bluuebunny/crossref_metadata_2025 huggingface dataset:
Developmental expression of syndecan, an integral membrane proteoglycan, correlates with cell differentiation
Authors: Trautman Michael S. , Kimelman Judy , Bernfield Merton
Published: April 2021

Syndecan is an integral membrane proteoglycan that behaves as a matrix receptor by binding cells to interstitial matrix and associating intracellularly with the actin cytoskeleton. Using immunohistology, we have now localized this proteoglycan during the morphogenesis of various derivatives of the surface ectoderm in mouse embryos. Syndecan is expressed on ectodermal epithelia, but is selectively lost from the cells that differentiate into the localized placodes that initiate lens, nasal, otic and vibrissal development. The loss is transient on presumptive ear, nasal and vibrissal epithelia; the derivatives of the differentiating ectodermal cells that have lost syndecan subsequently re-express syndecan. In contrast, syndecan is initially absent from the mesenchyme underlying the surface ectoderm, and is transiently expressed when the surface ectoderm loses syndecan. These results demonstrate that expression of syndecan is developmentally regulated in a distinct spatiotemporal pattern. On epithelia, syndecan is lost at a time and, location that correlates with epithelial cell differentiation and, on mesenchyme, syndecan is acquired when the cells aggregate in proximity to the epithelium. This pattern of change with morphogenetic events is unique and not duplicated by other matrix molecules or adhesion receptors.
Temporal and regional differences in the expression pattern of distinct retinoic acid receptor-β transcripts in the chick embryo
Authors: Smith Susan M. , Eichele Gregor
Published: April 2021

Retinoic add (RA) is a signaling molecule apparently involved in a variety of morphogenetic processes, such as patterning of developing and regenerating vertebrate limbs. RA binds to specific intracellular receptors that constitute a multigene family. RA receptors (RAR) bind to the regulatory region of specific target genes and thereby control the expression of these genes. Here we report the sequence and spatiotemporal expression pattern of RAR-β from chick. Northern blots of RNA from whole embryos and from limb buds reveal the presence of transcripts of 3.2, 3.4, and 4.6 kb in size. Using two riboprobes, one that hybridizes to all three RAR-β mRNAs and a second one, specific for the 4.6 kb transcript, we found by in situ hybridization a differential distribution of RAR-β transcripts in limb bud mesenchyme, in craniofacial mesenchyme and in hindbrain neuroectoderm. In the hindbrain the 4.6 kb mRNA exhibits an anterior boundary of expression at the level of the constriction between rhombomeres 5 and 6. Examination of neural plate stage embryos by in situ hybridization indicates that this boundary of expression is already defined by this stage. In addition to having several RA receptors that are expressed with distinct spatial patterns in the embryo, our data indicate that the expression pattern of transcripts derived from a single receptor gene can also be differentially expressed, thus providing another level for regulating RA action.
Retinoic acid receptors and cellular retinoid binding proteins : II. Their differential pattern of transcription during early morphogenesis in mouse embryos
Authors: Ruberte Esther , Dolle Pascal , Chambon Pierre , Morriss-Kay Gillian
Published: April 2021

In situ hybridization with 35S-labefled RNA probes was used to study the distribution of transcripts of genes coding for the retinoic acid receptors, RAR-α, βand -γ, and the cellular binding proteins for retinoic acid (CRABP I) and retinol (CRBP I), in mouse embryos during the period of early morphogenesis. Primary mesenchyme formation was associated with CRBP I labelling of both epiblast and mesenchyme of the primitive streak, while the CRABP probe labelled the migrating primary mesenchyme cells. Neural crest cell emigration and migration were associated with CRABP labelling of both neural epithelium (excluding the floor plate) and neural crest cells, while CRBP I expression was restricted to basal and apical regions of the epithelium (excluding the floor plate). The strongest neuroepithelial signal for CRABP was in the preotic hindbrain. RAR-β was present in presomitic stage embryos, being expressed at highest levels in the lateral regions. RAR-α was associated with crest cell emigration and migration, while RAR-γ was present in the primitive streak region throughout the period of neurulation. There was a change from RAR-β to RAR-γ expression at the junction between closed and open neural epithelium at the caudal neuropore. RAR-α and RAR-β were expressed at specific levels of the hindbrain and in the spinal cord. These distribution patterns are discussed in relation to segmental expression patterns of other genes, and to maturational changes in the caudal neuropore region. The CRABP transcript distribution patterns correlated well with known target tissues of excess retinoid-induced teratogenesis (migrating pri-mary mesenchyme and neural crest cells, preotic hindbrain), providing further support for our hypoth-esis that cells expressing CRABP are those that cannot tolerate high levels of RA for their normal developmen-tal function.
The origin of postembryonic neuroblasts in the ventral nerve cord of Drosophila melanogaster
Authors: Prokop Andreas , Technau Gerhard M.
Published: April 2021

Embryonic and postembryonic neuroblasts in the thoracic ventral nerve cord of Drosophila melanogaster have the same origin. We have traced the development of threefold-labelled single precursor cells from the early gastrula stage to late larval stages. The technique allows in the same individual monitoring of progeny cells at embryonic stages (in vivo) and differentially staining embryonic and postembryonic progeny within the resulting neural clone at late postembryonic stages. The analysis reveals that postembryonic cells always appear together with embryonic cells in one clone. Further-more, BrdU labelling suggests that the embryonic neuroblast itself rather than one of its progeny resumes proliferation as a postembryonic neuroblast. A second type of clone consists of embryonic progeny only.
Xenopus Myf-5 marks early muscle cells and can activate muscle genes ectopically in early embryos
Authors: Hopwood N. D. , Pluck A. , Gurdon J. B.
Published: April 2021

