bluuebunny/crossref_metadata_2025 huggingface dataset:
Expressing antisense Po RNA in Schwann cells perturbs myelination
Authors: Owens Geoffrey C. , Boyd Carol J.
Published: April 2021

Primary Schwann cells were infected in vitro with a recombinant retrovirus expressing a dominant selectable marker, neomycin phosphotransferase (conferring resistance to the drug G418), and antisense Po RNA under the control of the human β-actin promoter. A proportion of the G418-resistant cells failed to form myelin when cocultured with dorsal root ganglion neurons under conditions that promote Schwann cell differentiation. These cells expressed high levels of Po antisense RNA. Among the impaired cells, the majority had segregated and ensheathed individual axons but had not differentiated further. They did not express Po but did express myelin-associated glycoprotein and galacto cerebroside. A minority of partially inhibited Schwann cells were also observed that elaborated thin myelin sheaths containing variable numbers of compacted and noncompacted lamellae. These data indicate that restricting the level of Po expression inhibits spiralling of the Schwann cell membrane and subsequent compaction.
Retinoic acid induces NGF-dependent survival response and high-affinity NGF receptors in immature chick sympathetic neurons
Authors: Rodriguez-Tébar Alfredo , Rohrer Hermann
Published: April 2021

An important step in the development of peripheral sensory and sympathetic neurons is the onset of the survival response and dependence on the presence of nerve growth factor (NGF) or other neurotrophic factors. We have recently observed that immature sympathetic neurons from 7-day-old chick embryos are unable to become NGF-responsive in vitro and we have now used these cells to identify molecules that induce NGF-dependent neuronal survival. We found that retinoic acid (RA) induces the ability of these cells to survive in the presence of NGF. At RA concentrations of 10−9–10−8M virtually all neurons survived in the presence of NGF. RA was found to also induce the biologically active, high-affinity NGF receptor: highaffinity receptors were undetectable on dissociated E7 sympathetic neurons and were observed in vitro only in RA-treated neurons. These findings suggest that the induction of high-affinity NGF receptors may be sufficient to activate the survival response in sympathetic neurons and imply an important role for RA during neuron differentiation in the peripheral nervous system.
Interactions of different vegetal cells with mesomeres during early stages of sea urchin development*
Authors: Khaner Oded , Wilt Fred
Published: April 2021

It has been known from results obtained in the classical experiments on sea urchin embryos that cell isolation and transplantation showed extensive interactions between the early blastomeres and/or their descendants. In the experiments reported here a systematic reexamination of recombination of mesomeres and their progeny (which come from the animal hemisphere) with various vegetal cells derived from blastomeres of the 32- and 64-cell stage was carried out. Cells were marked with lineage tracers to follow which cell gave rise to what structures, and newly available molecular markers have been used to analyze different structures characteristic of regional differentiation. Large micromeres form spicules and induce gut and pigment cells in mesomeres, conforming to previous results. Small micromeres, a cell type not heretofore examined, gave rise to no recognizable structure and had very limited ability to evoke poorly differentiated gut tissue in mesomeres. Macromeres and their descendants, Veg 1 and Veg 2, form primarily what their normal fate dictated, though both did have some capacity to form spicules, presumably by formation from secondary mesenchyme. Macromeres and their descendants were not potent inducers of vegetal structures in animal cells, but they suppress the latent ability of mesomeres to form vegetal structures. The results lead us to propose that the significant interactions during normal development may be principally suppressive effects of mesomeres on one another and of adjacent vegetal cells on mesomeres.
Functional tagging of regulatory elements in the plant genome
Authors: Topping Jennifer F. , Wei Wenbin , Lindsey Keith
Published: April 2021

In comparison with animals, relatively few plant genes have been identified that have been shown to be under organ-, tissue- or cell-type-specific regulation. In this paper, we describe how the β-glucuronidase (GUS) reporter gene (gusA or uidA), fused to a weak promoter (a truncated (−90 bp) CaMV35S promoter), can be used to identify tissue-specific markers in transgenic tobacco plants. The rationale was that the expression of gusA would be determined primarily by position effect. Quantitative analysis revealed that, of 184 −90−gus transgenic plants, 73 % exhibited gusA gene activation in leaf tissue, and the level of GUS enzyme activity varied over a 300-fold range within the population. In comparison, transformation with a promoterless gusA gene resulted in GUS expression in 78 % of all plants analyzed (in leaf and/or root) and expression levels were three-fold or more lower. Qualitative GUS analysis of single locus −90-gus transformants revealed differential expression in diverse tissues. The spatial pattern of GUS activity was unique to individual transformants, was a reflection of differential gusA gene transcription, and was stably transmissible to progeny. Evidence for preferential expression in roots not only of the −90-gus, but also the promoterless gusA gene is presented. The value of the −90 bp promoter-gusA sequence, which is termed an ‘interposon’, as a tool both to identify native enhancer sequences in situ and to investigate position effects in plants, is discussed.
Well-defined growth factors promote cardiac development in axolotl mesodermal explants
Authors: Muslin Anthony J. , Williams Lewis T.
Published: April 2021

