Divergent evolution of a bifunctional de novo protein

Primordial proteins, the evolutionary ancestors of modern sequences, are presumed to have been minimally active and nonspecific. Following eons of selective pressure, these early progenitors evolved into highly active and specific proteins. While evolutionary trajectories from poorly active and multifunctional generalists toward highly active specialists likely occurred many times in evolutionary history, such pathways are difficult to reconstruct in natural systems, where primordial sequences are lost to time. To test the hypothesis that selection for enhanced activity leads to a loss of promiscuity, we evolved a de novo designed bifunctional protein. The parental protein, denoted Syn‐IF, was chosen from a library of binary patterned 4‐helix bundles. Syn‐IF was shown previously to rescue two different auxotrophic strains of E. coli: ΔilvA and Δfes. These two strains contain deletions for proteins with very different biochemical functions; IlvA is involved in isoleucine biosynthesis, while Fes is involved in iron assimilation. In two separate experiments, Syn‐IF, was evolved for faster rescue of either ΔilvA or Δfes. Following multiple rounds of mutagenesis, two new proteins were selected, each capable of rescuing the selected function significantly faster than the parental protein. In each case, the evolved protein also lost the ability to rescue the unselected function. In both evolutionary trajectories, the original bifunctional generalist was evolved into a monofunctional specialist with enhanced activity.     

Dazap2 is required for FGF-mediated posterior neural patterning, independent of Wnt and Cdx function

The organization of the embryonic neural plate requires coordination of multiple signal transduction pathways, including fibroblast growth factors (FGFs), bone morphogenetic proteins (BMPs), and WNTs. Many studies have suggested that a critical component of this process is the patterning of posterior neural tissues by an FGF-caudal signaling cascade. Here, we have identified a novel player, Dazap2, and show that it is required in vivo for posterior neural fate. Loss of Dazap2 in embryos resulted in diminished expression of hoxb9 with a concurrent increase in the anterior marker otx2. Furthermore, we found that Dazap2 is required for FGF dependent posterior patterning; surprisingly, this is independent of Cdx activity. Furthermore, in contrast to FGF activity, Dazap2 induction of hoxb9 is not blocked by loss of canonical Wnt signaling. Functionally, we found that increasing Dazap2 levels alters neural patterning and induces posterior neural markers. This activity overcomes the anteriorizing effects of noggin, and is downstream of FGF receptor activation. Our results strongly suggest that Dazap2 is a novel and essential branch of FGF-induced neural patterning.     

Expression of Hoxa2 in cells entering chondrogenesis impairs overall cartilage development

Vertebrate Hox genes act as developmental architects by patterning embryonic structures like axial skeletal elements, limbs, brainstem territories, or neural crest derivatives. While active during the patterning steps of development, these genes turn out to be down-regulated in specific differentiation programs like that leading to chondrogenesis. To investigate why chondrocyte differentiation is correlated to the silencing of a Hox gene, we generated transgenic mice allowing Cre-mediated conditional misexpression of Hoxa2 and induced this gene in Collagen 2 alpha 1-expressing cells committed to enter chondrogenesis. Persistent Hoxa2 expression in chondrogenic cells resulted in overall chondrodysplasia with delayed cartilage hypertrophy, mineralization, and ossification but without proliferation defects. The absence of skeletal patterning anomaly and the regular migration of precursor cells indicated that the condensation step of chondrogenesis was normal. In contrast, closer examination at the differentiation step showed severely impaired chondrocyte differentiation. In addition, this inhibition affected structures independently of their embryonic origin. In conclusion, for the first time here, by a cell-type specific misexpression, we precisely uncoupled the patterning function of Hoxa2 from its involvement in regulating differentiation programs per se and demonstrate that Hoxa2 displays an anti-chondrogenic activity that is distinct from its patterning function.     

Patterning of the early Middle Paleolithic occupations at Douara Cave and its implications for settlement dynamics in the Palmyra basin,Syria

This paper examines spatial patterning and settlement dynamics of early Middle Paleolithic hominins at Douara Cave, located on the northern edge of the Syrian Desert. The analyzed material came from our 1984 excavations of Horizons IVB–IVD, techno-typologically assigned to the Tabun D-type Levantine Mousterian (ca. 250 to 130 ka). Two findings are reported. One is the existence of spatial organization in the cave interior. Analysis of the field records shows that the occupation floor of the early Middle Paleolithic at Douara was well-organized into specific activity areas with a focal area of intensive activities close to the back wall. This suggests that the organized use of space known at late Middle Paleolithic sites like Tor Faraj, Jordan, is also applicable to the early Middle Paleolithic. Second, this paper discusses the functional role of this cave within the regional settlement system. A range of features characterizing its living floor(s) point to a very low occupational intensity, undoubtedly reflecting adaptation and particular land use patterns in the arid environments of this region. Moreover, this pattern, along with the division of interior space, seems to have remained consistent through multiple early Middle Paleolithic levels (IVB–IVD). These observations suggest that Douara Cave was a short-term camp embedded in a regional settlement system in the arid environments of this period.     

