bluuebunny/crossref_metadata_2025 huggingface dataset:
Combined RT-qPCR and pyrosequencing of a Spike glycoprotein polybasic cleavage motif can uncover pediatric SARS-CoV-2 infections associated with heterogeneous presentation
Authors: Weil Patrick Philipp , Hentschel Jacqueline , Schult Frank , Pembaur Anton , Ghebremedhin Beniam , Mboma Olivier , Heusch Andreas , Reuter Anna-Christin , Müller Daniel , Wirth Stefan , Aydin Malik , Jenke Andreas C. W. , Postberg Jan
Published: April 2021

Background

Reverse transcription of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (+)RNA genome and subgenomic RNAs (sgRNAs) and subsequent quantitative polymerase chain reaction (RT-qPCR) is the reliable diagnostic gold standard for COVID-19 diagnosis and the identification of potential spreaders. Apart from clinical relevance and containment, for specific questions, it might be of interest to (re)investigate cases with low SARS-CoV-2 load, where RT-qPCR alone can deliver conflicting results, even though these cases might neither be clinically relevant nor significant for containment measures, because they might probably not be infectious. In order to expand the diagnostic bandwidth for non-routine questions, particularly for the reliable discrimination between negative and false-negative specimens associated with high CT values, we combined the RT-qPCR workflow with subsequent pyrosequencing of a S-gene amplicon. This expansion can help to confirm SARS-CoV-2 infections without the demand of confirmative antibody testing, which requires to summon patients again for blood sampling few to several weeks after symptom onset.

Results

We successfully established a combined RT-qPCR and S-gene pyrosequencing method which can be optionally exploited after routine diagnostics. This allows a reliable interpretation of RT-qPCR results in specimens with relatively low viral loads and close to the detection limits of qPCR. After laboratory implementation, we tested the combined method in a large pediatric cohort from two German medical centers (n=769). Pyrosequencing after RT-qPCR enabled us to uncover 5 previously unrecognized cases of pediatric SARS-CoV-2-associated diseases, mainly exhibiting mild and heterogeneous presentation—apart from a single case of multisystem inflammatory syndrome in children (MIS-C) associated with SARS-CoV-2, who was hospitalized in the course of the study.

Conclusions

The proposed protocol allows a specific and sensitive confirmation of SARS-CoV-2 infections close to the detection limits of RT-qPCR. The tested biotinylated primers do not negatively affect the RT-qPCR pipeline and thus can be optionally applied to enable deeper inspection of RT-qPCR results by subsequent pyrosequencing. Moreover, due to the incremental transmission of SARS-CoV-2 variants of concern, we note that the used strategy can uncover (Spike) P681H allowing the pre-selection of SARS-CoV-2 B.1.1.7 candidate specimens for deep sequencing.
Femoral mechanical performance of precocial and altricial birds: a simulation study
Authors: Wei Xinsen , Zhang Zihui
Published: April 2021

Background

As the major load-bearing structures, bones exhibit various properties related to mechanical performance to adapt to different locomotor intensities. The habits and ontogenetic changes of locomotion in animals can, thus, be explored by assessing skeletal mechanical performance.

Methods

In this study, we investigated the growing femoral mechanical performance in an ontogenetic series of Cabot’s Tragopans (Tragopan caboti) and Pigeons (Columba livia domestica). Micro-computed tomography-based finite element analysis was conducted to evaluate the stress, strain, and strain energy density (SED) of femora under axial and radial loading.

Results

Femora deflected medio-laterally and dorso-ventrally under axial and radial loading, respectively. Femora deformed and tensed more severely under radial loading than axial loading. In adult individuals, Cabot’s Tragopans had lower strain and SED than pigeons. During ontogeny, the strain and SED of pigeons decreased sharply, while Cabot’s Tragopans showed moderately change. The structural properties of hatchling pigeons are more robust than those of hatchling Cabot’s Tragopans.

Conclusions

Limb postures have dominant effect on skeletal deformation. The erect posture is preferred by large mammals and birds to achieve a high safety factor of bones during locomotion. Adult Cabot’s Tragopans have stronger femora than pigeons, reflecting a better bone adaption to the terrestrial locomotion of the studied pheasant species. Changes in strain and SED during growth reflect the marked difference in locomotor ability between precocial and altricial hatchlings. The femora of hatchling Cabot’s Tragopans were built with better energy efficiency than deformation resistance, enabling optimized mechanical performance. In contrast, although weak in mechanical function at the time of hatching, pigeon femora were suggested to be established with a more mature structural design as a prerequisite for rapid growth. These results will be helpful for studies regarding developmental patterns of fossil avian species.
The role of co-infections and secondary infections in patients with COVID-19
Authors: Feldman Charles , Anderson Ronald
Published: April 2021

Background

It has been recognised for a considerable time-period, that viral respiratory infections predispose patients to bacterial infections, and that these co-infections have a worse outcome than either infection on its own. However, it is still unclear what exact roles co-infections and/or superinfections play in patients with COVID-19 infection.