The effect of growth factors on the formation of cardiac mesoderm in the urodele, Ambystoma mexicanum (axolotl), has been examined using an in vitro explant system. It has previously been shown that cardiac mesoderm is induced by pharyngeal endoderm during neurula stages in urodeles. In this study, explants of prospective cardiac mesoderm from early neurula stage embryos rarely formed beating cardiac tissue in culture. When transforming growth factor beta-1 (TGF-β1) or platelet-derived growth factor BB (PDGF) was added to such explants, the frequency of heart tissue formation increased markedly. The addition of other growth factors to these explants did not enhance cardiac mesoderm formation. The addition of basic fibroblast growth factor (bFGF) to prospective heart mesoderm derived from later stage embryos resulted in a decreased tendency to form cardiac tissue. These results suggest that growth factors analogous to TGF-β1 PDGF, and bFGF may regulate the initial stages of vertebrate cardiac development in vivo.
Retinoic acid modifies the pattern of cell differentiation in the central nervous system of neurula stage Xenopus embryos
Authors: i Altaba A. Ruiz , Jessell T. M.
Published: April 2021

Neural cell markers have been used to examine the effect of retinoic acid (RA) on the development of the central nervous system (CNS) of Xenopus embryos. RA treatment of neurula stage embryos resulted in a concentration-dependent perturbation of anterior CNS development leading to a reduction in the size of the forebrain, midbrain and hindbrain. In addition the overt segmental organization of the hindbrain was abolished by high concentrations of RA. The regional expression of two cell-specific markers, the homeobox protein Xhox3 and the neurotransmitter serotonin was also examined in embryos exposed to RA. Treatment with RA caused a concentration-dependent change in the pattern of expression of Xhox3 and serotonin and resulted in the ectopic appearance of immunoreactive neurons in anterior regions of the CNS, including the forebrain. Collectively, our results extend previous studies by showing that RA treatment of embryos at the neurula stage inhibits the development of anterior regions of the CNS while promoting the differentiation of more posterior cell types. The relevance of these findings to the possible role of endogenous retinoids in the determination of neural cell fate and axial patterning is discussed.
Hepatic Nuclear Factor 1 (HNF1) shows a wider distribution than products of its known target genes in developing mouse
Authors: Blumenfeld Marta , Maury Martine , Chouard Tanguy , Yaniv Moshe , Condamine Hubert
Published: April 2021

Hepatic nuclear factor 1 (HNF1) is a highly diverged homeoprotein that is crucial for transcription of many liver-specific genes including albumin. In particular, a minimal promoter, consisting of an HNF1-binding-site and a TATA box, is highly active only in hepatoma cell lines. The expression of the HNF1 and albumin genes has been examined in mouse embryos by in situ hybridization. At 10.5 days of gestation, the HNF1 mRNA was detected in both the hepatic primordia and visceral endoderm of the yolk sac whereas the albumin transcript was present only in the nascent liver. At later stages of development, HNF1 was detected in liver, in the epithelial cells of most of the digestive tract and in the cortex of the kidney, whereas albumin was again found only in the liver. The presence of HNF1 protein in adult kidney was demonstrated by immunodetection in gelretardation assays and western blot analysis. These experiments show that, even though the HNF1 homeoprotein is essential for expression of many liver-specific genes, it cannot, by itself, force high expression levels of these genes, in non-hepatic tissues.
Role of c-kit in mouse spermatogenesis: identification of spermatogonia as a specific site of c-kit expression and function
Authors: Yoshinaga Kazuya , Nishikawa Satomi , Ogawa Minetaro , Hayashi Shin-Ichi , Kunisada Takahiro , Fujimoto Toyoaki , Nishikawa Shin-Ichi
Published: April 2021

Recent studies have shown that the dominant white spotting (W) locus encodes the proto-oncogene c-kit, a member of the tyrosine kinase receptor family. One symptom of mice bearing mutation within this gene is sterility due to developmental failure of the primordial germ cells during early embryogenesis. To elucidate the role of the c-kit in gametogenesis, we used an anti-c-kit monoclonal antibody, ACK2, as an antagonistic blocker for c-kit function to interfere with the development of male and female germ cells during postnatal life. ACK2 enabled us to detect the expression of c-kit in the gonadal tissue and also to determine the functional status of c-kit, which is expressed on the surface of a particular cell lineage. Consistent with our immunohistochemical findings, the intravenous injection of ACK2 into adult mice caused a depletion in the differentiating type A spermatogonia from the testis during 24 – 36 h, while the undifferentiated type A spermatogonia were basically unaffected. Intraperitoneal injections of ACK2 into prepuberal mice could completely block the mitosis of mature (differentiating) type A spermatogonia, but not the mitosis of the gonocytes and primitive type A spermatogonia, or the meiosis of spermatocytes. Our results indicate that the survival and/or proliferation of the differentiating type A spermatogonia requires c-kit, but the primitive (undifferentiated) type A spermatogonia or spermatogenic stem cells are independent from c-kit. Moreover, the antibody administration had no significant effect on oocyte maturation despite its intense expression of c-kit.
A normally attractive cell interaction is repulsive in two C. elegans mesodermal cell migration mutants
Authors: Stern Michael J. , Horvitz H. Robert
Published: April 2021