Single‐cell dynamics of chromatin activity during cell lineage differentiation in Caenorhabditis elegans embryos

Elucidating the chromatin dynamics that orchestrate embryogenesis is a fundamental question in developmental biology. Here, we exploit position effects on expression as an indicator of chromatin activity and infer the chromatin activity landscape in every lineaged cell during Caenorhabditis elegans early embryogenesis. Systems‐level analyses reveal that chromatin activity distinguishes cellular states and correlates with fate patterning in the early embryos. As cell lineage unfolds, chromatin activity diversifies in a lineage‐dependent manner, with switch‐like changes accompanying anterior–posterior fate asymmetry and characteristic landscapes being established in different cell lineages. Upon tissue differentiation, cellular chromatin from distinct lineages converges according to tissue types but retains stable memories of lineage history, contributing to intra‐tissue cell heterogeneity. However, the chromatin landscapes of cells organized in a left–right symmetric pattern are predetermined to be analogous in early progenitors so as to pre‐set equivalent states. Finally, genome‐wide analysis identifies many regions exhibiting concordant chromatin activity changes that mediate the co‐regulation of functionally related genes during differentiation. Collectively, our study reveals the developmental and genomic dynamics of chromatin activity at the single‐cell level.     

Cell placement and guidance on substrates for neurochip interfaces

Interface devices such as integrated planar patch‐clamp chips are being developed to study the electrophysiological activity of neuronal networks grown in vitro. The utility of such devices will be dependent upon the ability to align neurons with interface features on the chip by controlling neuronal placement and by guiding cell connectivity. In this paper, we present a strategy to accomplish this goal. Patterned chemical modification of SiN surfaces with poly‐d‐lysine transferred from PDMS stamps was used to promote adhesion and guidance of cryo‐preserved primary rat cortical neurons. We demonstrate that these neurons can be positioned and grown over microhole features which will ultimately serve as patch‐clamp interfaces on the chip. Biotechnol. Bioeng. 2010; 105: 368–373. © 2009 Wiley Periodicals, Inc.     

Spatial regulation of segment polarity gene expression in the anterior terminal region of the Drosophila blastoderm embryo

The effects of mutations in five anterior gap genes (hkb, tll, otd, ems and btd) on the spatial expression of the segment polarity genes, wg and hh, were analyzed at the late blastoderm stage and during subsequent development. Both wg and hh are normally expressed at blastoderm stage in two broad domains anterior to the segmental stripes of the trunk region. At the blastoderm stage, each gap gene acts specifically to regulate the expression of either wg or hh in the anterior cephalic region: hkb, otd and btd regulate the anterior blastoderm expression of wg, while tll and ems regulate hh blastoderm expression. Additionally, btd is required for the first segmental stripe (mandibular segment) of both hh and wg at blastoderm stages. The subsequent segmentation of the cephalic segments (preantennal, antennal and intercalary) appears to be dependent on the overlap of the wg and hh cephalic domains as defined by these gap genes at the blastoderm stage. None of these five known gap genes are required for the activation of the labral segment domains of hh and wg, which are presumably either activated directly by maternal pathways or by an unidentified gap gene.     

Metamerism,morphogenesis, and the expression of carabelli and other dental traits in humans

The patterning cascade model of tooth morphogenesis has emerged as a useful tool in explaining how tooth shape develops and how tooth evolution may occur. Enamel knots, specialized areas of dental epithelium where cusps initiate, act as signaling centers that direct the growth of surrounding tissues. For a new cusp to form, an enamel knot must form beyond the inhibition fields of other enamel knots. The model predicts that the number and size of cusps depends on the spacing between enamel knots, reflected in the spacing between cusps. Recently, work by our group demonstrated that the model predicted Carabelli trait expression in human first molars. Here we test whether differences in Carabelli trait expression along the molar row can also be predicted by the model. Crown areas and intercusp distances were measured from dental casts of 316 individuals with a digital microscope. Although absolute cusp spacing is similar in first and second molars, the smaller size and more triangular shape of second molars results in larger cusp spacing relative to size and, likely, less opportunity for the Carabelli trait to form. The presence and size of the hypocone (HY) and a range of small accessory cusps in a larger sample of 340 individuals were also found to covary with the Carabelli trait in a complex way. The results of this study lend further support to the view that the dentition develops, varies, and evolves as a single functional complex. Am J Phys Anthropol, 2013. © 2013 Wiley Periodicals, Inc.     

Asynchronous mitotic domains during blastoderm formation inMusca domestica L. (Diptera)

Summary We describe the mitotic cleavage patterns during blastoderm stage of the house flyMusca domestica L. Nuclear divisions up to mitotic stage 11 are apparently synchronous. Beginning with stage 12, nuclear divisions in the posterior third of the embryo lag behind, resulting first in a parasynchronous and finally in an asynchronous cleavage pattern. Thus a stage exists where all nuclei in the anterior region have completed 14 nuclear division cycles, while those in the posterior region have completed only 13 cycles. The border region between these nuclei is well defined and lies at 35% EL (egg length), the expression border of a gap gene. This border region is about 4–5 nuclei wide and shows a specialized mitotic behaviour.