bluuebunny/crossref_metadata_2025 huggingface dataset:
Epithelial-mesenchymal interactions are required for msx 1 and msx 2 gene expression in the developing murine molar tooth
Authors: Jowett Adrian K. , Vainio Seppo , Ferguson Mark W. J. , Sharpe Paul T. , Thesleff Irma
Published: April 2021

Duplication of the msh-like homeobox gene of Drosophila may be related to the evolution of the vertebrate head. The murine homologues of this gene, msx 1 and msx 2 are expressed in the developing craniofacial complex including the branchial arches, especially in regions of epithelial-mesenchymal organogenesis including the developing tooth.

By performing in vitro recombination experiments using homochronic dental and non-dental epithelial and mesenchymal tissues from E10 to E18 mouse embryos, we have found that the maintenance of homeobox gene expression in the tooth is dependent upon tissue interactions. In homotypic recombinants, dental-type tissue interactions occur, leading to expression of both genes in a manner similar to that seen during in vivo development. msx 1 is expressed exclusively in mesenchyme, both in the dental papilla and follicle. msx 2 is expressed in the dental epithelium and only in the mesenchyme of the dental papilla. In heterotypic recombinants, the dental epithelium is able to induce msx 1 expression in non-dental mesenchyme, this potential being lost at the bell stage. In these recombinants msx 2 was induced by presumptive dental epithelium prior to the bud stage but not thereafter. The expression of msx 1 and msx 2 in dental mesenchyme requires the presence of epithelium until the early bell stage. However, whereas non-dental, oral epithelium is capable of maintaining expression of msx 1 in dental mesenchyme throughout tooth development, induction of msx 2 was temporally restricted suggesting regulation by a specific epithelial-mesenchymal interaction related to the inductive events of tooth formation.

msx 1 and msx 2, as putative transcription factors, may play a role in regulating the expression of other genes during tooth formation. We conclude that expression of msx 1 in jaw mesenchyme requires a non-specific epithelial signal, whereas msx 2 expression in either epithelium or mesenchyme requires reciprocal interactions between specialized dental cell populations.
Ontogeny of hyaluronan secretion during early mouse development
Authors: Brown Jeremy J. G. , Papaioannou Virginia E.
Published: April 2021

The ontogeny of hyaluronan (HA) secretion during early mouse embryogenesis has been investigated using a biotin-labelled HA-binding complex from cartilage proteoglycan. HA is first secreted by visceral endoderm cells of the early egg cylinder on day 5.5 post coitum (p.c.), predominantly into the expanding yolk cavity. On day 6.5 p.c., HA is present in both the yolk and proamniotic cavities, but pericellular staining is restricted to the visceral endoderm and a population of embryonic ectoderm cells at the antimesometrial end of the proamniotic cavity. By the primitive streak stage, HA is secreted into the ectoplacental, exocoelomic, amniotic and yolk cavities, whilst the only cells exhibiting pericellular staining are those of the embryonic and extraembryonic mesoderm, including the allantois. Comparisons of HA-staining patterns of cultured whole blastocysts, microdissected trophectoderm fragments and immuno-surgically isolated inner cell masses, revealed no trophoblast-associated HA secretion during outgrowth in vitro but significant synthetic activity by the endodermal derivatives of differentiating inner cell masses. To identify the cell lineages responsible for secretion of HA into the embryonic cavities and to investigate the origin of the HA observed around migrating mesoderm cells, day 7.5 p.c. primitive streak stage conceptuses were dis-sected into their various embryonic and extraembryonic cell lineages. HA secretion was observed after shortterm suspension culture of mesoderm, embryonic ectoderm and embryonic endoderm, but was undetectable in fragments of ectoplacental cone, parietal yolk sac (primary giant trophoblast and parietal endoderm), extraembryonic ectoderm or extraembryonic endoderm. The level of synthesis by the HA-positive tissues was markedly enhanced by culture in medium containing serum, compared with that obtained following culture in medium supplemented with a defined serum substitute containing insulin, transferrin, selenous acid and linoleic acid. This suggests that additional growth factors, present in serum but absent from the serum substitute, are required for optimal HA synthesis by the HA-secreting tissues in vitro, and probably also in vivo. The implications of these events for implantation and the development of peri- and early post-implantation mouse embryos are discussed, and a new role for HA in the initial formation and expansion of the embryonic cavities is proposed.
Conservation of regulatory elements controlling hairy pair-rule stripe formation
Authors: Langeland James A. , Carroll Sean B.
Published: April 2021

