bluuebunny/crossref_metadata_2025 huggingface dataset:
Hedgehog is required for murine yolk sac angiogenesis
Authors: Byrd Noah , Becker Sandy , Maye Peter , Narasimhaiah Roopa , St-Jacques Benoit , Zhang Xiaoyan , McMahon Jill , McMahon Andrew , Grabel Laura
Published: April 2021

Blood islands, the precursors of yolk sac blood vessels, contain primitive erythrocytes surrounded by a layer of endothelial cells. These structures differentiate from extra-embryonic mesodermal cells that underlie the visceral endoderm. Our previous studies have shown that Indian hedgehog (Ihh) is expressed in the visceral endoderm both in the visceral yolk sac in vivo and in embryonic stem (ES) cell-derived embryoid bodies. Differentiating embryoid bodies form blood islands, providing an in vitro model for studying vasculogenesis and hematopoiesis. A role for Ihh in yolk sac function is suggested by the observation that roughly 50% of Ihh–/– mice die at mid-gestation, potentially owing to vascular defects in the yolk sac. To address the nature of the possible vascular defects, we have examined the ability of ES cells deficient for Ihh or smoothened (Smo), which encodes a receptor component essential for all hedgehog signaling, to form blood islands in vitro. Embryoid bodies derived from these cell lines are unable to form blood islands, and express reduced levels of both PECAM1, an endothelial cell marker, and α-SMA, a vascular smooth muscle marker. RT-PCR analysis in the Ihh–/– lines shows a substantial decrease in the expression of Flk1 and Tal1, markers for the hemangioblast, the precursor of both blood and endothelial cells, as well as Flt1, an angiogenesis marker. To extend these observations, we have examined the phenotypes of embryo yolk sacs deficient for Ihh or Smo. Whereas Ihh–/– yolk sacs can form blood vessels, the vessels are fewer in number and smaller, perhaps owing to their inability to undergo vascular remodeling. Smo–/– yolk sacs arrest at an earlier stage: the endothelial tubes are packed with hematopoietic cells, and fail to undergo even the limited vascular remodeling observed in the Ihh–/– yolk sacs. Our study supports a role for hedgehog signaling in yolk sac angiogenesis.
Notch1 is required for neuronal and glial differentiation in the cerebellum
Authors: Lütolf Simone , Radtke Freddy , Aguet Michel , Suter Ueli , Taylor Verdon
Published: April 2021

The mechanisms that guide progenitor cell fate and differentiation in the vertebrate central nervous system (CNS) are poorly understood. Gain-of-function experiments suggest that Notch signaling is involved in the early stages of mammalian neurogenesis. On the basis of the expression of Notch1 by putative progenitor cells of the vertebrate CNS, we have addressed directly the role of Notch1 in the development of the mammalian brain. Using conditional gene ablation, we show that loss of Notch1 results in premature onset of neurogenesis by neuroepithelial cells of the midbrain-hindbrain region of the neural tube. Notch1-deficient cells do not complete differentiation but are eliminated by apoptosis, resulting in a reduced number of neurons in the adult cerebellum. We have also analyzed the effects of Notch1 ablation on gliogenesis in vivo. Our results show that Notch1 is required for both neuron and glia formation and modulates the onset of neurogenesis within the cerebellar neuroepithelium.
Cell polarity and gastrulation inC. elegans
Authors: Nance Jeremy , Priess James R.
Published: April 2021

Gastrulation in C. elegans embryos involves formation of a blastocoel and the ingression of surface cells into the blastocoel. Mutations in the par-3 gene cause abnormal separations between embryonic cells, suggesting that the PAR-3 protein has a role in blastocoel formation. In normal development, PAR proteins localize to either the apical or basal surfaces of cells prior to blastocoel formation; we demonstrate that this localization is determined by cell contacts. Cells that ingress into the blastocoel undergo an apical flattening associated with an apical concentration of non-muscle myosin. We provide evidence that ingression times are determined by genes that control cell fate, though interactions with neighboring cells can prevent ingression.
X-chromosome silencing in the germline ofC. elegans
Authors: Kelly William G. , Schaner Christine E. , Dernburg Abby F. , Lee Min-Ho , Kim Stuart K. , Villeneuve Anne M. , Reinke Valerie
Published: April 2021

Germline maintenance in the nematode C. elegans requires global repressive mechanisms that involve chromatin organization. During meiosis, the X chromosome in both sexes exhibits a striking reduction of histone modifications that correlate with transcriptional activation when compared with the genome as a whole. The histone modification spectrum on the X chromosome corresponds with a lack of transcriptional competence, as measured by reporter transgene arrays. The X chromosome in XO males is structurally analogous to the sex body in mammals, contains a histone modification associated with heterochromatin in other species and is inactivated throughout meiosis. The synapsed X chromosomes in hermaphrodites also appear to be silenced in early meiosis, but genes on the X chromosome are detectably expressed at later stages of oocyte meiosis. Silencing of the sex chromosome during early meiosis is a conserved feature throughout the nematode phylum, and is not limited to hermaphroditic species.
Deletion of the ecdysis-triggering hormone gene leads to lethal ecdysis deficiency
Authors: Park Yoonseong , Filippov Valery , Gill Sarjeet S. , Adams Michael E.
Published: April 2021