We have cloned a Xenopus cDNA that encodes a homologue of the human myogenic factor, Myf-5. Xenopus Myf-5 (XMyf5) transcripts first accumulate in the prospective somite region of early gastrulae. The pattern of XMyf5 expression is similar to that of the Xenopus MyoD (XMyoD) gene, except that XMyf5 transcripts are largely restricted to posterior somitic mesoderm even before any somites have formed. Transient ectopic expression of XMyf5 activates cardiac actin and XMyoD genes in animal cap cells, but does not cause full myogenesis, even in combination with XMyoD. These results suggest that XMyf5 acts together with XMyoD as one of the set of genes regulating the earliest events of myogenesis, additional factors being required for complete muscle differentiation.
Basic FGF and TGF-β1 influence commitment to melanogenesis in neural crest-derived cells of avian embryos
Authors: Stocker Kate M. , Sherman Larry , Rees Sean , Ciment Gary
Published: April 2021

In previous studies, we showed that neural crest (NC)-derived cells from embryonic quail dorsal root ganglia (DRG) and peripheral nerve (PN), which do not normally give rise to melanocytes, become committed to melanogenesis following treatment in culture with the phorbol ester drug 12-O-tetradecanoyl phorbol-13-acetate (TPA). These and other observations support the notion that melanocytes and Schwann cells are derived from a common bipotent intermediate in the neural crest lineage – the melanocyte/Schwann cell progenitor. In this study, we test the possibility that peptide growth factors found in the embryonic environment might act similarly to TPA to influence the fates of these cells. DRG and PN expiants were cultured in medium supplemented with a variety of growth factors, and then the cultures were examined for the presence of pigment cells. We found that basic fibroblast growth factor (bFGF), but not various other growth factors, induced pigmentation in about 20 % of these cultures. When low concentrations of TPA were included in the culture medium, bFGF augmented the TPA-induced pigmen-tation, significantly increasing the proportion of pigmented cultures. These effects of bFGF were age-dependent, and could be blocked by addition of a bFGF-neutralizing antibody to the culture medium. In contrast to these stimulatory effects of bFGF, transforming growth factor-βl (TGF-β1) was found to inhibit the TPA- or bFGF-induced pigmentation of DRG cultures. These data suggest, therefore, that at least some NC-derived cells are responsive to bFGF and TGF-β1, and that these growth factors may play an important role in the control of NC cell fate.
Positioning and maintenance of embryonic body wall muscle attachments in C. elegans requires the mup-1 gene
Authors: Goh Phuay-Yee , Bogaert Thierry
Published: April 2021

As part of a general study of genes specifying a pattern of muscle attachments, we identified and genetically characterised mutants in the mup-1 gene. The body wall muscles of early stage mup-1 embryos have a wild-type myofilament pattern but may extend ectopic processes. Later in embryogenesis, some body wall muscles detach from the hypodermis. Genetic analysis suggests that mup-1 has both a maternal and a zygotic component and is not required for postembryonic muscle growth and attachment, mup-1 mutants are suppressed by mutations in several genes that encode extracellular matrix components. We propose that mup-1 may encode a cell surface/extracellular matrix molecule required both for the positioning of body wall muscle attachments in early embryogenesis and the subsequent maintenance of these attachments to the hypodermis until after cuticle synthesis.
The spatial and temporal distribution of polarizing activity in the flank of the pre-limb-bud stages in the chick embryo
Authors: Hornbruch Amata , Wolpert Lewis
Published: April 2021

The presence of polarizing activity in the limb buds of developing avian embryos determines the pattern of the anteroposterior axis of the limbs in the adult. Maps of the spatial distribution and the strength of the signal within limb buds of different stages are well documented. Polarizing activity can also be found in Hensen’s node in the early embryo.

We have mapped the distribution of polarizing activity as it emerges from Hensen’s node and spreads into the flank tissue of the embryo. There is a clear change in the local pattern of expression of polarizing activity between stage 8 and 18. Almost no activity is measured for stages 8 and 9. More or less uniform levels of around 10 % are spread along the flank lateral to the unsegmented somitic mesoderm from somite position 12 to 22 in stage 10 embryos. Some 6 to 8 h later at stage 12, there is a distinct peak of activity at somite position 18, the middle of the wing field. This peak increases at stages 13 to 15 and its position traverses to the posterior edge of the wing field. Full strength of activity is reached shortly before the onset of limb bud formation at stage 16 to 17. Stages 16 to 18 were investigated for polarizing activity in the wing and the leg field. Low levels of polarizing activity are present in the anterior leg field at stages 16 and 17 but have disappeared by stage 18 and all activity is confined to the posterior part of the leg bud.
A member of the RXR nuclear receptor family is expressed in neural-crest-derived cells of the developing chick peripheral nervous system
Authors: Rowe Annie , Eager Nicholas S. C. , Brickell Paul M.
Published: April 2021