The hairy (h) gene is one of two pair-rule loci whose striped expression is directly regulated by combinations of gap proteins acting through discrete upstream regulatory fragments, which span several kilobases. We have undertaken a comparative study of the molecular biology of h pair-rule expression in order to identify conserved elements in this complex regulatory system, which should provide important clues concerning the mechanism of stripe formation. A molecular comparison of the h locus in Drosophila virilis and Drosophila melanogaster reveals a conserved overall arrangement of the upstream regulatory elements that control individual pair-rule stripes. We demonstrate that upstream fragments from D. virilis will direct the proper expression of stripes in D. melanogaster, indicating that these are true functional homologs of the stripe-producing D. melanogaster regulatory elements, and that the network of trans-acting proteins that act upon these regulatory elements is highly conserved. We also demonstrate that the spatial relationships between specific h stripes and selected gap proteins are highly conserved. We find several tracts of extensive nucleotide sequence conservation within homologous stripe-specific regulatory fragments, which have facilitated the identification of functional subelements within the D. melanogaster regulatory fragment for h stripe 5. Some of the conserved nulceotide tracts within this regulatory fragment contain consensus binding sites for potential trans-regulatory (gap and other) proteins, while many appear devoid of known binding sites. This comparative approach, coupled with the analysis of reporter gene expression in gap mutant embryos suggests that the Kr and gt proteins establish the anterior and posterior borders of h stripe 5, respectively, through spatial repression. Other, as yet unidentified, proteins are certain to play a role in stripe activation, presumably acting through other conserved sequence tracts.
RNA and protein localisations of TGFβ2 in the early mouse embryo suggest an involvement in cardiac development
Authors: Dickson Marion C. , Slager Hans G. , Duffie Elizabeth , Mummery Christine L. , Akhurst Rosemary J.
Published: April 2021

We have performed a detailed analysis of the localisations of RNAs for TGFβ2 and β3, and of TGFβ2 protein in mouse embryos from 6.5 to 9.5 days post coitum, using in situ hybridisation and immunohistochemistry on serial sections, and whole-mount in situ hybridisation to complete embryos. TGFβ3 RNA was not seen in any of the tissue sections, but very low levels of the RNA were seen by whole-mount in situ hybridisation around the outflow tract of the heart at 8.5 days post coitum. TGFβ2 RNA is expressed at high levels in all cells with the potential to differentiate into cardiomyocytes. Additionally, the foregut endoderm, juxtaposed to the heart, and the neuroepithelium at the rostral extremity of the foregut, express very high levels of TGFβ2 RNA, between 8.5 and 9.5 days post coitum. As cardiomyogenesis proceeds, TGFβ2 RNA levels diminishes within the myocytes, with a concomitant increase in staining for TGFβ2 protein. TGFβ2 protein staining of cardiomyocytes persists throughout development and in the adult, in the absence of detectable levels of the corresponding RNA. Superimposed upon this myocardial pattern of expression, there is an upregulation of TGFβ2 RNA in the myocardium of the outflow tract and atrioventricular canal between 8.5 and 9.5 days post coitum, which returns to low levels by 11.5 days post coitum. The results are discussed in terms of a potential role of TGFβ2 in controlling cardiomyogenesis and in induc-tive interactions leading to cardiac cushion tissue formation.
Actin depolymerisation induces process formation on MAP2-transfected non-neuronal cells
Authors: Edson Kathryn , Weisshaar Brigitte , Matus Andrew
Published: April 2021

We have previously shown that microtubules in non-neuronal cells form long, stable bundles after transfection with the embryonic neuronal microtubule-associated protein MAP2c. In this study, we found that treating MAP2c-transfected cells with the actin depolymerising drug cytochalasin B led to the outgrowth of microtubule-containing processes from the cell surface. This effect was specific to MAP2c and did not occur in untransfected cells whose microtubules had been stabilised by treatment with taxol. The outgrowth and retraction of these processes during repeated cycles of cytochalasin addition and removal was followed by video time-lapse microscopy and was suggestive of a physical interaction between compressive forces exerted by the MAP2c-stabilised microtubule bundles and tensile forces originating in the cortical actin network. We suggest that MAP2c confers three properties on cellular microtubules that are essential for process out-growth: stability, bundling and stiffness. The latter probably arises from the linking together of neighbouring tubulin subunits by three closely spaced tubulin-binding motifs in the MAP2 molecule that limits their motion relative to one another and thus reduces the flexibility of the polymer. Similar multimeric tubulin-binding domains in other proteins of the MAP2 class, including tau in axons and MAP4 in glial cells, may play the same role in the development and support of asymmetric cell morphology. Axial bundles of microtubules are found in growing neurites but not in growth cones, suggesting that the regulated expression of these MAP-induced properties makes an important contribution to the establishment of a stable process behind the advancing growth cone.
Activins are expressed in preimplantation mouse embryos and in ES and EC cells and are regulated on their differentiation
Authors: Albano Rodolpho M. , Groome Nigel , Smith J. C.
Published: April 2021