At the end of each developmental stage, insects perform a stereotypic behavioral sequence leading to ecdysis of the old cuticle. While ecdysis-triggering hormone (ETH) is sufficient to trigger this sequence, it has remained unclear whether it is required. We show that deletion of eth, the gene encoding ETH in Drosophila, leads to lethal behavioral and physiological deficits. Null mutants (eth–) fail to inflate the new respiratory system on schedule, do not perform the ecdysis behavioral sequence, and exhibit the phenotype buttoned-up, which is characterized by incomplete ecdysis and 98% mortality at the transition from first to second larval instar. Precisely timed injection of synthetic DmETH1 restores all deficits and allows normal ecdysis to occur. These findings establish obligatory roles for eth and its gene products in initiation and regulation of the ecdysis sequence. The ETH signaling system provides an opportunity for genetic analysis of a chemically coded physiological and behavioral sequence.
Msx2is an immediate downstream effector ofPax3in the development of the murine cardiac neural crest
Authors: Kwang Stanford J. , Brugger Sean M. , Lazik Arthur , Merrill Amy E. , Wu Lan-Ying , Liu Yi-Hsin , Ishii Mamoru , Sangiorgi Frank O. , Rauchman Michael , Sucov Henry M. , Maas Richard L. , Maxson Robert E.
Published: April 2021

The neural crest plays a crucial part in cardiac development. Cells of the cardiac subpopulation of cranial neural crest migrate from the hindbrain into the outflow tract of the heart where they contribute to the septum that divides the pulmonary and aortic channels. In Splotch mutant mice, which lack a functional Pax3 gene, migration of cardiac neural crest is deficient and aorticopulmonary septation does not occur. Downstream genes through which Pax3 regulates cardiac neural crest development are unknown. Here, using a combination of genetic and molecular approaches, we show that the deficiency of cardiac neural crest development in the Splotch mutant is caused by upregulation of Msx2, a homeobox gene with a well-documented role as a regulator of BMP signaling. We provide evidence, moreover, that Pax3 represses Msx2 expression via a direct effect on a conserved Pax3 binding site in the Msx2 promoter. These results establish Msx2 as an effector of Pax3 in cardiac neural crest development.
Distinct regulatory cascades for head and trunk myogenesis
Authors: Mootoosamy Roy C. , Dietrich Susanne
Published: April 2021

Most head muscles arise from the pre-otic axial and paraxial head mesoderm. This tissue does not form somites, yet expresses the somitic markers Lbx1, Pax7 and Paraxis in a regionalised fashion. The domain set aside by these markers provides the lateral rectus muscle, the most caudal of the extrinsic eye muscles. In contrast to somitic cells that express Lbx1, lateral rectus precursors are non-migratory. Moreover, the set of markers characteristic for the lateral rectus precursors differs from the marker sets indicative of somitic muscle precursors. This suggests distinct roles for Lbx1/Pax7/Paraxis in the development of head and trunk muscles. When grafted to the trunk, the pre-otic head mesoderm fails to activate Lbx1, Pax7 or Paraxis. Likewise, somites grafted into the region of the lateral rectus precursors fail to activate the lateral rectus marker set. This suggests that distinct regulatory cascades act in the development of trunk and head muscles, possibly reflecting their distinct function and evolution.
Meis family proteins are required for hindbrain development in the zebrafish
Authors: Choe Seong-Kyu , Vlachakis Nikolaos , Sagerström Charles G.
Published: April 2021

Meis homeodomain proteins function as Hox-cofactors by binding Pbx and Hox proteins to form multimeric complexes that control transcription of genes involved in development and differentiation. It is not known what role Meis proteins play in these complexes, nor is it clear which Hox functions require Meis proteins in vivo. We now show that a divergent Meis family member, Prep1, acts as a Hox co-factor in zebrafish. This suggests that all Meis family members have at least one shared function and that this function must be carried out by a conserved domain. We proceed to show that the Meinox domain, an N-terminal conserved domain shown to mediate Pbx binding, is sufficient to provide Meis activity to a Pbx/Hox complex. We find that this activity is separable from Pbx binding and resides within the M1 subdomain. This finding also presents a rational strategy for interfering with Meis activity in vivo. We accomplish this by expressing the Pbx4/Lzr N-terminus, which sequesters Meis proteins in the cytoplasm away from the nuclear transcription complexes. Sequestering Meis proteins in the cytoplasm leads to extensive loss of rhombomere (r) 3- and r4-specific gene expression, as well as defective rhombomere boundary formation in this region. These changes in gene expression correlate with impaired neuronal differentiation in r3 and r4, e.g. the loss of r3-specific nV branchiomotor neurons and r4-specific Mauthner neurons. We conclude that Meis family proteins are essential for the specification of r3 and r4 of the hindbrain.
Limb type-specific regulation ofbric a braccontributes to morphological diversity
Authors: Chu Jessie , Dong P. D. Si , Panganiban Grace
Published: April 2021