Main body

This was an extensive review of the current literature regarding co-infections and superinfections in patients with SARS-CoV-2 infection. The definitions used were those of the Centers for Disease Control and Prevention (US), which defines coinfection as one occurring concurrently with the initial infection, while superinfections are those infections that follow on a previous infection, especially when caused by microorganisms that are resistant, or have become resistant, to the antibiotics used earlier. Some researchers have envisioned three potential scenarios of bacterial/SARS-CoV-2 co-infection; namely, secondary SARS-CoV-2 infection following bacterial infection or colonisation, combined viral/bacterial pneumonia, or secondary bacterial superinfection following SARS-CoV-2. There are a myriad of published articles ranging from letters to the editor to systematic reviews and meta-analyses describing varying ranges of co-infection and/or superinfection in patients with COVID-19. The concomitant infections described included other respiratory viruses, bacteria, including mycobacteria, fungi, as well as other, more unusual, pathogens. However, as will be seen in this review, there is often not a clear distinction made in the literature as to what the authors are referring to, whether true concomitant/co-infections or superinfections. In addition, possible mechanisms of the interactions between viral infections, including SARS-CoV-2, and other infections, particularly bacterial infections are discussed further. Lastly, the impact of these co-infections and superinfections in the severity of COVID-19 infections and their outcome is also described.

Conclusion

The current review describes varying rates of co-infections and/or superinfections in patients with COVID-19 infections, although often a clear distinction between the two is not clear in the literature. When they occur, these infections appear to be associated with both severity of COVID-19 as well as poorer outcomes.
Mapping the transcriptomic changes of endothelial compartment in human hippocampus across aging and mild cognitive impairment
Authors: Guebel Daniel V. , Torres Néstor V. , Acebes Ángel
Published: April 2021

Compromise of the vascular system has important consequences on cognitive abilities and neurodegeneration. The identification of the main molecular signatures present in the blood vessels of human hippocampus could provide the basis to understand and tackle these pathologies. As direct vascular experimentation in hippocampus is problematic, we achieved this information by computationally disaggregating publicly available whole microarrays data of human hippocampal homogenates. Three conditions were analyzed: ‘Young Adults’, ‘Aged’, and ‘aged with Mild Cognitive Impairment’ (MCI). The genes identified were contrasted against two independent data-sets. Here we show that the endothelial cells from the Younger Group appeared in an ‘activated stage’. In turn, in the Aged Group, the endothelial cells showed a significant loss of response to shear stress, changes in cell adhesion molecules, increased inflammation, brain-insulin resistance, lipidic alterations, and changes in the extracellular matrix. Some specific changes in the MCI group were also detected. Noticeably, in this study the features arisen from the Aged Group (high tortuosity, increased bifurcations, and smooth muscle proliferation), pose the need for further experimental verification to discern between the occurrence of arteriogenesis and/or vascular remodeling by capillary arterialization.

This article has an associated First Person interview with the first author of the paper.
A widely distributed antigen developmentally regulated in the nervous system
Authors: Pourquié Olivier , Coltey Monique , Thomas Jean-Léon , Le Douarin Nicole M.
Published: April 2021

We have identified a glycoprotein (BEN) of 95 –100 ×103Mr using a monoclonal antibody. This protein is transiently expressed at the cell surface of the peripherally projecting neurons, i.e. motoneurons of the spinal cord and cranial nuclei, sensory neurons of the dorsal root and cranial sensory ganglia and sympathetic, parasympathetic and enteric neurons. In vitro cultures of dorsal root and sympathetic ganglia have shown that BEN is expressed on neurons but not on glial cells. On motor and sensory neurons, BEN first appears at the level of the cell body just after withdrawal from the cell cycle. Soon afterwards, expression of the antigen extends to the elongating axon. After a few days, BEN is no longer expressed by the motor and sensory neurons, disappearing first from the cell body and then progressively from the fibres. The loss of expression is concomitant with the onset of intense proliferation of satellite and Schwann cells. This modulated expression within the nervous sytem is unlike that of any surface glycoprotein so far described in vertebrates. Preliminary biochemical analysis indicates that, although it bears the adhesion-associated epitope HNK-1, BEN does not share characteristics with any previously described axonal glycoprotein. Consequently, we speculate that this glycoprotein might be a novel molecule implicated in selective adhesion phenomena, such as axonal fasciculation.
Pax2, a new murine paired-box-containing gene and its expression in the developing excretory system
Authors: Dressler Gregory R. , Deutsch Urban , Chowdhury Kamal , Nornes Howard O. , Gruss Peter
Published: April 2021

The murine genome contains multiple genes with protein domains homologous to the Drosophila paired box, present in certain segmentation genes. At least one of these murine paired box (Pax) genes is associated with a developmental mutation. This report, in conjunction with the accompanying paper, describes a second member of this gene family, Pax2, that is also expressed during embryogenesis. Two overlapping cDNA clones were isolated and sequenced. At least two forms of the Pax2 protein can be deduced from the cDNA sequence. In addition to the highly conserved paired domain, an octapeptide sequence is located downstream. Expression of Pax2 is primarily restricted to the developing embryo in the excretory and central nervous systems. The transient nature of Pax2 expression during kidney organogenesis correlates with polarization and induction of epithelial structures and may indicate an important morphogenetic role for this gene.
Spatially and temporally restricted expression of Pax2 during murine neurogenesis
Authors: Nornes Howard O. , Dresslert Gregory R. , Knapik Elzbieta W. , Deutsch Urban , Gruss Peter
Published: April 2021