In wild-type Caenorhabditis elegans hermaphrodites, two bilaterally symmetric sex myoblasts (SMs) migrate anteriorly to Sank the precise center of the gonad, where they divide to generate the muscles required for egg laying (J. E. Sulston and H. R. Horvitz (1977) Devi Biol. 56, 110–156). Although this migration is largely independent of the gonad, a signal from the gonad attracts the SMs to their precise final positions (J. H. Thomas, M. J. Stern and H. R. Horvitz (1990) Cell 62, 1041–1052). Here we show that mutations in either of two genes, egl-15 and egl-17, cause the premature termination of the migrations of the SMs. This incomplete migration is caused by the repulsion of the SMs by the same cells in the somatic gonad that are the source of the attractive signal in wild-type animals.
Demonstration of a phagocytic cell system belonging to the hemopoietic lineage and originating from the yolk sac in the early avian embryo
Authors: Cuadros Miguel A. , Coltey Pierre , Nieto M. Carmen , Martin Claude
Published: April 2021

It is well established that hemopoietic cells arising from the yolk sac invade the avian embryo. To study the fate and role of these cells during the first 2.5-4.5 days of incubation, we constructed yolk sac chimeras (a chick embryo grafted on a quail yolk sac and vice versa) and immunostained them with antibodies specific to cells of quail hemangioblastic lineage (MB1 and QH1). This approach revealed that endothelial cells of the embryonic vessels are of intraembryonic origin. In contrast, numerous hemopoietic cells of yolk sac origin were seen in embryos ranging from 2.5 to 4.5 days of incubation. These cells were already present within the vessels and in the mesenchyme at the earliest developmental stages analyzed. Two hemopoietic cell types of yolk sac origin were distinguishable, undifferentiated cells and macro-phage-like cells. The number of the latter cells increased progressively as development proceeded, and they showed marked acid phosphatase activity and phagocytic capacity, as revealed by the presence of numerous phagocytic inclusions in their cytoplasm. The macro-phage-like cells were mostly distributed in the mesen-chyme and also appeared within some organ primordia such as the neural tube, the liver anlage and the nephric rudiment. Comparison of the results in the two types of chimeras and the findings obtained with acid phosphatase/MB1 double labelling showed that some hemopoietic macrophage-like cells of intraembryonic origin were also present at the stages considered. These results support the existence in the early avian embryo of a phagocytic cell system of blood cell lineage, derived chiefly from the yolk sac. Cells belonging to this system perform phagocytosis in cell death and may also be involved in other morphogenetic processes.
Differential co-expression of long and short form type IX collagen transcripts during avian limb chondrogenesis in ovo
Authors: Swiderski Ruth E. , Solursh Michael
Published: April 2021

Using RNA blot analysis of developmentally staged avian limb buds, we demonstrate that transcripts of several cartilage marker genes appear in limb tissue prior to overt chondrogenesis. Type II collagen mRNA, cartilage proteoglycan core protein mRNA, α2(IX) collagen mRNA, and transcripts of the short form α1(IX) collagen chain derived from the downstream promoter are co-expressed in limb tissue approximately 24–36 hours before the appearance of the respective polypeptides in differentiating cartilagenous tissue. Transcripts of the long form α1(IX) collagen chain derived from the upstream promoter appear somewhat later in development; nearly coincident with the immunolocalization of type IX collagen in the cartilage elements of the limb. The spatial distribution of type n and type IX collagen transcripts was analyzed by in situ hybridization. Type II collagen and the long form α1(IX) collagen transcripts co-localized in the chondrogenic elements of the developing forelimb. In contrast, short form α1(IX) collagen transcripts which lack the 5’ region encoding the NC4 globular amino-terminal domain were distributed throughout the non-chondrogenic, non-myogenic mesenchymal regions of the limb and were not detectable above background levels in the limb chondrogenic elements. The precocious appearance of several cartilage marker gene transcripts prior to chondrogenesis suggests that multiple levels of gene regulation including alternative promoter use, alternative RNA splicing, alternative poly adenylation, and other post-transcriptional as well as translational mechanisms are active prior to, and during avian limb chondrogenesis.
β-D xyloside alters dermatan sulfate proteoglycan synthesis and the organization of the developing avian corneal stroma
Authors: Hahn Rita A. , Birk David E.
Published: April 2021

Corneal transparency is dependent upon the development of an organized extracellular matrix containing small diameter collagen fibrils with regular spacing, organized as orthogonal lamellae. Proteoglycan-collagen interactions have been implicated in the regulation of collagen fibrillogenesis and matrix assembly. To determine the role of dermatan sulfate proteoglycan in the development and organization of the secondary corneal stroma, its synthesis was disrupted using β-D xyloside. The secondary corneal stroma contains two different proteoglycans, dermatan sulfate and keratan sulfate proteoglycan. β-D xyloside interferes with xylose-me-diated O-linked proteoglycan synthesis, and thus disrupts dermatan sulfate proteoglycan synthesis. Corneal keratan sulfate proteoglycan, a mannose-mediated N-linked proteoglycan, should not be altered. Biochemical analysis of corneas treated both in vitro and in ovo revealed a reduced synthesis of normally glycosylated dermatan sulfate proteoglycans and an increased synthesis of free xyloside-dermatan sulfate glycosaminoglycans. Keratan sulfate proteoglycan synthesis was unaltered in both cases. Corneal stromas were studied using histochemistry and electron microscopy after in ovo treatment with β-D xyloside. The observed biochemical alterations in dermatan sulfate proteoglycans translated into disruptions in the organization of β-D xyloside-treated stromas. There was a reduction in the histochemical staining of proteoglycans, but no alteration in collagen fibril diameter. In addition, focal alterations in collagen fibril packing, and a disruption of lamellar organization were observed in β-D xyloside-treated corneas. These data suggest that dermatan sulfate proteoglycans are not involved in the regulation of corneal collagen fibril diameter, but are important in the fibril-fibril spacing as well as in lamellar organization, and cohesiveness.
The Drosophila retinoid X receptor homolog ultraspiracle functions in both female reproduction and eye morphogenesis
Authors: Oro Anthony E. , Mckeown Michael , Evans Ronald M.
Published: April 2021