The insect antenna and leg are considered homologous structures, likely to have arisen via duplication and divergence from an ancestral limb. Consistent with this, the antenna and leg are derived from primordia with similar developmental potentials. Nonetheless, the adult structures differ in both form and function. In Drosophila, one conspicuous morphological difference is that the antenna has fewer distal segments than the leg. We propose that this is due in part to the variations in the regulation of bric a brac. bric a brac is required for joint formation, and loss of bric a brac function leads to fusion of distal antennal and leg segments, resulting in fewer total segments. Here, we address how bric a brac is regulated to generate the mature expression patterns of two concentric rings in the antenna versus four concentric rings in the leg. We find that bric a brac expression is activated early throughout most of the Distal-less domain in both antenna and leg and subsequently is restricted to the distal portion and into rings. Although bric a brac expression in the antenna and in all four tarsal rings of the leg requires Distal-less, only the proximal three tarsal rings are Spineless-dependent. Thus bric a brac is regulated differentially even within a single appendage type. The restriction of bric a brac expression to the distal portion of the Distal-less domain is a consequence of negative regulation by distinct sets of genes in different limb types. In the leg, the proximal boundary of bric a brac is established by the medial-patterning gene dachshund, but dachshund alone is insufficient to repress bric a brac, and the expression of the two genes overlaps. In the antenna, the proximal boundary of bric a brac is established by an antenna-specifying gene, homothorax, in conjunction with dachshund and spalt, and there is much less overlap between the bric a brac and the dachshund domains. Thus tissue-specific expression of other patterning genes that differentially repress bric a brac accounts for antenna-leg differences in bric a brac pattern. We propose that the limb type-specific variations in expression of bric a brac repressors contribute to morphological variations by controlling distal limb segment number.
JAK signaling is somatically required for follicle cell differentiation inDrosophila
Authors: McGregor Jennifer R. , Xi Rongwen , Harrison Douglas A.
Published: April 2021

Janus kinase (JAK) pathway activity is an integral part of signaling through a variety of ligands and receptors in mammals. The extensive re-utilization and pleiotropy of this pathway in vertebrate development is conserved in other animals as well. In Drosophila melanogaster, JAK signaling has been implicated in embryonic pattern formation, sex determination, larval blood cell development, wing venation, planar polarity in the eye, and formation of other adult structures. Here we describe several roles for JAK signaling in Drosophila oogenesis. The gene for a JAK pathway ligand, unpaired, is expressed specifically in the polar follicle cells, two pairs of somatic cells at the anterior and posterior poles of the developing egg chamber. Consistent with unpaired expression, reduced JAK pathway activity results in the fusion of developing egg chambers. A primary defect of these chambers is the expansion of the polar cell population and concomitant loss of interfollicular stalk cells. These phenotypes are enhanced by reduction of unpaired activity, suggesting that Unpaired is a necessary ligand for the JAK pathway in oogenesis. Mosaic analysis of both JAK pathway transducers, hopscotch and Stat92E, reveals that JAK signaling is specifically required in the somatic follicle cells. Moreover, JAK activity is also necessary for the initial commitment of epithelial follicle cells. Many of these roles are in common with, but distinct from, the known functions of Notch signaling in oogenesis. Consistent with these data is a model in which Notch signaling determines a pool of cells to be competent to adopt stalk or polar fate, while JAK signaling assigns specific identity within that competent pool.
Micromere lineages in the glossiphoniid leechHelobdella
Authors: Huang Françoise Z. , Kang Dongmin , Ramirez-Weber Felipe-Andres , Bissen Shirley T. , Weisblat David A.
Published: April 2021

In leech embryos, segmental mesoderm and ectoderm arise from teloblasts by lineages that are already relatively well characterized. Here, we present data concerning the early divisions and the definitive fate maps of the micromeres, a group of 25 small cells that arise during the modified spiral cleavage in leech (Helobdella robusta) and contribute to most of the nonsegmental tissues of the adult. Three noteworthy results of this work are as follows. (1) The c′′′ and dm′ clones (3d and 3c in traditional nomenclature) give rise to a hitherto undescribed network of fibers that run from one end of the embryo to the other. (2) The clones of micromeres b′′ and b′′′ (2b and 3b in traditional nomenclature) die in normal development; the b′′ clone can be rescued to assume the normal c′′ fate if micromere c′′ or its clone are ablated in early development. (3) Two qualitative differences in micromere fates are seen between H. robusta (Sacramento) and another Helobdella sp. (Galt). First, in Helobdella sp. (Galt), the clone of micromere b′′ does not normally die, and contributes a subset of the cells arising exclusively from c′′ in H. robusta (Sacramento). Second, in Helobdella sp. (Galt), micromere c′′′ makes no definitive contribution, whereas micromere dm′ gives rise to cells equivalent to those arising from c′′′ and dm′ in H. robusta (Sacramento).
Elevated transforming growth factor β2 enhances apoptosis and contributes to abnormal outflow tract and aortic sac development in retinoic X receptor α knockout embryos
Authors: Kubalak Steven W. , Hutson D. Reneé , Scott Karen K. , Shannon Rebecca A.
Published: April 2021