The expression of the murine paired-box-containing gene, Pax2, is examined in the developing central nervous system by in situ hybridization. Pax2 expression is detected along the boundaries of primary divisions of the neural tube. Initially, Pax2 is expressed in the ventricular zone in two compartments of cells on either side of the sulcus limitans and along the entire rhombencephalon and spinal cord. At later times, Pax2 is restricted to progeny cells that have migrated to specific regions of the intermediate zone. In the eye, Pax2 expression is restricted to the ventral half of the optic cup and stalk and later to the optic disc and nerve. In the ear, expression is restricted to regions of the otic vesicle that form neuronal components. The transient and restricted nature of Pax2 expression suggests that this murine segmentation gene homologue may also establish compartmental boundaries and contribute to the specification of neuronal identity, as do certain Drosophila segmentation genes.
Autonomous determination of anterior structures in the early Drosophila embryo by the bicoid morphogen
Authors: Driever Wolfgang , Siegel Vivian , Nüsslein-Volhard Christiane
Published: April 2021

A small number of maternal effect genes determine anterior-posterior pattern in the Drosophila embryo. Embryos from females mutant for the maternal gene bicoid lack head and thorax, bcd mRNA becomes localized to the anterior tip of the egg during oogenesis and is the source for the morphogen gradient of bcd protein.

Here we show that in vitro transcribed bicoid mRNA that has its own leader sequences substituted by the Xenopus β-globin 5′ untranslated sequences is translated more efficiently than bicoid mRNA with the natural 5′ mRNA leader when tested in vitro and in Drosophila Schneider cells. When injected into bicoid mutant embryos, only the bcd mRNA with the ′-globin leader sequence, substituted for the natural leader, is able to induce anterior development. We used P-transformation to show that sequences in the 5′ leader are neither necessary for localization of the transcript nor for the translational block of the bcd mRNA during oogenesis.

For our injection experiments, we used only one of the identified splicing forms of bcd mRNA. The bcd protein species derived from this mRNA is able to induce anterior development at any position along the anterior-posterior axis. Thus bicoid protein can induce development of head and thorax independent of any other specifically localized morphogenetic factor. Our findings further support the notion that the concentration gradient of bcd protein, and not the existence of different forms of bcd protein, is responsible for specifying subregions of the embryo.
Tentacle morphogenesis in hydra: II. Formation of a complex between a sensory nerve cell and a battery cell
Authors: Hobmayer Engelbert , Holstein Thomas W. , David Charles N.
Published: April 2021

Differentiation of sensory nerve cells in tentacles of Hydra magnipapillata was Investigated using the monoclonal antibody NV1. NV1+ sensory nerve cells form specific complexes with battery cells in tentacles. NV1+ cells can only be formed by differentiation from interstitial cell precursors. These precursors complete a terminal cell cycle in the distal gastric region at the base of tentacles; differentiation from the S/G>2 boundary to expression of the NV1 antigen requires 30 h. During this time, precursors move from the distal gastric region into the tentacles, differentiate to morphologically fully formed nerve cells and then begin expressing NV1 antigen. The neuropeptide head activator stimulates NV1+ differentiation in S-phase of the precursor’s cell cycle.
Developmental expression of c-kit, a proto-oncogene encoded by the W locus
Authors: Orr-Urtreger Avi , Avivi Aaron , Zimmer Yitzhak , Givol David , Yarden Yosef , Lonai Peter
Published: April 2021

Developmental expression of the c-kit proto-oncogene, a receptor tyrosine kinase encoded by the W locus, was investigated by in situ hybridization in normal mouse embryos. Early after implantation transcripts were detectable only in the maternal placenta ( days p.c.). Subsequently ( days p.c.) numerous ectodermal (neural tube, sensory placodes) and endodermal (embryonic gut) derivatives expressed c-kit. Later transcripts were detected also in the blood islands of the yolk sac and in the embryonic liver, the main sites of embryonic hemopoiesis. Around midgestation, transcripts accumulated in the branchial pouches and also in primordial germ cells of the genital ridges. This complex pattern of expression remained characteristic also later in gestation, when c-kit was expressed in highly differentiated structures of the craniofacial area, in presumptive melanoblasts and in the CNS. In the adult ovary, maternal c-kit transcripts were detected. They were present in the oocytes of both immature and mature ovarian follicles, but not in the male germ line, where c-kit expression may be down regulated. Thus, c-kit activity is complex and appears in multiple tissues including those that also display defects in mutations at the W locus where c-kit is encoded. Correlation between W phenotypes and c-kit expression, as well as the regulation of the complex and multiple expression of polypeptide growth factors and receptors, is discussed.
The formation of basal body domains in the membrane skeleton of Tetrahymena
Authors: Williams Norman E. , Honts Jerry E. , Kaczanowska Janina
Published: April 2021

Differentiated regions within the membrane skeleton are described around basal bodies in the ciliary rows of Tetrahymena. These domains, approximately 1μm in diameter, are characterized by a relatively dense ultra-structure, the presence of a family of proteins called K antigens (Mr 39– 44 × 103) that are recognized by mAb 424A8, and the apparent exclusion of major membrane skeleton proteins that are present in most other regions of the cell (MT 135, 125 ×103). Mature basal body domains are asymmetric, reflecting the polarity of the cell as a whole. A similar differentiation of the membrane skeleton occurs in the oral apparatus, except here the K antigens surround four clusters of basal bodies (from which this cell takes its name) rather than the individual basal bodies. The development of new basal body domains in the cell cycle is described, with similarities and differences noted between somatic and oral regions of the cell. It is concluded that the capacity of this cell for precise topographic regulation of molecular events in the membrane skeleton makes it a useful model for the study of cortical differentiation in cells generally.
PLCζ: a sperm-specific trigger of Ca2+ oscillations in eggs and embryo development
Authors: Saunders Christopher M. , Larman Mark G. , Parrington John , Cox Llewellyn J. , Royse Jillian , Blayney Lynda M. , Swann Karl , Lai F. Anthony
Published: April 2021