ultraspiracle (usp) encodes the Drosophila cognate of RXR, the human retinoid X receptor. To examine how RXR subfamily members function in development, we have undertaken a phenotypic analysis of usp mutants. usp is required at multiple stages of development for functions that occur in a wide variety of tissues, usp is required in the eye-antennal imaginal disc for normal eye morphogenesis and in the somatic and germline tissues of adult females for fertilization, eggshell morphogenesis and embryonic development. An unusual sunken eye phenotype with marked ventral-dorsal polarity appears to be caused by a lack of usp function in the imaginal disc cells that reside between the eye and antennal anlage. The usp functions include cell autonomous and non-cell autonomous components, suggesting that usp controls the production of factors important for both cell-cell communication and cellular differentiation. These usp signalling pathways have mechanistic parallels to steroid and retinoid action in developing vertebrate tissues.
Gynandromorphs of Drosophila suggest one common primordium for the somatic cells of the female and male gonads in the region of abdominal segments 4 and 5
Authors: Szabad János , Nöthiger ROLF
Published: April 2021

In mosaic gonads of gynandromorphs of Drosophila, the amount of female and the amount of male somatic tissues add up to roughly one unit. This suggests that the somatic component of the gonads in males and females derives from a single common primordium, i.e. testes and ovaries appear to be homologous. Fate-mapping places this primordium ventrally of the sternites into the mesodermal region of the fourth and fifth abdominal segment. This location is corroborated by the observation that defects in and around abdominal segment 4 and absence of the gonads are strongly correlated in animals damaged by the mutation osk301. Gonads were mosaic with a frequency of 10.5% which indicates that the gonadal primordium originates from about 10 progenitor cells, and together with other evidence, suggests that these progenitor cells are located within a single segment (or parasegment).
Imprinting of phosphoribosyltransferases during preimplantation development of the mouse mutant, Hprtb-m3
Authors: Moore T. F. , Whittingham D. G.
Published: April 2021

The measurement of the activity of the X-linked enzyme HPRT has been widely used as an indicator of X-chromosome activity during preimplantation development in the mouse. More recently, the concomitant measure-ment of the activity of the autosomally-encoded enzyme APRT has been used in an attempt to decrease the variability inherent in the measurement of enzyme activity from minute samples such as preimplantation embryos.

In this study the use of the HPRT-deficient mouse mutant, Hprtb-m3, allowed the unequivocal identification of the parental origin of HPRT activity measured in embryos derived from crosses between wild-type mice, and mice which were homozygous or hemizygous for the Hprtb-m3 allele. Results were similar to those of a previous study, where oocyte-encoded HPRT activity accounted for about 10% of total HPRT activity at 76 hours post human chorionic gonadotrophin injection and the paternally-derived Hprt allele was shown to be transcriptionally active by the late 2-cell stage.

In contrast to other studies, differential expression of the two Hprt alleles was detected during the preim-plantation period, in embryos derived from crosses between wild-type and HPRT-deficient mice. Evidence was also found for the existence of an X-linked locus which influences the amount of APRT activity in the unfertilized oocyte. We propose that the expression pat-tern of this locus may be influenced by its parental origin.
The role of the teashirt gene in trunk segmental identity in Drosophila
Authors: Röder Laurence , Vola Christine , Kerridge Stephen
Published: April 2021

The phenotypes of different mutant combinations of teashirt (tsh) and homeotic genes together with their regulatory interactions are described in order to gain insight into tsh gene function. We show that when tsh, Scr, Antp and BX-C genes are missing, the ventral part of the trunk (or thorax and abdomen) is transformed to anterior head identity showing that tsh is a homeotic gene. These genes act synergistically to suppress the expression of the procephalic gene labial (lab) in subsets of cells in each segment of the trunk. Transcripts from the tsh gene always accumulate in segments destined to acquire trunk identities. tsh gene activity is required for the normal function of the Antp and BX-C genes, which modulate in part the expression of tsh. As a whole, our results suggest that tsh plays an essential dual role, during embryogenesis, for determining segmental identity of the trunk. First, tsh is required critically for the identity of the anterior prothorax. Second, tsh is required globally for segmental identity throughout the entire trunk whereas the “classical” homeotic genes have more specific roles. Our results are consistent with the idea that tsh is defining the ground state of the Drosophila trunk region seen in the absence of the Antp and BX-C genes.
Perturbation of developmental gene expression in rat liver by fibric acid derivatives: lipoprotein lipase and α-fetoprotein as models
Authors: Staels Bart , Auwerx Johan
Published: April 2021