Septation of the single tubular embryonic outflow tract into two outlet segments in the heart requires the precise integration of proliferation, differentiation and apoptosis during remodeling. Lack of proper coordination between these processes would result in a variety of congenital cardiac defects such as those seen in the retinoid X receptor α knockout (Rxra–/–) mouse. Rxra–/– embryos exhibit lethality between embryonic day (E) 13.5 and 15.5 and harbor a variety of conotruncal and aortic sac defects making it an excellent system to investigate the molecular and morphogenic causes of these cardiac malformations. At E12.5, before the embryonic lethality, we found no qualitative difference between wild type and Rxra–/– proliferation (BrdU incorporation) in outflow tract cushion tissue but a significant increase in apoptosis as assessed by both TUNEL labeling in paraffin sections and caspase activity in trypsin-dispersed hearts. Additionally, E12.5 embryos demonstrated elevated levels of transforming growth factor β2 (TGFβ2) protein in multiple cell lineages in the heart. Using a whole-mouse-embryo culture system, wild-type E11.5 embryos treated with TGFβ2 protein for 24 hours displayed enhanced apoptosis in both the sinistroventralconal cushion and dextrodorsalconal cushion in a manner analogous to that observed in the Rxra–/–. TGFβ2 protein treatment also led to malformations in both the outflow tract and aortic sac. Importantly, Rxra–/– embryos that were heterozygous for a null mutation in the Tgfb2 allele exhibited a partial restoration of the elevated apoptosis and of the malformations. This was evident at both E12.5 and E13.5. The data suggests that elevated levels of TGFβ2 can (1) contribute to abnormal outflow tract morphogenesis by enhancing apoptosis in the endocardial cushions and (2) promote aortic sac malformations by interfering with the normal development of the aorticopulmonary septum.
HEN1functions pleiotropically inArabidopsisdevelopment and acts in C function in the flower
Authors: Chen Xuemei , Liu Jun , Cheng Yulan , Jia Dongxuan
Published: April 2021

Four classes of floral homeotic MADS domain proteins specify the identities of the four organ types in an Arabidopsis flower. While the activities of the MADS domain proteins are essentially confined to the flower or to the inflorescence, several genes, such as APETALA2, HUA1 and HUA2, also act outside the flower in addition to their organ identity functions inside the flower. We identified a new gene, HUA ENHANCER 1 (HEN1) from a sensitized genetic screen in the hua1-1 hua2-1 background that is compromised in floral homeotic C function. We showed that HEN1, like the C function gene AGAMOUS, acts to specify reproductive organ identities and to repress A function. HEN1 also shares AG’s non-homeotic function in controlling floral determinacy. HEN1 may achieve these functions by regulating the expression of AG. hen1 single mutants exhibit pleiotropic phenotypes such as reduced organ size, altered rosette leaf shape and increased number of coflorescences, during most stages of development. Therefore, HEN1, like the A function gene AP2, plays multiple roles in plant development as well as acting in organ identity specification in the flower. HEN1 codes for a novel protein and is expressed throughout the plant.
Cues from neuroepithelium and surface ectoderm maintain neural crest-free regions within cranial mesenchyme of the developing chick
Authors: Golding Jon P. , Dixon Monica , Gassmann Martin
Published: April 2021

Within the developing vertebrate head, neural crest cells (NCCs) migrate from the dorsal surface of the hindbrain into the mesenchyme adjacent to rhombomeres (r)1 plus r2, r4 and r6 in three segregated streams. NCCs do not enter the intervening mesenchyme adjacent to r3 or r5, suggesting that these regions contain a NCC-repulsive activity.

We have used surgical manipulations in the chick to demonstrate that r3 neuroepithelium and its overlying surface ectoderm independently help maintain the NCC-free zone within r3 mesenchyme. In the absence of r3, subpopulations of NCCs enter r3 mesenchyme in a dorsolateral stream and an ectopic cranial nerve forms between the trigeminal and facial ganglia. The NCC-repulsive activity dissipates/degrades within 5-10 hours of r3 removal. Initially, r4 NCCs more readily enter the altered mesenchyme than r2 NCCs, irrespective of their maturational stage. Following surface ectoderm removal, mainly r4 NCCs enter r3 mesenchyme within 5 hours, but after 20 hours the proportions of r2 NCCs and r4 NCCs ectopically within r3 mesenchyme appear similar.
The Sox-domain containing geneDichaete/fish-hookacts in concert withvndandindto regulate cell fate in theDrosophilaneuroectoderm
Authors: Zhao Guoyan , Skeath James B.
Published: April 2021

In the Drosophila embryonic central nervous system, neural stem cells, called neuroblasts, acquire fates in a position-specific manner. Recent work has identified a set of genes that functions along the dorsoventral axis to enable neuroblasts that develop in different dorsoventral domains to acquire distinct fates. These genes include the evolutionarily conserved transcription factors ventral nerve cord defective and intermediate neuroblasts defective, as well as the Drosophila EGF receptor. We show that the Sox-domain-containing gene Dichaete/fish-hook also plays a crucial role to pattern the neuroectoderm along the DV axis. Dichaete is expressed in the medial and intermediate columns of the neuroectoderm, and mutant analysis indicates that Dichaete regulates cell fate and neuroblast formation in these domains. Molecular epistasis tests, double mutant analysis and dosage-sensitive interactions demonstrate that during these processes, Dichaete functions in parallel with ventral nerve cord defective and intermediate neuroblasts defective, and downstream of EGF receptor signaling to mediate its effect on development. These results identify Dichaete as an important regulator of dorsoventral pattern in the neuroectoderm, and indicate that Dichaete acts in concert with ventral nerve cord defective and intermediate neuroblasts defective to regulate pattern and cell fate in the neuroectoderm.
Parasegmental organization of the spider embryo implies that the parasegment is an evolutionary conserved entity in arthropod embryogenesis
Authors: Damen Wim G. M.
Published: April 2021