Retinoic acid (RA) affects differentiation and morphogenesis in various developmental systems and is believed to act through nuclear RA receptors that belong to the steroid/thyroid hormone family of ligand-binding transcription factors. Three closely related receptors, RARα, -β and -γ, with distinct expression patterns, have been identified and a fourth receptor, hRXR-α, which responds to RA but which has low homology to RAR-α, -β and -γ, was recently discovered. Here we report the isolation of a cDNA clone encoding a chicken homologue of hRXR-α (cRXR) and show that a cRXR transcript of 2.5 kb is expressed in a range of embryonic chick tissues. By in situ hybridization to sections from stage 24 and stage 27 chick embryos, we show that cRXR transcripts are expressed at high levels in the liver and in elements of the developing peripheral nervous system derived from the neural crest, including dorsal root ganglia, cranial ganglia, enteric ganglia and peripheral nerve tracts. At stage 16, in the posterior trunk region, cRXR transcripts are expressed by cells in the neural crest and in neural crest cells migrating into the sclerotome, indicating that neural crest cells express cRXR transcripts before overt differentiation into peripheral nervous tissue. This distribution suggests a novel role for RA in the developing peripheral nervous system, mediated by cRXR. In addition, it identifies cRXR as a marker for a specific population of neural-crest-derived cells.
Tyrosine phosphorylation of p34cdc2 and p42 during meiotic maturation of Xenopus oocyte: Antagonistic action of okadaic acid and 6-DMAP
Authors: Jessus Catherine , Rime Héléne , Haccard Olivier , Lint Johan Van , Goris Jozef , Merlevede Wilfried , Ozon RenÉ
Published: April 2021

The tyrosine phosphorylation/dephosphorylation of p34cdc2 was estimated by immunoblotting with antiphosphotyrosine antibody during meiotic maturation of Xenopus oocytes. At the time of germinal vesicle breakdown (GVBD), p34cdc2 is tyrosine dephosphorylated whereas a p42 protein, which might correspond to a MAP2 kinase, becomes tyrosine phosphorylated. No modification in the level of tyrosine phosphorylation of either proteins was noticed during the whole maturation process from GVBD until metaphase H. When added to prophase oocytes, 6-DMAP (6-dimethyl-aminopurine) blocks GVBD, M-phase-promoting factor (MPF) activation and Hl-histone, kinase activation induced by either progesterone, MPF transfer or okadaic acid microinjection. In each case, the tyrosine déphosphorylation reaction of p34cdc2 is inhibited. In meiosis I oocytes (just after the initiation of GVBD), 6-DMAP provokes the rephosphorylation of p34cdc2 on tyrosine residue(s), inactivation of MPF and Hl-histone kinase and re-entry of the cell into an interphase-like state. These processes are reversible by simply removing the agent. In contrast to the observations in prophase oocytes, okadaic acid is able to reverse the inhibitory effect of 6-DMAP in meiosis I oocytes on MPF and Hl-histone kinase activities and to initiate dephosphorylation of p34cdc2 on tyrosyl residue(s) even in the presence of 6-DMAP. Altogether, our results show that 6-DMAP and okadaic acid antagonistically control in vivo the level of tyrosine phosphorylation of p34cdc2.
Migration and proliferation of cultured neural crest cells in W mutant neural crest chimeras
Authors: Huszar D. , Sharpe A. , Jaenisch R.
Published: April 2021

Chimeric mice, generated by aggregating preimplantation embryos, have been instrumental in the study of the development of coat color patterns in mammals. This approach, however, does not allow for direct experimental manipulation of the neural crest cells, which are the precursors of melanoblasts. We have devised a system that allows assessment of the developmental potential and migration of neural crest cells in vivo following their experimental manipulation in vitro. Cultured C57B1/6 neural crest cells were microinjected in utero into neurulating Balb/c or W embryos and shown to contribute efficiently to pigmentation in the host animal. The resulting neural crest chimeras showed, however, different coat pigmentation patterns depending on the genotype of the host embryo. Whereas Balb/c neural crest chimeras showed very limited donor cell pigment contribution, restricted largely to the head, W mutant chimeras displayed extensive pigmentation throughout, often exceeding 50 % of the coat. In contrast to Balb/c chimeras, where the donor melanoblasts appeared to have migrated primarily in the characteristic dorsoventral direction, in W mutants the injected cells appeared to migrate in the longitudinal as well as the dorsoventral direction, as if the cells were spreading through an empty space. This is consistent with the absence of a functional endogenous melanoblast population in W mutants, in contrast to Balb/c mice, which contain a full complement of melanocytes. Our results suggest that the IV mutation disturbs migration and/or proliferation of endogenous melanoblasts. In order to obtain information on clonal size and extent of intermingling of donor cells, two genetically marked neural crest cell populations were mixed and coinjected into W embryos. In half of the tricolored chimeras, no co-localization of donor crest cells was observed, while, in the other half, a fine intermingling of donor-derived colors had occurred. These results are consistent with the hypothesis that pigmented areas in the chimeras can be derived from extensive proliferation of a few donor clones, which were able to colonize large territories in the host embryo. We have also analyzed the development of pigmentation in neural crest cultures in vitro, and found that neural tubes explanted from embryos carrying wt or weak IV alleles produced pigmented melanocytes while more severe W genotypes were associated with deficient pigment formation in vitro.
The ability of hamster oolemma to fuse with spermatozoa: Its acquisition during oogenesis and loss after fertilization
Authors: Zuccotti Maurizio , Yanagimachi Ryuzo , Yanagimachi Hiroko
Published: April 2021