Liver lipoprotein lipase (LPL) and α-fetoprotein (AFP) gene expression show similar developmental patterns. Both mRNAs are abundantly expressed in neonatal rat liver and gradually disappear upon ageing. Treatment with fibric acid derivatives, such as fenofibrate, not only delays the developmental extinction of the LPL gene, but also increases LPL mRNA levels in neonatal rat liver. Similarly, the developmental extinction of the AFP gene in the liver is clearly delayed after fenofibrate. In adult rat liver, fibric acid derivatives transcriptionally reinduce a mRNA with similar size as LPL, but no effect on AFP mRNA was detected. Sequence comparison of clones isolated from a fenofibrate-induced cDNA library demonstrates that the fenofibrate-(re)induced mRNA in adult rat liver is encoding for LPL. The induction of LPL after fenofibrate is tissue-specific, since heart and adipose tissue LPL mRNA levels remain unchanged. In conclusion, fibric acid derivatives modulate develop-mental expression patterns in rat liver, and may selec-tively reinduce the expression of extinct genes in adult rat liver.
Inhibin α- and βA -subunit immunoreactivity in the chicken embryo during morphogenesis
Authors: Bläuer Merja , Kohonen Juhani , Leivonen Ilkka , Vilja Pekka , Tuohimaa Pentti
Published: April 2021

Antibodies against synthetic peptides selected from the amino acid sequences of human inhibin α- and β β β A-subunits were used to examine the distribution of inhibin subunit immunoreactivity in chicken embryos during the first week of development. Inhibin α.-subunit immunoreactivity was localized in skeletal and smooth muscle myoblasts as well as developing cardiac muscle cells. In somites, immunostaining was seen exclusively in myotomes. The appearance of α.-subunit immunore-activity was correlated with myogenic differentiation; immunoreactivity was not seen in non-differentiated mesenchymal cells or in terminally differentiated adult muscle cells. In cardiac muscle, some immunopositive myocytes were seen also in the adult. In the adult heart, the Purkinje fibers were strongly immunoreactive, suggesting a possible role of the immunoreactive protein in the impulse-conducting function of these specialized cells. Inhibin α.-subunit immunoreactivity was also seen in the visceral and parietal cells of the Bowman’s capsule in both mesonephric and metanephric kidneys. In addition to mesodermal derivatives, α.-subunit immunoreactivity was localized in neuroepithelial cells and axons in the developing central nervous system. Immunoblotting with anti-α.(1-32) revealed two protein bands with Mr values of 50 000 and 32 000 in cytosol samples of whole embryos under nonreducing conditions. In reduced samples an approximately 14 000 Mr protein species was detected.

Inhibin β A-subunit immunoreactivity was detected only in chondrocytes, suggesting that the immunoreactive protein might represent a chicken homologue of the various cartilage and bone morphogenetic proteins expressed in mammals.
Developmental modulation of myosin expression by thyroid hormone in avian skeletal muscle
Authors: Gardahaut M. F. , Fontaine-Perus J. , Rouaud T. , Bandman E. , Ferrand R.
Published: April 2021

It is well established that a rise in circulating thyroid hormone during the second half of chick embryo development significantly influences muscle weight gain and bone growth. We studied thyroid influence on differentiation in slow anterior latissimus dorsi (ALD) and fast posterior latissimus dorsi (PLD) muscles of embryos rendered hypothyroid by hypophysectomy or administration of an anti-thyroid drug. The expression of native myosins and myosin light chains (MLCs) was studied by electrophoretic analysis, and the myosin heavy chain (MHC) was characterized by immunohistochemistry. The first effects of hypothyroid status were observed at day 21 of embryonic development (stage 46 according to Hamburger and Hamilton). Analysis of myosin isoform expression in PLD muscles of hypothyroid embryos showed persistence of slow migrating native myosins and slow MLCs as well as inhibition of neonatal fast MHC expression, indicating retarded differentiaton of this muscle. In ALD muscle, hypothyroidism maintained fast embryonic MHC and induced noticeable amounts of fast MLCs, thus delaying slow muscle differentiation. Our results suggest that thyroid hormones play a role in modulating the appearance of neonatal fast MHC and the disappearance of isomyosins transiently present during embryogenesis. However, T3 supplemental treatment would seem to compensate in part for the effects of hypothyroidism induced by hypophysectomy, suggesting that thyroid hormone might interfere with other factors also accounting for the observed effects.
Ventrolateral regionalization of Xenopus laevis mesoderm is characterized by the expression of α-smooth muscle actin
Authors: Saint-Jeannet Jean-Pierre , Levi Giovanni , Girault Jeanne-Marie , Koteliansky Victor , Thiery Jean-Paul
Published: April 2021