Upon fertilisation by sperm, mammalian eggs are activated by a series of intracellular Ca2+ oscillations that are essential for embryo development. The mechanism by which sperm induces this complex signalling phenomenon is unknown. One proposal is that the sperm introduces an exclusive cytosolic factor into the egg that elicits serial Ca2+ release. The ‘sperm factor’ hypothesis has not been ratified because a sperm-specific protein that generates repetitive Ca2+ transients and egg activation has not been found. We identify a novel, sperm-specific phospholipase C, PLCζ, that triggers Ca2+ oscillations in mouse eggs indistinguishable from those at fertilisation. PLCζ removal from sperm extracts abolishes Ca2+ release in eggs. Moreover, the PLCζ content of a single sperm was sufficient to produce Ca2+ oscillations as well as normal embryo development to blastocyst. Our results are consistent with sperm PLCζ as the molecular trigger for development of a fertilised egg into an embryo.
Evolving role of Antennapedia protein in arthropod limb patterning
Authors: Shiga Yasuhiro , Yasumoto Ryusuke , Yamagata Hideo , Hayashi Shigeo
Published: April 2021

Evolutional changes in homeotic gene functions have contributed to segmental diversification of arthropodan limbs, but crucial molecular changes have not been identified to date. The first leg of the crustacean Daphnia lacks a prominent ventral branch found in the second to fourth legs. We show here that this phenotype correlates with the loss of Distal-less and concomitant expression of Antennapedia in the limb primordium. Unlike its Drosophila counterpart, Daphnia Antennapedia represses Distal-less in Drosophila assays, and the protein region conferring this activity was mapped to the N terminal region of the protein. The results imply that Dapnia Antennapedia specifies leg morphology by repressing Distal-less, and this activity was acquired through a change in protein structure after separation of crustaceans and insects.
Embryonic retinoic acid synthesis is required for forelimb growth and anteroposterior patterning in the mouse
Authors: Niederreither Karen , Vermot Julien , Schuhbaur Brigitte , Chambon Pierre , Dollé Pascal
Published: April 2021

Numerous studies, often performed on avian embryos, have implicated retinoic acid (RA) in the control of limb bud growth and patterning. Here we have investigated whether the lack of endogenous RA synthesis affects limb morphogenesis in mutant mouse embryos deficient for the retinaldehyde dehydrogenase 2 (Raldh2/Aldh1a2). These mutants, which have no detectable embryonic RA except in the developing retina, die at E9.5-E10 without any evidence of limb bud formation, but maternal RA supplementation through oral gavage from E7.5 can extend their survival. Such survivors exhibit highly reduced forelimb rudiments, but apparently normal hindlimbs. By providing RA within maternal food, we found both a stage- and dose-dependency for rescue of forelimb growth and patterning. Following RA supplementation from E7.5 to 8.5, mutant forelimbs are markedly hypoplastic and lack anteroposterior (AP) patterning, with a single medial cartilage and 1-2 digit rudiments. RA provided until E9.5 significantly rescues forelimb growth, but cannot restore normal AP patterning. Increasing the RA dose rescues the hypodactyly, but leads to lack of asymmetry of the digit pattern, with abnormally long first digit or symmetrical polydactyly. Mutant forelimb buds are characterized by lack of expression or abnormal distal distribution of Sonic hedgehog (Shh) transcripts, sometimes with highest expression anteriorly. Downregulation or ectopic anterior expression of Fgf4 is also seen. As a result, genes such as Bmp2 or Hoxd genes are expressed symmetrically along the AP axis of the forelimb buds, and/or later, of the autopod. We suggest that RA signaling cooperates with a posteriorly restricted factor such as dHand, to generate a functional zone of polarizing activity (ZPA).
Cell coherence during production of the presomitic mesoderm and somitogenesis in the mouse embryo
Authors: Eloy-Trinquet Sophie , Nicolas Jean-François
Published: April 2021

In this study, we investigated (in the early mouse embryo) the clonal properties of precursor cells which contribute to the segmented myotome, a structure derived from the somites. We used the laacZ method of single cell-labelling to visualise clones born before segmentation and bilateralisation. We found that clones which contribute to several segments both unilateral and bilateral were regionalised along the mediolateral axis and that their mediolateral position was maintained in successive adjacent segments. Furthermore, clones contributed to all segments, from their most anterior to their most posterior borders. Therefore, it appears that mediolateral regionalisation of myotomal precursor cells is a property established before bilateralisation of the presomitic mesoderm and that coherent clonal growth accompanies cell dispersion along both the mediolateral and anteroposterior axes. These findings in the mouse correlate well with what is known in the chick, suggesting conservation of the mode of production and distribution of the cells of the presomitic mesoderm. However, in addition, we also found that the mediolateral contribution of a clone is already determined in the pool of self-renewing cells that produces the myotomal precursor cells and thus that this pool is itself regionalised. Finally, we found that bilateral clones exhibit symmetry in right and left sides in the embryo at all levels of the mediolateral axis of the myotome. All these properties indicate synchrony and symmetry of formation of the presomitic mesoderm on both sides of the embryo leading to formation of a static embryonic structure with few cell movements. We suggest that sequential production of groups of cells with an identical clonal origin for both sides of the embryo from a single pool of self-renewing cells, coupled with aquisition of static cell behaviour, could play a role in colinearity of expression of Hox genes and in the segmentation system of higher vertebrates.
Oskar anchoring restricts pole plasm formation to the posterior of theDrosophilaoocyte
Authors: Vanzo Nathalie F. , Ephrussi Anne
Published: April 2021

