antitumor efficacy.