Mesodermal patterning in the amphibian embryo has been extensively studied in its dorsal aspects, whereas little is known regarding its ventrolateral regionalization due to a lack of specific molecular markers for derivatives of this type of mesoderm. Since smooth muscles (SM) are thought to arise from lateral plate mesoderm, we have analyzed the expression of an - actin isoform specific for SM with regard to mesoderm patterning. Using an antibody directed against -SM actin that recognized specifically this actin isoform in Xenopus, we have found that the expression of -SM actin is restricted to visceral and vascular SM with a transient expression in the heart. The overall expression of the -SM actin appears restricted to the ventral aspects of the differentiating embryo. -SM actin expression appears to be activated following mesoderm induction in animal cap derivatives. Moreover, at the gastrula stage, SM precursor cells are regionalized since they will only differentiate from ventrolateral marginal zone explants. Using the animal cap assay, we have found that -SM actin expression is specifically induced in treated animal cap with bFGF or a low concentration of XTC-MIF, which induce ventral structures, but not with a high concentration of XTC-MIF, which induces dorsal structures. Altogether, these results establish that -SM actin is a reliable marker for ven-trolateral mesoderm. We discuss the importance of this novel marker in studying mesoderm regionalization.
Reorganization of the cytoskeleton during Drosophila oogenesis: implications for axis specification and intercellular transport
Authors: Theurkauf William E. , Smiley Scott , Wong Mei Lie , Alberts Bruce M.
Published: April 2021

Inhibitor studies have implicated microtubules in at least three important developmental processes during Drosophila oogenesis: oocyte determination and growth during stages 1 through 6, positioning of the anterior determinant bicoid mRNA during stages 9 through 12, and ooplasmic streaming during stages 10b through 12. We have used fluorescence cytochemistry together with laser scanning confocal microscopy to identify distinct microtubule structures at each of the above three periods that are likely to be involved in these processes. During stages 1 through 7, maternal components synthesized in nurse cells are transported through cytoplasmic bridges to the oocyte. At this time, microtubules that appear to originate in the oocyte pass through these cytoplasmic bridges into the adjacent nurse cells; these microtubules are likely to serve as a polarized scaffold on which maternal RNAs and proteins are transported. During stages 7 and 8, microtubules in the oocyte cortex reorganize to form an anterior-to-posterior gradient, suggesting a role for microtubules in the localization of morphogenetic determinants. Finally, when ooplasmic streaming begins during stage 10 b, it is accompanied by the assembly of subsurface microtubule arrays that spiral around the oocyte; these arrays disassemble as the oocyte matures and streaming stops. During ooplasmic streaming, many vesicles are closely associated with the subsurface microtubules, suggesting that streaming is driven by vesicle translocation along microtubules. We believe that actin plays a secondary role in each of these morphogenetic events, based on our parallel studies of actin organization during each of the above stages of oogenesis.
Regional differences in retinoid release from embryonic neural tissue detected by an in vitro reporter assay
Authors: Wagner M. , Han B. , Jessell T. M.
Published: April 2021

Retinoic acid and related retinoids have been suggested to contribute to the pattern of cell differentiation during vertebrate embryonic development. To identify cell groups that release morphogenetically active retinoids, we have developed a reporter assay that makes use of a retinoic acid inducible response element (RARE) to drive lacZ or luciferase reporter genes in stably transfected cell lines. This reporter gene assay allows detection of retinoids released from embryonic tissues over a range equivalent to that induced by femtomole amounts of retinoic acid. We have used this assay first to determine whether the floor plate, a cell group that has polarizing properties in neural tube and limb bud differentiation, is a local source of retinoids within the spinal cord. We have also examined whether the effects of exogenously administered retinoic acid on anteroposterior patterning of cells in the developing central nervous system correlate with differences in retinoid release from anterior and posterior neural tissue. We find that the release of morphogenetically active retinoids from the floor plate is only about 1.5-fold that of the dorsal spinal cord, which does not have neural tube or limb polarizing activity. These results suggest that the spatial distribution of retinoid release from spinal cord tissues differs from that of the neural and limb polarizing activity. This assay has also shown that retinoids are released from the embryonic spinal cord at much greater levels than from the forebrain. This result, together with previous observations that the development of forebrain structures is suppressed by low concentrations of retinoic acid, suggest that the normal development of forebrain structures is dependent on the maintenance of low concentrations of retinoids in anterior regions of the embryonic axis. This assay has also provided initial evidence that other embryonic tissues with polarizing properties in vivo release retinoids in vitro.
Mesoderm-inducing factors and the control of gastrulation
Authors: Smith J. C. , Howard J. E.
Published: April 2021

One of the reasons that we know so little about the control of vertebrate gastrulation is that there are very few systems available in which the process can be studied in vitro. In this paper, we suggest that one suitable system might be provided by the use of mesoderm-inducing factors. In amphibian embryos such as Xenopus laevis, gastrulation is driven by cells of the mesoderm, and the mesoderm itself arises through an inductive interaction in which cells of the vegetal hemisphere of the embryo emit a signal which acts on overlying equatorial cells. Several factors have recently been discovered that modify the pattern of mesodermal differentiation or induce mesoderm from presumptive ectoderm. Some of these mesoderm-inducing factors will also elicit gastrulation movements, which provides a powerful model system for the study of gastrulation, because a population of cells that would not normally undertake the process can be induced to do so. In this paper, we use mesoderm-inducing factors to attempt to answer four questions. How do cells know when to gastrulate? How do cells know what kind of gastrulation movement to undertake? What is the cellular basis of gastrulation? What is the molecular basis of gastrulation?
Evolution of developmental decisions and morphogenesis: the view from two camps
Authors: Raff Rudolf A.
Published: April 2021