To determine when the growing hamster oocyte gains the ability to fuse with the spermatozoon, oocytes at various stages of development were collected from ovaries, and zona-pellucida-free oocytes were inseminated in vitro with acrosome-reacted spermatozoa. Very small primary oocytes were unable to fuse with spermatozoa. Oocytes first became competent to fuse with spermatozoa when they had grown to about 20 μm in diameter. The acquisition of fusibility coincided with the first appearance of zona pellucida material and oolemma microvilli. The fusibility of the oolemma increased as the oocyte grew, reaching a maximum when the oocyte reached the metaphase of the second meiosis. The fusibility of the oolemma was reduced drastically after fertilization, and was lost completely by the 8-cell stage. The appearance and subsequent disappearance of a putative fusion-mediating molecule in the oolemma is proposed. Since this molecule is fairly resistant to proteinase digestion, at least in the hamster, it could be a cryptic protein or a glycolipid.
Formation and regeneration of rhombomere boundaries in the developing chick hindbrain
Authors: Guthrie Sarah , Lumsden Andrewc
Published: April 2021

Development in the chick hindbrain is founded on a segmented pattern. Groups of cells are allocated to particular segmental levels early in development, the cells of each segment (rhombomere) mixing freely with each other, but not with those of adjacent segments. After rhombomere formation, cells in the boundary regions become increasingly specialised. Rhombomeres are thus separate territories that will ultimately pursue different developmental fates. We are investigating the mechanisms that establish and maintain the pattern of rhombomeres and their boundaries. Donor-to-host transplantation experiments were used to confront tissue from different axial levels within the hindbrain. The frequency of boundary regeneration and patterning in the hindbrain was then assessed, based on gross morphology, arrangement of motor neurons and immunohistochemistry. We found that when rhombomeres from adjacent positions or positions three rhombomeres distant from one another were confronted, a normal boundary was invariably reconstructed. Juxtaposition of rhombomere 5 with 7 also yielded a new boundary. By contrast, donor and host tissue of the same positional origin combined without forming a boundary. The same result was obtained in combinations of rhombomeres 3 and 5. Confrontation of tissue from even-numbered rhombomeres 4 with 6 or 2 with 4 also failed to regenerate a boundary in the majority of cases. These results suggest that cell surface properties vary according to rhombomeric level in the hindbrain, and may support the idea of a two-segment periodicity.
Separate elements cause lineage restriction and specify boundaries of Hox-1·1 expression
Authors: Puschel Andreas W. , Balling Rudi , Gruss Peter
Published: April 2021

The Hox genes are a class of putative developmental control genes that are thought to be involved in the specification of positional identity along the anteroposterior axis of the vertebrate embryo. It is apparent from their expression pattern that their regulation is dependent upon positional information. In a previous analysis of the Hox-1·1 promoter in transgenic mice, we identified sequences that were sufficient to establish transgene expression in a specific region of the embryo. The construct used, however, did not contain enough regulatory sequences to reproduce all aspects of Hox-1·1 expression. In particular, neither a posterior boundary nor a restriction of expression to prevertebrae was achieved. Here we show correct regulation by Hox-1·1 sequences in transgenic mice and identify the elements responsible for different levels of control. Concomitant with the subdivision of mesodermal cells into different lineages during gastrulation and organogenesis, Hox-1·1 expression is restricted to successively smaller sets of cells. Distinct elements are required at different stages of development to execute this developmental programme. One position-responsive element (130 bp nontranslated leader) was shown to be crucial for the restriction of expression not only along the anteroposterior axis of the embryo, setting the posterior border, but also along the dorsoventral axis of the neural tube and to the lineage giving rise to the prevertebrae. Thus, Hox-1 ·1 expression is established in a specific region of the embryo and in a specific lineage of the mesoderm by restricting the activity of the promoter by the combined effect of several regulatory elements.
Cell rearrangement during gastrulation ofXenopus:direct observation of cultured explants
Authors: Wilson Paul , Keller Ray
Published: April 2021

We have analyzed cell behavior in the organizer region of the Xenopus laevis gastrula by making high resolution time-lapse recordings of cultured expiants. The dorsal marginal zone, comprising among other tissues prospective notochord and somitic mesoderm, was cut from early gastrulae and cultured in a way that permits high resolution microscopy of the deep mesodermal cells, whose organized intercalation produces the dramatic movements of convergent extension. At first, the expiants extend without much convergence. This initial expansion results from rapid radial intercalation, or exchange of cells between layers. During the second half of gastrulation, the expiants begin to converge strongly toward the midline while continuing to extend vigorously. This second phase of extension is driven by mediolateral cell intercalation, the rearrangement of cells within each layer to lengthen and narrow the array. Toward the end of gastrulation, fissures separate the central notochord from the somitic mesoderm on each side, and cells in both tissues elongate mediolaterally as they intercalate. A detailed analysis of the spatial and temporal pattern of these behaviors shows that both radial and mediolateral intercalation begin first in anterior tissue, demonstrating that the anteriorposterior timing gradient so evident in the mesoderm of the neurula is already forming in the gastrula. Finally, time-lapse recordings of intact embryos reveal that radial intercalation takes places primarily before involution, while mediolateral intercalation begins as the mesoderm goes around the lip. We discuss the significance of these findings to our understanding of both the mechanics of gastrulation and the patterning of the dorsal axis.
Developmental expression of the axonal glycoprotein TAG-1: differential regulation by central and peripheral neurons in vitro
Authors: Karagogeos D. , Morton S. B. , Casano F. , Dodd J. , Jessell T. M.
Published: April 2021