Localization of the maternal determinant Oskar at the posterior pole of Drosophila melanogaster oocyte provides the positional information for pole plasm formation. Spatial control of Oskar expression is achieved through the tight coupling of mRNA localization to translational control, such that only posterior-localized oskar mRNA is translated, producing the two Oskar isoforms Long Osk and Short Osk. We present evidence that this coupling is not sufficient to restrict Oskar to the posterior pole of the oocyte. We show that Long Osk anchors both oskar mRNA and Short Osk, the isoform active in pole plasm assembly, at the posterior pole. In the absence of anchoring by Long Osk, Short Osk disperses into the bulk cytoplasm during late oogenesis, impairing pole cell formation in the embryo. In addition, the pool of untethered Short Osk causes anteroposterior patterning defects, owing to the dispersion of pole plasm and its abdomen-inducing activity throughout the oocyte. We show that the N-terminal extension of Long Osk is necessary but not sufficient for posterior anchoring, arguing for multiple docking elements in Oskar. This study reveals cortical anchoring of the posterior determinant Oskar as a crucial step in pole plasm assembly and restriction, required for proper development of Drosophila melanogaster.
Bmp signaling is required for development of primary lens fiber cells
Authors: Faber Sonya C. , Robinson Michael L. , Makarenkova Helen P. , Lang Richard A.
Published: April 2021

We have investigated the role of Bmp signaling in development of the mouse lens using three experimental strategies. First, we have shown that the Bmp ligand inhibitor noggin can suppress the differentiation of primary lens fiber cells in explant culture. Second, we have expressed a dominant-negative form of the type 1 Bmp family receptor Alk6 (Bmpr1b – Mouse Genome Informatics) in the lens in transgenic mice and shown that an inhibition of primary fiber cell differentiation can be detected at E13.5. Interestingly, the observed inhibition of primary fiber cell development was asymmetrical and appeared only on the nasal side of the lens in the ventral half. Expression of the inhibitory form of Alk6 was driven either by the αA-cystallin promoter or the ectoderm enhancer from the Pax6 gene in two different transgenes. These expression units drive transgene expression in distinct patterns that overlap in the equatorial cells of the lens vesicle at E12.5. Despite the distinctions between the transgenes, they caused primary fiber cell differentiation defects that were essentially identical, which implied that the equatorial lens vesicle cells were responding to Bmp signals in permitting primary fiber cells to develop. Importantly, E12.5 equatorial lens vesicle cells showed cell-surface immunoreactivity for bone-morphogenetic protein receptor type 2 and nuclear immunoreactivity for the active, phosphorylated form of the Bmp responsive Smads. This indicated that these cells had the machinery for Bmp signaling and were responding to Bmp signals. We conclude that Bmp signaling is required for primary lens fiber cell differentiation and, given the asymmetry of the differentiation inhibition, that distinct differentiation stimuli may be active in different quadrants of the eye.
rasp, a putative transmembrane acyltransferase, is required for Hedgehog signaling
Authors: Micchelli Craig A. , The Inge , Selva Erica , Mogila Vladic , Perrimon Norbert
Published: April 2021

Members of the Hedgehog (Hh) family encode secreted molecules that act as potent organizers during vertebrate and invertebrate development. Post-translational modification regulates both the range and efficacy of Hh protein. One such modification is the acylation of the N-terminal cysteine of Hh. In a screen for zygotic lethal mutations associated with maternal effects, we have identified rasp, a novel Drosophila segment polarity gene. Analysis of the rasp mutant phenotype, in both the embryo and wing imaginal disc demonstrates that rasp does not disrupt Wnt/Wingless signaling but is specifically required for Hh signaling. The requirement of rasp is restricted only to those cells that produce Hh; hh transcription, protein levels and distribution are not affected by the loss of rasp. Molecular analysis reveals that rasp encodes a multipass transmembrane protein that has homology to a family of membrane bound O-acyl transferases. Our results suggest that Rasp-dependent acylation is necessary to generate a fully active Hh protein.
Low density detergent-insoluble membrane ofXenopuseggs: subcellular microdomain for tyrosine kinase signaling in fertilization
Authors: Sato Ken-ichi , Iwasaki Tetsushi , Ogawa Keiko , Konishi Masako , Tokmakov Alexander A. , Fukami Yasuo
Published: April 2021