Spiders belong to the chelicerates, which is a basal arthropod group. To shed more light on the evolution of the segmentation process, orthologs of the Drosophila segment polarity genes engrailed, wingless/Wnt and cubitus interruptus have been recovered from the spider Cupiennius salei. The spider has two engrailed genes. The expression of Cs-engrailed-1 is reminiscent of engrailed expression in insects and crustaceans, suggesting that this gene is regulated in a similar way. This is different for the second spider engrailed gene, Cs-engrailed-2, which is expressed at the posterior cap of the embryo from which stripes split off, suggesting a different mode of regulation. Nevertheless, the Cs-engrailed-2 stripes eventually define the same border as the Cs-engrailed-1 stripes. The spider wingless/Wnt genes are expressed in different patterns from their orthologs in insects and crustaceans. The Cs-wingless gene is expressed in iterated stripes just anterior to the engrailed stripes, but is not expressed in the most ventral region of the germ band. However, Cs-Wnt5-1 appears to act in this ventral region. Cs-wingless and Cs-Wnt5-1 together seem to perform the role of insect wingless. Although there are differences, the wingless/Wnt-expressing cells and en-expressing cells seem to define an important boundary that is conserved among arthropods. This boundary may match the parasegmental compartment boundary and is even visible morphologically in the spider embryo. An additional piece of evidence for a parasegmental organization comes from the expression domains of the Hox genes that are confined to the boundaries, as molecularly defined by the engrailed and wingless/Wnt genes. Parasegments, therefore, are presumably important functional units and conserved entities in arthropod development and form an ancestral character of arthropods. The lack of by engrailed and wingless/Wnt-defined boundaries in other segmented phyla does not support a common origin of segmentation.
Identification of a developmental transition in plasmodesmatal function during embryogenesis inArabidopsis thaliana
Authors: Kim Insoon , Hempel Frederick D. , Sha Kyle , Pfluger Jennifer , Zambryski Patricia C.
Published: April 2021

Plasmodesmata provide routes for communication and nutrient transfer between plant cells by interconnecting the cytoplasm of adjacent cells. A simple fluorescent tracer loading assay was developed to monitor patterns of cell-to-cell transport via plasmodesmata specifically during embryogenesis. A developmental transition in plasmodesmatal size exclusion limit was found to occur at the torpedo stage of embryogenesis in Arabidopsis; at this time, plasmodesmata are down-regulated, allowing transport of small (approx. 0.5 kDa) but not large (approx. 10 kDa) tracers. This assay system was used to screen for embryo-defective mutants, designated increased size exclusion limit of plasmodesmata(ise), that maintain dilated plasmodesmata at the torpedo stage. The morphology of ise1 and ise2 mutants discussed here resembled that of the wild-type during embryo development, although the rate of their embryogenesis was slower. The ISE1 gene was mapped to position 13 cM on chromosome I using PCR-based biallelic markers. ise2 was found to be allelic to the previously characterized mutant emb25 which maps to position 100 cM on chromosome I. The results presented have implications for intercellular signaling pathways that regulate embryonic development, and furthermore represent the first attempt to screen directly for mutants of Arabidopsis with altered size exclusion limit of plasmodesmata.
Defining epithelial-mesenchymal transitions in animal development
Authors: Sheng Guojun
Published: April 2021

Over 50 years after its discovery in early chick embryos, the concept of epithelial-mesenchymal transition (EMT) is now widely applied to morphogenetic studies in both physiological and pathological contexts. Indeed, the EMT field has witnessed exponential growth in recent years, driven primarily by a rapid expansion of cancer-oriented EMT research. This has led to EMT-based therapeutic interventions that bear the prospect of fighting cancer, and has given developmental biologists new impetus to investigate EMT phenomena more closely and to find suitable models to address emerging EMT-related questions. Here, and in the accompanying poster, I provide a brief summary of the current status of EMT research and give an overview of EMT models that have been used in developmental studies. I also highlight dynamic epithelialization and de-epithelialization events that are involved in many developmental processes and that should be considered to provide a broader perspective of EMT. Finally, I put forward a set of criteria to separate morphogenetic phenomena that are EMT-related from those that are not.
Reproductive history determines Erb
b
2 locus amplification, WNT signalling and tumour phenotype in a murine breast cancer model
Authors: Ordonez Liliana D. , Melchor Lorenzo , Greenow Kirsty R. , Kendrick Howard , Tornillo Giusy , Bradford James , Giles Peter , Smalley Matthew J.
Published: April 2021