TAG-1 is a 135000 Mr axonal glycoprotein of the immunoglobulin superfamily that promotes axon extension in vitro. One distinguishing feature of TAG-1 is its transient expression on subsets of axons in the developing nervous system. To examine the mechanisms that regulate TAG-1, we have monitored the expression of this protein by developing central and peripheral neurons in vitro. TAG-1 was detected on the surface of a subset of Ell to E13 spinal cord neurons in vitro and was also released by these neurons. Expression of TAG-1 on the cell surface was transient but it was possible to detect a released form of TAG-1 at all times in vitro. Spinal cord neurons isolated from older embryos did not express surface TAG-1 when they regenerated axons in vitro. Changes in the environment of spinal cord neurons did not alter the time course of TAG-1 expression, suggesting that regulation of the protein is cell autonomous. In contrast to these results with spinal cord neurons, surface expression of TAG-1 by DRG neurons persisted in vitro and adult DRG neurons re-expressed TAG-1 when grown in vitro. The cell surface and released forms of TAG-1 therefore appear to be regulated differently by central and peripheral neurons.
Interactions between fused, a segment-polarity gene in Drosophila, and other segmentation genes
Authors: Limbourg-Bouchon Bernadette , Busson Denise , Lamour-Isnard Claudie
Published: April 2021

Fused (fu) is a segment polarity gene whose product is maternally required in the posterior part of each segment. To define further the role of fused and determine how it interacts with other segmentation genes, we examined the phenotypes obtained by combining fused with mutations of pair rule, homeotic and other segment polarity loci. When it was possible, we also looked at the distribution of corresponding proteins in fused mutant embryos. We observed that fused;naked (fu;nkd) double mutant embryos display a phenotypic suppression of simple mutant phenotypes: both naked cuticle and denticle belts, which would normally have been deleted by one of the two mutants alone, were restored. In fused, mutant embryos, engrailed (en) and wingless (wg) expression was normal until germ band extension, but partially and completely disappeared respectively during germ band retraction. In the fu;nkd double mutant embryo, en was expressed as in nkd mutant at germ band extension, but later this expression was restricted and became normal at germ band retraction. On the contrary, wg expression disappeared as in fu simple mutant embryos. We conclude that the requirements for fused, naked and wingless activities for normal segmental patterning are not absolute, and propose mechanisms by which these genes interact to specify anterior and posterior cell fates.
Dye-coupling and the formation and fate of the hypoblast in the teleost fish embryo, Barbus conchonius
Authors: Gevers Petra , Timmermans Lucy P. M.
Published: April 2021

The present report describes Lucifer Yellow (LY) transfer between the syncytial layer of the yolk cell (YSL) and blastodermal cells during epiboly in the teleost fish Barbus conchonius. The fate of a group of labeled cells is described until germ layer formation.

At the onset of epiboly, LY seems to be transferred from the YSL to all blastodermal cells. Between 10 % and 40 % epiboly, dye-coupling appears to be restricted to the marginal region. Within 60 min individually labeled cells are distributed among unlabeled cells within the blastoderm. Between 40 % and 60 % epiboly, we observed a ring-shaped group of labeled cells, which probably have involuted during early gastrulation. Consequently, this cell group may correlate with the leading edge of the hypoblast layer within the germ ring. At 60 % epiboly and later, the blastodermal cells are dye-uncoupled from the YSL.

A gradual translocation of the ring-shaped hypoblast towards a dorsally located bar-like structure is observed between 50 % and 100 % epiboly. At 100 % epiboly, fluorescent cells were located in contact with the YSL within the embryo proper, with the brightest fluorescence in the future head region. The translocation is due to dorsalwards convergent cell movements during the gastrulation process. The appearance of the hypoblast as a dye-coupled cell layer may correlate with some restriction in cell fate since the hypoblast differs in fate from the epiblast.
Hypoxanthine phosphoribosyl transferase deficiency, haematopoiesis and fertility in the mouse
Authors: Ansell J. D. , Samuel K. , Whittingham D. G. , Patek C. E. , Hardy K. , Handyside A. H. , Jones K. W. , Muggleton-HARRIS A. L. , Taylor A. H. , Hooper M. L.
Published: April 2021

We have looked for effects of deficiency in hypoxanthine phosphoribosyl transferase (HPRT) in the mouse comparable to non-behavioural consequences of HPRT-deficiency in humans. HPRT-deficient humans show abnormalities in haematopoiesis and, in heterozygotes, there is strong selection in haematopoietic tissues against HPRT-deficient cells arising as a result of X-chromosome inactivation. We have examined two situations in mice in which HPRT- and HPRT+ cells occur in the same individual. First, in chimaeras resulting from the injection of HPRT- embryonal stem cells into HPRT+ blastocysts the fate of HPRT- and HPRT+ cell populations was monitored by their expression of different isozymes of glucose phosphate isomerase and also, in those chimaeras that resulted from injecting the male ES cells into female blastocysts, by in situ hybridisation using a Y-chromosome-specific repetitive DNA probe. There was a small statistically significant selection against the HPRT- population in haematopoietic tissues in both XX↔->XY and XY<-*XY chimaeras. Second, in female mice doubly heterozygous for HPRT-deficiency and for an electrophoretic variant of the X-linked enzyme phosphoglycerate kinase, there was a similar small statistically significant selection against the HPRT- population in haematopoietic tissues. While further work is required to establish whether this selection is a consequence of the HPRT mutation, it is clear that any selection against cells in the haematopoietic system as a consequence of HPRT-deficiency is at most small compared with the effect seen in humans.