Protein-tyrosine phosphorylation plays an important role in egg activation signaling at fertilization. We show that in Xenopus, fertilization stimulates a rapid and transient tyrosine phosphorylation of egg proteins, as revealed by immunoblotting with anti-phosphotyrosine antibody. Immunofluorescent microscopic analysis demonstrated that the phosphorylation occurs in cortical area of the egg animal hemisphere. To further characterize subcellular compartment for fertilization-dependent tyrosine kinase signaling, we isolated low density detergent-insoluble membrane (LD-DIM) fraction from Xenopus eggs. The egg LD-DIM was enriched in cholesterol and GM1 ganglioside. It also contained signaling molecules such as Xyk (Xenopus egg Src), Gqα, Ras, integrin β1 and CD9. Fertilization stimulated tyrosine phosphorylation of Xyk and some other LD-DIM proteins. Remarkably, sperm stimulated tyrosine phosphorylation of the LD-DIM proteins in vitro. The sperm-dependent phosphorylation was sensitive to the tyrosine kinase inhibitors PP2 and genistein. We found that pretreatment of eggs with methyl-β-cyclodextrin, a cholesterol-binding substance, led to a decrease in cholesterol, Xyk and sperm-induced tyrosine phosphorylation in LD-DIM. In methyl-β-cyclodextrin-treated eggs, sperm-induced Ca2+ transient and first cell division were also inhibited. These findings suggest that the egg LD-DIM might serve as subcellular microdomain for tyrosine kinase signaling in Xenopus egg fertilization.
Grunge, related to human Atrophin-like proteins, has multiple functions inDrosophiladevelopment
Authors: Erkner Alfrun , Roure Agnès , Charroux Bernard , Delaage Michèle , Holway Nicolas , Coré Nathalie , Vola Christine , Angelats Corinne , Pagès Françoise , Fasano Laurent , Kerridge Stephen
Published: April 2021

We have carried out a genetic screen designed to isolate regulators of teashirt expression. One of these regulators is the Grunge gene, which encodes a protein with motifs found in human arginine-glutamic acid dipeptide repeat, Metastasis-associated-like and Atrophin-1 proteins. Grunge is the only Atrophin-like protein in Drosophila, whereas several exist in humans. We provide evidence that Grunge is required for the proper regulation of teashirt but also has multiple activities in fly development. First, Grunge is crucial for correct segmentation during embryogenesis via a failure in the repression of at least four segmentation genes known to regulate teashirt. Second, Grunge acts positively to regulate teashirt expression in proximoventral parts of the leg. Grunge has other regulatory functions in the leg, including the patterning of ventral parts along the entire proximodistal axis and the proper spacing of bristles in all regions.
Proper timing of a quiescence period in precursor prospermatogonia is required for stem cell pool establishment in the male germline
Authors: Du Guihua , Oatley Melissa J. , Law Nathan C. , Robbins Colton , Wu Xin , Oatley Jon M.
Published: April 2021

The stem cell-containing undifferentiated spermatogonial population in mammals, which ensures continual sperm production, arises during development from prospermatogonial precursors. Although a period of quiescence is known to occur in prospermatogonia prior to postnatal spermatogonial transition, the importance of this has not been defined. Here, using mouse models with conditional knockout of the master cell cycle regulator Rb1 to disrupt normal timing of the quiescence period, we found that failure to initiate mitotic arrest during fetal development leads to prospermatogonial apoptosis and germline ablation. Outcomes of single-cell RNA-sequencing analysis indicate that oxidative phosphorylation activity and inhibition of meiotic initiation are disrupted in prospermatogonia that fail to enter quiescence on a normal timeline. Taken together, these findings suggest that key layers of programming are laid down during the quiescent period in prospermatogonia to ensure proper fate specification and fitness in postnatal life.
FGF signaling acts on different levels of mesoderm development within Spiralia
Authors: Andrikou Carmen , Hejnol Andreas
Published: April 2021

FGF signaling is involved in mesoderm induction in members of deuterostomes (e.g. tunicates, hemichordates), but not in flies and nematodes, in which it has a role in mesoderm patterning and migration. However, we need comparable studies in other protostome taxa in order to decipher whether this mesoderm-inducing function of FGF extends beyond the lineage of deuterostomes. Here, we investigated the role of FGF signaling in mesoderm development in three species of lophophorates, a clade within the protostome group Spiralia. Our gene expression analyses show that the mesodermal molecular patterning is conserved between brachiopods and phoronids, but the spatial and temporal recruitment of transcription factors differs significantly. Moreover, the use of the inhibitor SU5402 demonstrates that FGF signaling is involved in different steps of mesoderm development, as well as in morphogenetic movements of gastrulation and axial elongation. Our findings suggest that the mesoderm-inducing role of FGF extends beyond the group of deuterostomes.
Nutrition and PI3K/Akt signaling are required for p38-dependent regeneration
Authors: Esteban-Collado José , Corominas Montserrat , Serras Florenci
Published: April 2021

Regeneration after damage requires early signals to trigger the tissue repair machinery. Reactive oxygen species (ROS) act as early signals that are sensed by the MAP3 kinase Ask1, which in turn activates by phosphorylation the MAP kinases p38 and JNK. The sustained or high activation of these kinases can result in apoptosis, whereas short or low activation can promote regeneration. Using the Ask1-dependent regeneration program, we demonstrate in Drosophila wing that PI3K/Akt signaling is necessary for Ask1 to activate p38, but not JNK. In addition, nutrient restriction or mutations that target Ser83 of the Drosophila Ask1 protein, a PI3K/Akt-sensitive residue, block regeneration. However, these effects can be reversed by the ectopic activation of p38, but not of JNK. Our results demonstrate that Ask1 controls the activation of p38 through Ser83, and that the phosphorylation of p38 during regeneration is nutrient sensitive. This mechanism is important for discriminating between p38 and JNK in the cells involved in tissue repair and regenerative growth.
PRSS50 is a testis protease responsible for proper sperm tail formation and function
Authors: Scovell Jason M. , Bournat Juan C. , Szafran Adam T. , Solis Minerva , Moore Joshua , Rivera Armando , Chen Ching H. , Zhang Jason , Wilken Nathan , Seth Abhishek , Jorgez Carolina J.
Published: April 2021