Understanding the mechanisms underlying tumour heterogeneity is key to the development of treatments that can target specific tumour subtypes. We have previously targeted CRE recombinase-dependent conditional deletion of the tumour suppressor genes Brca1, Brca2, p53 (also known as Trp53) and/or Pten to basal or luminal oestrogen receptor-negative (ER−) cells of the mouse mammary epithelium. We demonstrated that both the cell-of-origin and the tumour-initiating genetic lesions cooperate to influence mammary tumour phenotype. Here, we use a CRE-activated HER2 orthologue to specifically target HER2/ERBB2 oncogenic activity to basal or luminal ER− mammary epithelial cells and perform a detailed analysis of the tumours that develop. We find that, in contrast to our previous studies, basal epithelial cells are less sensitive to transformation by the activated NeuKI allele, with mammary epithelial tumour formation largely confined to luminal ER− cells. Histologically, most tumours that developed were classified as either adenocarcinomas of no special type or as metaplastic adenosquamous tumours. The former were typically characterized by amplification of the NeuNT/Erbb2 locus; in contrast, tumours displaying squamous metaplasia were enriched in animals that had been through at least one pregnancy and typically had lower levels of NeuNT/Erbb2 locus amplification but had activated canonical WNT signalling. Squamous changes in these tumours were associated with activation of the epidermal differentiation cluster. Thus, in this model of HER2 breast cancer, cell-of-origin, reproductive history, NeuNT/Erbb2 locus amplification and the activation of specific branches of the WNT signalling pathway all interact to drive inter-tumour heterogeneity.
Low-angle x-ray diffraction and electron-microscope studies of isolated cell membranes
Authors: Finean J. B. , Coleman R. , Green W. G. , Limbrick A. R.
Published: April 2021

A sequence of low-angle X-ray diffraction patterns obtained during the controlled drying of a preparation of rat erythrocyte ghosts has been interpreted with the aid of corresponding electron micrographs and of a parallel study of myelin isolated from guinea-pig brain. A diffraction pattern that persists down to a level of 10–20 % hydration of the sample is believed to arise from the close packing of native erythrocyte membranes. Each membrane is about 100 Å thick and it is suggested that it consists of a predominantly continuous bimolecular layer of lipid, with non-lipid components associated with both surfaces. Further changes in diffraction pattern which accompany continued drying could be interpreted either as a change from a lamellar to a hexagonal structure or as the formation of a multiphase system. Evidence is put forward to support the latter interpretation.
An ultrastructural study of interspecific cell fusion induced by inactivated sendai virus
Authors: Schneeberger E. E. , Harris H.
Published: April 2021

An electron-microscopic examination was made of the process of cell fusion induced by Sendai virus inactivated by ultraviolet light. Ehrlich ascites cells, HeLa cells, rabbit macrophages, rat lymphocytes and nucleated hen erythrocytes were chosen for study because it had previously been shown that these cells could be fused together, with varying degrees of facility, to form artificial heterokaryons. Cells which had large numbers of microvilli on their surfaces fused together more readily than those which had not, but the presence of microvilli was not essential for fusion to occur. Fusion appeared in all cases to be initiated by the formation of small cytoplasmic bridges between the cells ; but virus particles, although present elsewhere on the surface of the cells, were not detected at or near the cytoplasmic bridges. HeLa-hen erythrocyte heterokaryons were formed by the fusion of HeLa cells with red cell ghosts.
Movements of epithelial cell sheets in vitro
Authors: Vaughan R. B. , Trinkaus J. P.
Published: April 2021

Chick embryo epithelial cells, cultured in vitro on a plane glass surface, show ruffled membrane activity like that seen in fibroblasts. When similar epithelial cells come into contact, long-lasting adhesions form, which are associated with the cessation of membrane activity. Movement of epithelial cell sheets is correlated with adhesion to the substratum of some or all of the marginal cells. Such adhesion does not occur in the absence of membrane activity. Non-marginal cells ordinarily appear to be non-adherent to the substratum; occasionally, however, they become adherent when attachments to neighbouring cells in a sheet are broken. This behaviour is accompanied by marked membrane activity. Mobilization and orientation of marginal cells are the vital steps in movements of coherent cell sheets.
The role of calcium in the isolation of brush borders from epithelial cells of rat small intestine
Authors: Millington P. F. , Critchley D. R. , Tovell P. W. A.
Published: April 2021

A chelating agent such as EDTA or EGTA used with a dilute TRIS buffer at pH 7·2–7’5 was used in order to effect a good separation of brush borders from the epithelium of the small intestine. A good separation was not obtained in low concentrations of TRIS buffer or saline alone. Brush borders were not obtained when the calcium–chelate complex of EDTA or EGTA was used, and only a partial fractionation was obtained when the magnesium complex of EDTA was tried. The involvement of calcium was further illustrated by adding calcium salts directly to the fractionation medium ; separation was prevented when sufficient calcium had been added to saturate the chelating agent. It was found that there was no precise optimum concentration for EDTA but a separation could not be obtained below 2·4 mM/1. The effect of changing the pH of the buffer was also investigated and it was demonstrated that the onset of the ability to release brush borders coincided approximately with the ionization of the third acid radical of the chelating agent. This is in keeping with the suggested hypothesis that EDTA acts by chelating calcium ions.

From these and electron-microscope studies it is suggested that the binding of calcium ions is an important factor in the maintenance of the stability of the epithelial cell membrane.
Role of phragmoplast filaments in cell-plate formation
Authors: Bajer A. , Allen R. D.
Published: April 2021

With the differential interference microscope (Nomarski system), it is possible to observe the interaction of filaments of the phragmoplast with micron-size refractile particles and vesicles, very close to the resolution limit of the microscope, during formation of the cell plate. Filaments occupying the birefringent zone of the phragmoplast (i.e. remnants of the continuous and interzonal fibres of the mitotic spindle) shorten and thicken in their mid-region to become spindle-shaped. The thickenings later fuse laterally to form the cell plate. During the process of thickening, vesicles can be seen to move toward the plate in close association with the filaments on both sides of the phragmoplast. These observations, taken together with information from electron micrographs of thin sections of Haemanthus endosperm and other dividing cells, confirm the notion that the fibrillar component of the phragmoplast has a transport function. The sizes of particles entering the phragmoplast region by saltation suggest that they correspond to Golgi bodies, which have been suspected of producing vesicles that contribute to cell-plate formation. Neither light nor electron micrographs have yet provided any insight into the mechanism of transport of these vesicles.
The function and fine structure of the cephalic airflow receptor in schistocerca gregaria
Authors: Guthrie D.M.
Published: April 2021