In HPRT-deficient human males surviving beyond the normal age of puberty, there is testicular atrophy. However, we find no effect of HPRT-deficiency on the fertility of either male or female mice.

Thus, as with effects on behaviour, the consequences of HPRT-deficiency for haematopoiesis and testis development in the mouse are at most small compared with those in the human. We conclude that the reason for the difference in effects between the two species lies in a difference in purine-related intermediary metabolism per se, rather than in its interaction with brain amine biochemistry.
Ghox 4.7: a chick homeobox gene expressed primarily in limb buds with limb-type differences in expression
Authors: Mackem Susan , Mahon Kathleen A.
Published: April 2021

Homeobox genes play a key role in specifying the segmented body plan of Drosophila, and recent work suggests that at least several homeobox genes may play a regulatory role during vertebrate limb morphogenesis. We have used degenerate oligonucleotide primers from highly conserved domains in the homeobox motif to amplify homeobox gene segments from chick embryo limb bud cDNAs using the polymerase chain reaction. Expression of a large number of homeobox genes (at least 17) is detected using this approach. One of these genes contains a novel homeobox loosely related to the Drosophila Abdominal B class, and was further analyzed by determining its complete coding sequence and evaluating its expression during embryogenesis by in situ hybridization. Based on sequence and expression patterns, we have designated this gene as Ghox 4.7 and believe that it is the chick homologue of the murine Hox 4.7 gene (formerly Hox 5.6). Ghox 4.7 is expressed primarily in limb buds during development and shows a striking spatial restriction to the posterior zone of the limb bud, suggesting a role in specifying anterior-posterior pattern formation. In chick, this gene also displays differences in expression between wing and leg buds, raising the possibility that it may participate in specifying limb-type identity.
A regulatory region from the mouse Hox-2.2 promoter directs gene expression into developing limbs
Authors: Schughart Klaus , Bieberich Charles J. , Eid Roland , Ruddle Frank H.
Published: April 2021

To characterize cis-acting regulatory elements of the murine homeobox gene, Hox-2.2, transgenic mouse lines were generated that contained the LacZ reporter gene under the control of different fragments from the presumptive Hox-2.2 promoter. A promoter region of 3600 base pairs (bp) was identified, which reproducibly directed reporter gene expression into specific regions of developing mouse embryos. At 8.5 days postcoitum (p.c.) reporter gene activity was detected in posterior regions of the lateral mesoderm and, in subsequent developmental stages, expression of the LacZ gene was restricted to specific regions of the developing limb buds and the mesenchyme of the ventrolateral body region. This pattern of Hox-2.2-LacZ expression was found in all transgenic embryos that have been generated with the 3.6 kb promoter fragment (two founder embryos and embryos from five transgenic lines). In addition, embryos from two transgenic mouse lines expressed the reporter gene at low levels in the developing central nervous system (CNS). Our results are consistent with the idea that in addition to their presumptive role in CNS and vertebrae development, Hox-2.2 gene products are involved in controlling pattern formation in developing limbs.
A 3 kb sequence from the mouse cellular retinoic-acid-binding protein gene upstream region mediates spatial and temporal LacZ expression in transgenic mouse embryos
Authors: Wei Li-Na , Chen Gee-Jang , Chu Ya-Shu , Tsao Jen-Lan , Nguyen-Huu M. Chi
Published: April 2021

A 3233 base pair (bp) sequence of the 5′-flanking region of the mouse cellular retinoic-acid-binding protein (CRABP) gene is determined. From this region, a 3 kb fragment located 150 bp upstream from the transcriptional initiation site is isolated and fused to a LacZ reporter sequence. Transgenic mouse embryos of this fusion gene show spatially and temporally specific expression of LacZ protein and the expression of this fusion gene at the RNA level is confirmed by RNAase protection assays, which detect specific fusion transcripts in RNA samples from tissues of transgenic mouse embryos. In contrast, transgenic mouse embryos of a shorter fusion gene containing only 583 bp from the same upstream region of the mouse CRABP gene fused to the same reporter sequence show no LacZ activities. Thus, it is concluded that the 3 kb sequence, but not the 583 bp sequence, of the mouse CRABP gene contains information for its temporally and spatially specific expression in mouse embryos.
The switch from larval to adult globin gene expression in Xenopus laevis is mediated by erythroid cells from distinct compartments
Authors: Weber Rudolf , Blum Beat , Müller Peter Rudolf
Published: April 2021

The transition of hemoglobins during metamorphosis of Xenopus laevis involves replacement of the larval erythrocytes by adult ones, suggesting that the developmental control of this event depends upon the growth characteristics of the precursor cells. To identify the erythroid precursor cells and to investigate their developmental fate, we analyzed the distribution of stage-specific globin mRNAs by northern blotting in dorsal and ventral fragments of stage 32 embryos after in vitro culture as well as presumptive erythropoietic tissues of tadpoles during metamorphosis.