Multiple morphological abnormalities of the sperm flagella (MMAF) are a major cause of asthenoteratozoospermia. We have identified protease serine 50 (PRSS50) as having a crucial role in sperm development, because Prss50-null mice presented with impaired fertility and sperm tail abnormalities. PRSS50 could also be involved in centrosome function because these mice showed a threefold increase in acephalic sperm (head-tail junction defect), sperm with multiple heads (spermatid division defect) and sperm with multiple tails, including novel two conjoined sperm (complete or partial parts of several flagellum on the same plasma membrane). Our data support that, in the testis, as in tumorigenesis, PRSS50 activates NFκB target genes, such as the centromere protein leucine-rich repeats and WD repeat domain-containing protein 1 (LRWD1), which is required for heterochromatin maintenance. Prss50-null testes have increased IκκB, and reduced LRWD1 and histone expression. Low levels of de-repressed histone markers, such as H3K9me3, in the Prss50-null mouse testis may cause increases in post-meiosis proteins, such as AKAP4, affecting sperm formation. We provide important insights into the complex mechanisms of sperm development, the importance of testis proteases in fertility and a novel mechanism for MMAF.
Eukaryotic initiation factor 4A3 inhibits Wnt/β-catenin signaling and regulates axis formation in zebrafish embryos
Authors: Wang Bo , Rong Xiaozhi , Zhou Yumei , Liu Yunzhang , Sun Jiqin , Zhao Beibei , Deng Bei , Lu Lei , Lu Ling , Li Yun , Zhou Jianfeng
Published: April 2021

A key step in the activation of canonical Wnt signaling is the interaction between β-catenin and Tcf/Lefs that forms the transcription activation complex and facilitates the expression of target genes. Eukaryotic initiation factor 4A3 (EIF4A3) is an ATP-dependent DEAD box-family RNA helicase and acts as a core subunit of the exon junction complex (EJC) to control a series of RNA post-transcriptional processes. In this study, we uncover that EIF4A3 functions as a Wnt inhibitor by interfering with the formation of β-catenin/Tcf transcription activation complex. As Wnt stimulation increases, accumulated β-catenin displaces EIF4A3 from a transcriptional complex with Tcf/Lef, allowing the active complex to facilitate the expression of target genes. In zebrafish embryos, eif4a3 depletion inhibited the development of the dorsal organizer and pattern formation of the anterior neuroectoderm by increasing Wnt/β-catenin signaling. Conversely, overexpression of eif4a3 decreased Wnt/β-catenin signaling and inhibited the formation of the dorsal organizer before gastrulation. Our results reveal previously unreported roles of EIF4A3 in the inhibition of Wnt signaling and the regulation of embryonic development in zebrafish.
Germ cell determination and the developmental origin of germ cell tumors
Authors: Nicholls Peter K. , Page David C.
Published: April 2021

In each generation, the germline is tasked with producing somatic lineages that form the body, and segregating a population of cells for gametogenesis. During animal development, when do cells of the germline irreversibly commit to producing gametes? Integrating findings from diverse species, we conclude that the final commitment of the germline to gametogenesis – the process of germ cell determination – occurs after primordial germ cells (PGCs) colonize the gonads. Combining this understanding with medical findings, we present a model whereby germ cell tumors arise from cells that failed to undertake germ cell determination, regardless of their having colonized the gonads. We propose that the diversity of cell types present in these tumors reflects the broad developmental potential of migratory PGCs.
Sur le déterminisme de la qualitérégionale des régénérais d’appendices chezla Blatte, Blabera craniifer
Authors: Bullière Désiré
Published: April 2021