Electron micrographs of parts of the sense organ showed that the dendritic axis consisted of a large and a small envelope containing microtubules as their main inclusion. The envelopes are supported by a thick-walled tube believed to be part of the ist-tier sheath cells. The small envelope is segregated from the large envelope near its apex by a fold of the tube wall. The packing of the neurotubular array within the small envelope is both more dense and more regular than within the large envelope. The tube is separated by an extracellular space from the trichogen-tormogen cell. Sections through the apex of the dendrite reveal a homogeneous cap unlikely to be part of a structure continued into the upper region of the hair shaft. No ciliary structures were visible within the dendrite, whose microtubules pass into the neuron cell body proximally. Sections through the neuron cell body reveal branched mitochondria, and numerous microtubules.

Rates of discharge in sensory axons from these hair organs produced by deflexion of the hair shaft were found to be within the range 300–100 impulses/sec. There is an initial phase of rapid adaptation which gives place to a steady rate.

It is suggested that the fine structure of the receptor may indicate mechano-electrical transduction at a more proximal level than is believed to be the case in some other types of receptor.

The diaphragms that support the hair shaft laterally can be seen to be composed of fine cuticular strands.
The interpretation of electron micrographs of negatively stained native collagen
Authors: Cox R. W. , Grant R. A. , Kent C. M.
Published: April 2021

Deamination of native rat tail collagen followed by treatment with 1,2-cyclohexanedione removes the positive charge from the side chains of the lysine and hydroxylysine residues and blocks approximately 70 % of the positively charged side chains of the arginine residues. When such specimens are examined in the electron microscope after negative staining with 2 % potassium phosphotungstate at pH 6 ·8 –7 ·4 no change from the appearance of normal collagen is detectable. These findings suggest that electrostatic attraction does not occur between the stain and the side chains of the lysine, hydroxylysine and arginine residues of the collagen and justify the view that, at least with this stain and under these conditions, the effect is that of pure negative staining and not that of a mixture of positive and negative staining. Furthermore, the experiments indicate that possible electrostatic attractions between the positively charged side chains of lysine, hydroxylysine and arginine and the negatively charged side chains of residues such as aspartic acid and glutamic acid are relatively unimportant in the maintenance of the light cross-striations of negatively stained collagen. However, the loss of these light cross-striations after treatment of collagen with urea implies that they are mainly associated with intermolecular hydrophobic and, possibly, hydrogen bonding.
A study of terminal degeneration in the olfactory bulb of the rat
Authors: Pinching A. J. , Powell T. P. S.
Published: April 2021

An ultrastructural study of degeneration in axon terminals of the glomerular layer of the rat olfactory bulb is described, concentrating particularly on the sequence of degeneration in the olfactory nerve terminals and the long-term events in the degeneration process in several terminal types. Olfactory nerve terminal degeneration is divided into 5 stages, representing parts of the sequential changes taking place in the terminal after fibre section. The main features in the sequence are as follows: I. Swelling of the terminal and its vesicles. II. Initial shrinkage of the terminal, while vesicles remain swollen and some are distorted. III. Further shrinkage and darkening of the terminal, distortion of many of the vesicles and some mitochondrial swelling. IV. Extreme shrinkage of the terminal and loss of cytoplasmic detail; synaptic contact still intact or partially separated. V. Disappearance of the presynaptic terminal and persistence of the post-synaptic thickening.

The validity of the observation of the persistence of post-synaptic membrane thickenings is considered and evidence adduced in its favour, both qualitative and quantitative. Characteristics of the newly apposed profiles are described, including cisternae and alveolate vesicles; the extracellular material of the synaptic cleft is considered in the light of its association with persisting thickenings. The relevance of these associated structures is discussed in terms of function and development. Spontaneous degeneration of olfactory nerves and the degeneration of the vomeronasal nerves in the accessory olfactory bulb are described, as well as Stage V in the degeneration of other terminal types of the glomeruli, following various lesions; persistence of post-synaptic thickenings after the degeneration of terminals showing symmetrical membrane thickenings is included. Observations on the persistence of post-synaptic thickenings in various sites in the central and peripheral nervous systems are reviewed in the context of the present study; the problems arising out of the morphological identification of apposition or reinnervations are discussed.
A new junctional structure in the epithelia of insects of the order dictyoptera
Authors: Flower N. E.
Published: April 2021

The junctional complexes in the epithelia of insects of the order Dictyoptera have been investigated using the freeze-etch technique. As well as septate junctions, a new type of junction has been identified and the name ‘inverted gap junction’ proposed. The patch-like distribution of the inverted gap junctions basal to and often closely associated with septate junctions is very similar to the form of gap junctions and their relationship to tight junctions in vertebrates. This suggests that the inverted gap junctions, like normal gap junctions, could perform a communicating function between epithelial cells. The following features distinguish inverted gap junctions from normal gap junctions in freeze-etch preparations: (i) the arrays of particles and holes within inverted gap junctions appear on B- and A-type faces respectively, i.e. on the opposite faces to the particles and holes in gap junctions; (ii) the particles within inverted gap junctions appear to lie in rows which anastomose to form an irregular net, and not in an hexagonal array, as occurs in gap junctions.
A new specialization of the granular endoplasmic reticulum in the uterine glands of the pig in the later stages of pregnancy
Authors: Crombie Paula R.
Published: April 2021

A new specialization of the granular endoplasmic reticulum (GER) which develops within the uterine gland cells of the pig during the final third of pregnancy is described.