Modern developmental biology largely ignores evolution and instead focuses on use of standard model organisms to reveal general mechanisms of development Evolutionary biologists more widely hold developmental biology to be of major consequence in providing potential insights into evolution. Evolutionary insights can enlighten our views of developmental mechanisms as much as developmental data offer clearer views of mechanisms which underlie evolutionary change. However, insights have been limited by the long-term disengagement of the two fields dating to the rise of experimental embryology in the 1890s. Molecular genetics now provides a powerful tool to probe both gene function and evolutionary relationships, and a greater connection has become possible. The expansion of experimental organisms beyond the standard model animals used in most studies of development allows us to ask deeper questions about the interaction of development and evolution. This paper presents an analysis of the complementary uses of the resulting data in the two fields as they grope for accommodation. Analysis of the radical changes in early develoment seen in closely related sea urchins with alternate modes of development illustrate the complementarity of developmental and evolutionary data. These studies show that what have been thought to be constrained mechanisms of axial determination, cell lineage patterning, and gastrulation in fact evolve readily and provide the means for the rapid evolution of development
Brachyury - a gene affecting mouse gastrulation and early organogenesis
Authors: Beddington R. S. P. , Rashbass P. , Wilson V.
Published: April 2021

Mouse embryos that are homozygous for the Brachyury (T) deletion die at mid-gestation. They have prominent defects in the notochord, the allantois and the primitive streak. Expression of the T gene commences at the onset of gastrulation and is restricted to the primitive streak, mesoderm emerging from the streak, the head process and the notochord. Genetic evidence has suggested that there may be an increasing demand for T gene function along the rostrocaudal axis. Experiments reported here indicate that this may not be the case. Instead, the gradient in severity of the T defect may be caused by defective mesoderm cell movements, which result in a progressive accumulation of mesoderm cells near the primitive streak.

Embryonic stem (ES) cells which are homozygous for the T deletion have been isolated and their differentiation in vitro and in vivo compared with that of heterozygous and wild-type ES cell lines. In +/+ ↔ T/T ES cell chimeras the Brachyury phenotype is not rescued by the presence of wild-type cells and high level chimeras show most of the features characteristic of intact T/T mutants. A few offspring from blastocysts injected with T/T ES cells have been born, several of which had greatly reduced or abnormal tails. However, little or no ES cell contribution was detectable in these animals, either as coat colour pigmentation or by isozyme analysis. Inspection of potential +/+ ↔ T/T ES cell chimeras on the 11th or 12th day of gestation, stages later than that at which intact T/T mutants die, revealed the presence of chimeras with caudal defects. These chimeras displayed a gradient of ES cell colonisation along the rostrocaudal axis with increased colonisation of caudal regions. In addition, the extent of chimerism in ectodermal tissues (which do not invaginate during gastrulation) tended to be higher than that in mesodermal tissues (which are derived from cells invaginating through the primitive streak). These results suggest that nascent mesoderm cells lacking the T gene are compromised in their ability to move away from the primitive streak. This indicates that one function of the T gene may be to regulate cell adhesion or cell motility properties in mesoderm cells. Wild-type cells in +/+ ↔ T/T chimeras appear to move normally to populate trunk and head mesoderm, suggesting that the reduced motility in T/T cells is a cell autonomous defect
Dorsoventral development of the Drosophila embryo is controlled by a cascade of transcriptional regulators
Authors: Thisse Christine , Thisse Bernard
Published: April 2021

Maternal genes involved in dorsoventral (D/V) patterning of the Drosophila embryo interact to establish a stable nuclear concentration gradient of the Dorsal protein which acts as the morphogen along this axis. This protein belongs to the rel proto-oncogene and NF-KB transcriptional factor family and acts by controlling zygotic gene expression. In the ventral part of the embryo, dorsal specifically activates transcription of the gene twist and ventrally and laterally dorsal represses the expression of zerkn üllt, a gene involved in the formation of dorsal derivatives. The extent of dorsal action is closely related to the affinity and the number of dorsal response elements present in these zygotic gene promoters.

twist is one of the first zygotic genes necessary for mesoderm formation. It codes for a ‘b-HLH’ DNA-bind-ing protein which can dimerize and bind to DNA in vitro and to polytene chromosomes in vivo. In addition, in cultured cells twist has been shown to be a transcriptional activator. Thus, the first events of embryonic development along the D/V axis are controlled at the transcriptional level.
A gastrulation center in the ascidian egg
Authors: Jeffery William R.
Published: April 2021