The histological analysis shows that erythrocytes differentiate only in ventral fragments, suggesting that the ventral blood islands and most likely also the dorsolateral mesoderm are the primary sites of erythropoiesis. We also demonstrate that the first generations of erythrocytes, already express the predominating larval specific a-globin mRNAs. The globin mRNA patterns obtained from presumptive erythropoietic tissues suggest an important role of circulating precursor cells in larval erythropoiesis, whereas the liver appears to be the main site of formation and maturation of the adult erythrocytes.

Tentatively we propose that anuran erythropoiesis is dependent upon a self-perpetuating stem-cell line and that the larval and the adult erythrocytes are derived from successive generations of erythroid precursors, whose commitment may be imposed by the erythropoietic sites.
The expression of tenascin by neural crest cells and glia
Authors: Tucker Richard P. , Mckay Sharen E.
Published: April 2021

The extracellular matrix glycoprotein tenascin is concentrated in both the embryo and adult in regions where cell motility is taking place. For example, during avian neural crest morphogenesis tenascin is concentrated in the rostral half of the sclerotome, precisely where the neural crest cells themselves are found. Previous in vitro studies indicated that somite cells were the source of this tenascin, implying a role for tenascin in directing the ventral migration of neural crest cells and thus the establishment of the periodic arrangement of the PNS. In this study, we have used a cDNA probe to identify the source of tenascin found along the pathways of the neural crest using in situ hybridization. In tissue sections, individual cells found along the neural crest migratory pathways, both before entering the somites and within the somites, are strongly labelled by the tenascin cDNA. In vitro neural crest cells are more strongly labelled with the tenascin probe than somite cells. Finally, western blotting has been used to identify tenascin in culture medium conditioned by neural crest cells. This indicates that neural crest cells themselves are the source of much of the tenascin found lining their migratory pathways, and that interactions with somite cells may not be needed to induce the expression of tenascin. We have also studied the distribution of tenascin mRNA in the developing spinal cord and spinal ganglia. At embryonic days 7 and 10, tenascin cDNA hybridizes within cells that appear to be migrating from the ependymal layer to the white matter, as well as within cells in the dorsal roots. The hybridization is transient, as tenascin mRNA is no longer detected at E18. Thus, the role of tenascin in neural crest and CNS morphogenesis is not related to defining routes of migration, but perhaps to generating a local environment that supports motility.
Developmental expression of rat cardiac troponin I mRNA
Authors: Ausoni Simonetta , Nardi Costanza De , Moretti Paolo , Gorza Luisa , Schiaffino Stefano
Published: April 2021

We have isolated and sequenced a full-length cDNA clone of rat cardiac troponin I (Tnl). The amino acid sequence of rat cardiac Tnl is highly similar to that of other mammalian species in the portion of the molecule (residues 33–210) that is also homologous to skeletal muscle Tnl isoforms. In contrast, a lower degree of similarity is present in the cardiac Tnl-specific amino terminal extension (residues 1–32). This region contains a conserved serine residue that has been shown to be selectively phosphorylated by cAMP-dependent protein kinase. Cardiac Tnl mRNA is weakly expressed in the 18-day fetal heart and accumulates in neonatal and postnatal stages. No difference can be demonstrated between Tnl mRNAs present in fetal and postnatal heart by RNAase protection assays. The fetal and neonatal, but not the adult heart, contain significant amounts of slow skeletal Tnl transcripts, detected by oligonucleotide probes specific for the 5′- and 3′-untranslated regions of slow skeletal Tnl mRNA. In situ hybridization studies show that cardiac and slow skeletal Tnl mRNAs are coexpressed in the rat heart from embryonic day 11 throughout fetal and perinatal stages. Changes in troponin isoform expression during development may be responsible for the difference in calcium sensitivity and in the response to β-adrenergic stimulation between fetal and adult heart.
The Msh-like homeobox genes define domains in the developing vertebrate eye
Authors: Monaghan A. Paula , Davidson Duncan R. , Sime Christina , Graham Elizabeth , Baldock Richard , Bhattacharya Shomi S. , Hill Robert E.
Published: April 2021

The mouse Hox-7.1 gene has previously been shown to be related to the Drosophila Msh homeobox-containing gene. Here we report the isolation of a new member of this family which resides at an unlinked chromosomal location and has been designated Hox-8.1. Both Hox-7.1 and Hox-8.1 are expressed in the mouse embryo during the early stages of eye development in a distinct spatial and temporal relationship. Hox-8.1 is expressed in the surface ectoderm and in the optic vesicle before invagination occurs in regions corresponding to the prospective corneal epithelium and neural retina, respectively. Hox-7.1 is expressed after formation of the optic cup, marking the domain that will give rise to the ciliary body. The activity of these genes indicates that the inner layer of the optic cup is differentiated into three distinct compartments before overt cellular differentiation occurs. Our results suggest that these genes are involved in defining the region that gives rise to the inner layer of the optic cup and in patterning this tissue to define the iris, ciliary body and retina.
Expression of nerve growth factor (NGF) receptors in the developing inner ear of chick and rat
Authors: Bartheld Christopher S. Von , Patterson Susan L. , Heuer Josef G. , Wheeler Esther F. , Bothwell Mark , Rubel Edwin W.
Published: April 2021

The expression of nerve growth factor receptors (NGFRs) was studied in the developing inner ear with in situ hybridization in chick embryos and with immunocytochemistry in rat embryos to determine sites of possible functions of NGF or NGF-like molecules in inner ear development. NGFR expression in the chick otocyst and acoustic ganglion is compared with epithelial differentiation and the onset of afferent innervation as determined with fluorescent carbocyanine tracers.