The ‘tubular bodies ‘are interpreted as sheets of GER rolled up to form open-ended cylinders enclosing cytoplasm which is distinctly less electron-dense than the surrounding external cytoplasm, and in which large clear vesicles and free ribosomes are frequently found.

The tubular bodies are not present during the first two thirds of pregnancy, but by day 100 of gestation they are readily discerned. They increase in number within the last week of pregnancy and are still found in glands from the day 1 post-partum uterus. They are distributed, either singly or in groups of up to 30 or more units, throughout the cells. The cisternal space which surrounds them is continuous with that of the granular endoplasmic reticulum.

A tentative developmental sequence is suggested, based on morphological similarities between the wall structure of the early tubular body and the paired cistemae, which are seen scattered as single, short strands within the gland cells. The final form is achieved by modification of the inner apposed, ribosome-free cisternal membranes into the straightened-out, thickened membranes characteristic of the tubular body wall, together with a rolling-up of the ‘sheet’ of paired GER cistemae. Subsequent loss of the innermost unmodified cisternal membrane which is now in contact with the centrally enclosed cytoplasm results in the characteristic tubular body wall structure.

The characteristic appearance of the central cytoplasm and the presence of modified surrounding membranes suggests the possibility of a localized, modified internal environment required for some unspecified functional activity; possibly a synthetic process or some type of ionic regulation.
Action of rifampicin on the liverwort Marchantia polymorpha
Authors: Mache R. , Loiseaux S.
Published: April 2021

The effect of rifampicin was studied on a multicellular plant in comparison with its effect on unicellular organisms. Marchantía polymorpha L. is sensitive to rifampicin and was chosen as the material. The inhibitory dose on the growth of young gemmae was established. The effect of rifampicin on the ultrastructure of the cells was investigated and it was found to inhibit the formation of plastid ribosomes and dissociate existing ribosomes into their subunits, but probably does not inhibit the synthesis of some plastid proteins, which remain visible as small granules in the stroma. The effect of rifampicin on the chlorophyll content and on the absorption of C02 by intact thalli and isolated chloroplasts has also been studied. Little or no change was observed.
Microtubule-arms and propulsion of food particles inside a large feeding organelle in the ciliate Phascolodon vorticella
Authors: Tucker J. B.
Published: April 2021

Attention is drawn to the apparently invariable presence of rows of arm-bearing microtubules lining the sucking mouthparts of ciliates. Their arrangement inside the cytopharyngeal basket of Phascolodon is described and compared with that in the tentacles of suctorians and the sucking tubes and baskets of other ciliates. It is suggested that arms are involved in the propulsion of materials alongside microtubules when these ciliates ingest food. Mechanisms by which arms may accomplish this are outlined. The different functions of links and other intertubular materials in the several microtubular basket components of Phascolodon are considered. The form and arrangement of microfilaments and pointed, sharp-edged structures around the mouth are also described. The possibility that the pointed structures are used for biting is discussed.
Cytoplasmic nucleoloids of Lilium male reproductive cells contain rDNA transcripts and share features of development with nucleoli
Authors: Sato S. , Jones K. , Alche J. De Los Dios , Dickinson H. G.
Published: April 2021

Nucleolus-like inclusions in the meiocytes of Lilium sp. have been investigated at the light and electronmicroscope levels, with respect both to their composition and their formation during the meiotic divisions. Those present in the cytoplasm (cytoplasmic nucleoloids) first appear as small structures late in anaphase I. Coincidentally, small nuclear nucleoluslike inclusions (NLBs) develop on the surface of the chromosomes, and sometimes in the region between chromosomes as they are carried into the daughter nuclei. The cytoplasmic nucleoloids increase in size from late anaphase I to the dyad stage, but disappear before metaphase II. They redevelop late in anaphase II and persist, increasing in size, until the tetrad stage. The NLBs also increase in size over this period. Ultrastructural investigation indicates that both the nascent nucleoloids and NLBs are fibrous in texture as they arise in late anaphase. Elsewhere in the cytoplasm, however, large accumulations of amorphous electron-opaque material also aggregate. This material becomes associated with the nucleoloids and the NLBs during the period of their enlargement. Nucleoloids late in development, and post-telophase nucleoli are not invested by these amorphous masses. Interestingly, cytoplasmic nucleoloids and NLBs react in an identical fashion to a range of cytochemical treatments and, using in situ hybridisation at the electron-microscope level, they have been demonstrated to contain RNA sequences homologous with a wheat ribosomal gene probe. Cytoplasmic nucleoloids are thus interpreted as following an identical developmental pathway to nucleoli except in that, perhaps owing to high levels of rDNA transcription during meiosis, they condense in the cytoplasm rather than the nucleoplasm.