A gastrulation center is described in ascidian eggs. Extensive cytoplasmic rearrangements occur in ascidian eggs between fertilization and first cleavage. During ooplasmic segregation, a specific cytoskeletal domain (the myoplasm) is translocated first to the vegetal pole (VP) and then to the posterior region of the zygote. A few hours later, gastrulation is initiated by invagination of endoderm cells in the VP region of the 110-cell embryo. After the completion of gastrulation, the embryonic axis is formed, which includes induction of the nervous system, morphogenesis of the larval tail and differentiation of tail muscle cells. Microsurgical deletion or ultraviolet (UV) irradiation of the VP region during the first phase of myoplasmic segregation prevents gastrulation, nervous system induction and tail formation, without affecting muscle cell differentiation. Similar manipulations of unfertilized eggs or uncleaved zygotes after the second phase of segregation have no effect on development, suggesting that a gastrulation center is established by transient localization of myoplasm in the VP region. The function of the gastrulation center was investigated by comparing protein synthesis in normal and UV-irradiated embryos. About 5% of 433 labelled polypeptides detected in 2D gels were affected by UV irradiation. The most prominent protein is a 30 kDa cytoskeletal component (p30), whose synthesis is abolished by UV irradiation. p30 synthesis peaks during gastrulation, is affected by the same UV dose and has the same UV-sensitivity period as gastrulation. However, p30 is not a UV-sensitive target because it is absent during ooplasmic segregation, the UV-sensitivity period. Moreover, the UV target has the absorption maximum of a nucleic acid rather than a protein. Cell-free translation studies indicate that p30 is encoded by a maternal mRNA. UV irradiation inhibits the ability of this transcript to direct p30 synthesis, indicating that p30 mRNA is a UV-sensitive target The gastrulation center may function by sequestration or activation of maternal mRNAs encoding proteins that function during embryogenesis.
Differential expression of the HMG box factors TCF-1 and LEF-1 during murine embryogenesis
Authors: Oosterwegel Mariëtte , van de Wetering Marc , Timmerman Janneke , Kruisbeek Ada , Destree Olivier , Meijlink Frits , Clevers Hans
Published: April 2021

The recent identification of a number of T lymphocytespecific enhancers has allowed the cloning of several novel transcription factors. Two of these, TCF-1 and LEF-1, contain a virtually identical DNA-binding domain of the High Mobility Group (HMG-1) box type. TCF-1 and LEF-1 originate from a recent gene duplication event as evidenced by comparison with the chicken homologue, chTCF. We have now analyzed the differential expression of these two transcription factors. In a panel of lymphoid cell lines, TCF-1 was exclusively expressed in the T cell lineage. In contrast, LEF-1 mRNA was detected at equivalent levels in proand pre-B cells and in all T lineage cells.

In situ hybridization on murine embryos revealed that TCF-1 and LEF-1 were widely expressed at day 7.5 of gestation. At later stages, the expression patterns were complex and only partially overlapping. The expression of TCF-1 and LEF-1 coincided until day 10.5, when mRNAs were detected in limb buds, neural crest, pharyngeal arches and nasal process. At later time points (day 13.5 to 14.5), sites of overlapping expression included lung, the urogenital system, tooth buds, thymus and choroid plexus. Unique expression sites for TCF-1 included Reichert’s membrane and trophectodermderived cells, the ribs and thoracic prevertebrae, craniofacial structures, the adrenal gland and meninges. Unique LEF-1 expression was observed in the tail prevertebrae, brain and inner ear. Postnatally, expression of both genes could only be detected in lymphoid tissues. These observations suggest that TCF-1 and LEF-1 exert differential functions during murine embryogenesis.
Size regulation and morphogenesis: a cellular analysis of skeletogenesis in the sea urchin embryo
Authors: Ettensohn Charles A. , Malinda Katherine M.
Published: April 2021

The formation of the skeleton is a central event in sea urchin morphogenesis. The skeleton serves as a framework for the larval body and is the primary determinant of its shape. Previous studies have shown that the size of the skeleton is invariant despite wide experimentally induced variations in the number of skeleton forming primary mesenchyme cells (PMCs). In the present study, we have used PMC transplantation, fluorescent cell markers and confocal laser scanning microscopy to analyze cellular aspects of skeletal patterning. Labeling of embryos with 5-bromodeoxyuridine demonstrates that the entire embryonic phase of skeletal morphogenesis occurs in the absence of PMC division. During embryogenesis, skeletal rods elongate by one of two mechanisms; either preceded by a cluster (plug) of PMCs or by extending along an existing PMC filopodial cable. Elongation of skeletal rods occurs exclusively by the addition of new material at the rod tips, although radial growth (increase in rod thickness) occurs along the length of the rods. Photoablation of a distinctive region of ectoderm cells at the arm tip results in an inhibition of skeletal rod elongation, indicating that a local ectoderm-PMC interaction is required for skeletal growth. The regulation of skeletal patterning was also examined in embryos that had been microinjected with additional PMCs and in half-sized larvae derived from blastomeres isolated at the 2-cell stage. Microinjection of 50-100 PMCs into the blastocoel at the mesenchyme blastula stage leads to an increase in the numbers of PMCs along all skeletal rods and a two-fold increase in the number of cells in the plugs, yet no increase in the length of the skeletal rods. The length of the anal rods can, however, be increased by microinjecting developmentally ‘young’ PMCs into the arm tips of late stage embryos. We find that the rate of skeletal rod elongation is independent of both the mode of rod growth (chain or plug) and the number of PMCs in the plug at the growing rod tip. Instead, the rate of elongation appears to be strictly regulated by the quantity of ectodermal tissue present in the embryo. These studies provide new information concerning normal mechanisms of skeletal growth and patterning and lead us to propose a model for the regulation of skeleton size based upon an intrinsic PMC ‘clock’ and an ectoderm-derived signal that regulates the rate of skeletal rod elongation.