In the inner ear of the chick embryo, NGFR mRNA expression shows an alternating pattern in mesenchymal and epithelial tissues. NGFR mRNA is heavily expressed in the mesenchyme surrounding the otocyst (E2 –3), ceases at E3 –5, and reappears in a thin layer of mesenchymal cells surrounding the membraneous epithelia (E5 –13). In the otocyst epithelium, NGFR mRNA expression develops in one anterior and one posterior focus at E3 –4.5. NGFR mRNA is expressed in the primordia of the ampullary cristae (E5 –7) and possibly the anlage of the utricle; label transiently concentrates in the planum semilunatum of the cristae ampullares and in superior portions of the semicircular canals at E9, but is not seen in differentiating hair cells. In the acoustic ganglion, NGFR mRNA expression begins at E4; at the same time, the first peripheral acoustic nerve processes penetrate the otic epithelium (E4 –4.5). The acoustic ganglia remain weakly NGFR mRNA-labeled in the posthatch animal.

In the rat embryo, NGFR immunoreactivity is present in the auditory placode at E9, in the periotic mesenchyme at E9 –10, and in the medial half of the otocyst at E10 –11. At E12, epithelial NGFR expression becomes restricted anteriorly and posteriorly in a pattern similar to that of the chick otocyst and ceases at E13. NGFR immunoreactivity appears transiently in pillar cells of the cochlea in the third week of gestation. NGFR and NGFR mRNA is expressed after Ell in the acoustic ganglia. While NGFR transcripts are expressed in the cochlear ganglion cell bodies, NGFR protein becomes restricted to neuronal processes by the third week of gestation. The vestibular, but not the cochlear (spiral) ganglia remain NGFR-labeled in the adult rat.

Onset of NGFR mRNA expression in the acoustic ganglion during the period of afferent fiber ingrowth into the otocyst epithelium is consistent with the hypothesis that NGF-like molecules may have a neurotrophic function for acoustic ganglion cells. Transient expression of NGFRs in secretory cells of the vestibular endorgan and pillar cells in the organ of Corti implicate a role for neurotrophins in the differentiation of these epithelial cell types.
Nuclear translocation of fibroblast growth factor during Xenopus mesoderm induction
Authors: Shiurba Robert A. , Jing Naihe , Sakakura Teruyo , Godsave Susan F.
Published: April 2021

Mesoderm induction, the earliest inductive cell–cell interaction in vertebrate embryogenesis, is thought to be mediated by polypeptide growth factors including fibroblast growth factor (FGF). Here we present an immunocytochemical analysis of FGF during mesoderm induction in Xenopus laevis. Antibodies to both basic and acidic FGF were immunoreactive with oocytes and early embryos. Immunostaining was predominantly intracellular and was concentrated in the marginal zone and vegetal pole throughout cleavage and blastula stages. In addition, basic FGF (bFGF) antibodies showed intense nuclear staining in these regions, at and following the mid-blastula transition, when embryonic transcription begins. Acidic FGF (aFGF) also appeared in some nuclei at these stages. Taken together the evidence suggests that FGF is prepositioned in mesoderm-forming regions and is actively involved in mesoderm induction in vivo.
The cellular environment controls the expression of engrailed-like protein in the cranial neuroepithelium of quail-chick chimeric embryos
Authors: Gardner Charles A. , Barald Kate F.
Published: April 2021

We have previously shown that one of two chicken engrailed-like genes, chick En-2, is expressed in a restricted region of the early chick embryo brain: the mes/metencephalon (Gardner et al. 1988). In this study, we examine the role of the cellular environment in regulation of engrailed-like (En) protein expression in quail-chick chimeric embryos. Two types of transplant surgery were performed at the 9-15 somite stage to produce chimeric embryos. In the first, the mid-mesencephalic vesicle or caudal mesencephalic vesicle alar plate (which is En protein-positive) was transplanted from a quail embryo into an En protein-negative region of chick neuroepithelium, the prosencephalon (mMP and cMP grafts, respectively). In the second reciprocal surgery, prosencephalic alar plate which is En protein-negative, was transplanted into the En protein-positive mesencephalic vesicle (PM grafts). A polyclonal antiserum, aEnhb-1, which recognizes chick

En proteins (Davis et al. 1991) was used to identify En-positive cells 48 h after surgery. In mMP embryos, 71 % of integrated grafts had lost En expression (n=17). In contrast, in cMP grafts, 93% of integrated grafts continued to stain with the antiserum (n=14). In addition, in 86 % of these embryos, the graft induced adjacent chick host diencephalic cells to become En protein-positive as well. All PM grafts contained aEnhb-1 -positive cells; such cells never expressed this protein in their normal environment. These early changes in En protein expression correlate well with the morphological changes observed in similar graft surgeries assayed later in development. Thus, our results are consistent with the hypothesis that En genes play a role in the regionalization of the early cranial neuroepithelium.