Dans tous les phénomènes de régénération, il est bien évident que la qualité et l’organisation des tissus néoformés sont sous la dépendance des tissus anciens. Dans le cas de la régénération d’un appendice amputé, le moignon détermine la qualité régionale du régénérât. Mais la question se pose de savoir si le moignon agit par l’ensemble de ses organes laissés en place ou si seuls les tissus situés immédiatement en arrière de la section ont une action déterminante. En modifiant expérimentalement la qualité des tissus proches de la section on peut espérer obtenir une réponse à cette question. Les expériences de greffes hétéro-spécifiques, faites sur les Némertes par exemple (Bierne, 1967 a, b), montrent bien que le régénérât est sous la dépendance des tissus voisins de la section. Chez les Insectes, où les processus de régénération se déroulentà l’intérieur du segment de membre resté en place, les résultats obtenus jusqu’ici sont moins nets. Les expériences d’implantation de patte à la place de l’antenne faites par Urvoy (1963 a) et par Bohn (1965 b) conduisent à admettre un contrôle du régénérât par les tissus du greffon. Ce contrôle serait complet d’après Bohn et incomplet d’après Urvoy, qui pense à une influence du moignon antennaire sur le régénérât par l’intermédiaire du système nerveux de l’antenne. Les nerfs de l’antenne, en envahissant le régénérât du type patte conforme au greffon, le transformerait quelque peu dans le sens antenne. Les expériences d’implantation d’une partie de la patte prothoracique (P 1) sur un moignon de la patte métathoracique (P 3) ont été interprétées très différemment par leurs auteurs. Urvoy (1963 b), après avoir remplacé l’extrémité d’une hanche de patte métathoracique par l’extrémité d’une hanche prothoracique, conclut à la prédominance des tissus de l’hôte sur la qualité du régénérât. Bohn (1965 a), en greffant la partie distale de P x (tibia + tarse) sur l’extrémité du tibia de P 3, puis en sectionnant, après la première mue post-opératoire, le tarse de P 1 à sa base, conclut à l’influence prédominante du greffon sur la qualité du tarse régénéré. Ainsi, en ne considérant que le cas des Insectes, les résultats sont presque concordants lorsqu’il s’agit de régénération après greffe d’une partie d’appendice très différente du moignon porte-greffe (par exemple, patte sur antenne), mais divergent lorsqu’il s’agit de combinaisons entre parties d’appendices semblables (patte prothoracique sur patte méta-thoracique).
Interprétation des régénérais multiples chez les Insectes
Authors: Bulliére Désiré
Published: April 2021

Depuis très longtemps les auteurs se sont attachés à l’étude des régénérais surnuméraires chez les Insectes et ont cherché à reproduire expérimentalement ces malformations, afin d’en mieux comprendre la genèse. A la fin du 19° siècle, Bateson (1894) a essayé de trouver une explication aux variations naturelles en formulant des lois qui régissent ces variations. Bodenstein (1937, 1941) a, le premier, réussi à provoquer expérimentalement l’apparition de régénérais multiples chez Phryganidia california et P. cardini. A sa suite, Balazuc (1948, 1955) et plus récemment Penzlin (1965), Bart (1965 a, b, 1966) et Bohn (1965 a, b) ont tenté de donner une explication à la production expérimentale de régénérais multiples. Ces études, faites sur les Insectes, ne s’accordent pas toujours avec les conclusions de Della Valle (1913) et de Przibram (1921) qui ont obtenu des régénérats multiples chez les Batraciens urodèles. D’après Przibram, la section incomplète d’un membre conduit à l’élaboration d’un régénérât à partir de chacune des deux lèvres de la plaie, maintenues écartées artificiellement.
A light and electron microscopic study of cell behavior and microtubules in the embryonic chicken lens using Colcemid
Authors: Pearce Thomas L. , Zwaan Johan
Published: April 2021

The first sign of differentiation of the lens rudiment is a change in cellular shape, as in many other embryonic systems (Tilney, 1968 a). This occurs some time before the beginning of overt chemical differentiation, the appearance of specific proteins (Zwaan, 1968). From stage 11 (Hamburger & Hamilton, 1951), just after the optic cup contacts the surface ectoderm, to stage 13, before the onset of invagination, the lens Anlage is rapidly transformed from a cuboidal to a high columnar epithelium, a process described by McKeehan (1951) as palisading. This is slightly preceded in time by nuclear rearrangement. First irregular in outline and randomly oriented, the nuclei become smooth and oval, and aligned at right angles to the surface. They take up positions in the basal portions of the cells (McKeehan, 1951).
Dissociation of sensory ganglia from the embryonic chick by pronase and other dispersing agents
Authors: Banks Barbara E. C. , Banthorpe D. V. , Lamont D. Margaret , Pearce F. L. , Redding Katharine A. , Vernon C. A.
Published: April 2021

The gross effect of protein factors from mouse salivary glands and certain snake venoms on the growth, in tissue culture, of explants of sensory ganglia from chick embryos is well established (Levi-Montalcini, 1964, 1965; Angeletti, Calissano, Chen & Levi-Montalcini, 1967; Banks et al. 1968; Varon, Nomura & Shooter, 1967a, b). The effects of these substances, called nerve growth factors, on individual neurons in vitro have, however, been little investigated and no quantitative studies have been reported. In order to make such a study we required a method of making cell suspensions of high density from chick embryonic sensory ganglia: the target tissue normally used for the assay of nerve growth factors.
Mandibular growth retardation as a cause of cleft palate in mice homozygous for the chondrodysplasia gene
Authors: Seegmiller Robert E. , Fraser F. Clarke
Published: April 2021

Defective chondrogenesis in C57BL mice homozygous for the chondrodysplasia gene leads to deformity of limbs, ribs, trachea, mandible and palate. Since formation of the secondary palate depends upon coordinated development of several craniofacial structures, the presence of micrognathia and cleft palate in cho/cho newborn mice suggested a cause-and-effect relation between these two deformities. To determine whether or not lower jaw shortening coincided with the time of palate closure, heads from mutant and control littermates previously rated morphologically were examined in median sagittal section. Of six parameters analyzed, growth rates for mutant mandible and anterior vertical dimension were significantly less than those of controls from the beginning of control palate closure. Since there is evidence that intrinsic shelf force is normal, these observations suggested that, during palatogenesis, growth retardation of Meckel’s cartilage did not allow forward displacement of the tongue, and that the consequent failure to straighten the tongue impaired shelf movement. The data support the concept that growth of Meckel’s cartilage is necessary for normal palate formation.