Members of the activin family have been suggested to act as mesoderm-inducing factors during early amphib- ian development. Little is known, however, about meso- derm formation in the mammalian embryo, and as one approach to investigating this we have studied activin expression during early mouse development.

Activins are homo- or heterodimers of the βA or βB subunits of inhibin, itself a heterodimer consisting of one of the β subunits together with an α subunit. Our results indicate that the oocyte contains mRNA encoding all three subunits, and antibody staining demonstrates the presence of both α and β protein chains. From the fer- tilized egg stage onwards, α subunit protein cannot be detected, so the presence of β subunits reflects the pres- ence of activin rather than inhibin.

Maternal levels of activin protein decline during early cleavage stages but increase, presumably due to zygotic transcription (see below), in the compacted morula. By 3.5 days, only the inner cell mass (ICM) cells of the blas- tocyst express activin, but at 4.5 days the situation is reversed; activin expression is confined to the trophec- toderm. Using reverse transcription-PCR, neither βA nor βB mRNA was detectable at the two-cell stage but transcripts encoding both subunits were detectable at the morula stage, with βB mRNA persisting into the blastocyst.

We have also analyzed activin and inhibin expression in ES and EC cells. Consistent with the observation that activins are expressed in the ICM of 3.5-day blastocysts, we find high levels of βA and βB mRNA in all eight ES cell lines tested. F9 EC cells express only activin βB, together with low levels of the inhibin αchain. When ES and EC cells are induced to differentiate, levels of activin fall dramatically. These results are consistent with a role for activins in mesoderm formation and other steps of early mouse development.
Somite pattern regulation in the avian segmental plate mesoderm
Authors: Packard David S. , Zheng Rui-Zhen , Turner David C.
Published: April 2021

Previous experimental evidence suggested that the avian segmental pattern is already specified in the apparently unsegmented paraxial (segmental plate) mesoderm, but is susceptible to modification and reconstitution. We explored capacities of embryos to alter the specified pat-tern and restore it after disruption. In control experi-ments, right segmental plates of chicken or Japanese quail embryos were removed after about 48 hours of incubation and immediately replaced. Hensen’s node and the primitive streak were removed to halt further segmental plate formation and the embryos were cul-tured for about 18 hours more. Somite numbers on the operated and unoperated sides were nearly identical (r=0.904, n=31, P<0.001); no species differences were noted. Right segmental plates of chicken hosts were then replaced with right segmental plates from quail donors. The numbers of somites formed by donors and grafts were not significantly correlated (r=0.305, n=30, P<0.1), but the correlation between the graft and the host’s unoperated side was significant (r=0.666, n=30, P<0.001). The host is therefore able to alter the number of somites formed by the graft to one more compatible with the host’s pattern. From orthostereoscopic recon-tructions, it appeared that the location and size of somites could also be adjusted by the host. Similar results were obtained for tandem grafts of anterior halves of segmental plates and for grafts of minced seg-mental plates, though in the latter case contact with tissues near the midline was necessary for somite for-mation.
Expression of the Drosophila optomotor-blind gene transcript in neuronal and glial cells of the developing nervous system
Authors: Poeck Burkhard , Hofbauer Alois , Pflugfelder Gert O.
Published: April 2021

Mutations in the complex gene locus optomotor-blind (omb) can lead to defects in the development of both the optic lobes and external features of the adult fly. We describe here the expression of omb in the developing and adult nervous system using in situ hybridization. During embryogenesis, omb expression is first observed in the optic lobe anlagen. It later expands to a larger part of the developing larval brain and to the gnathal lobes. Cells in the ventral and peripheral nervous systems begin to express omb after completion of germ band extension. Later in embryonic development, expression declines and only persists in the antennomaxillary complex and in part of the brain hemispheres. During the larval and pupal stages, omb expression in the brain is confined to the developing optic lobes and contiguous regions of the central brain. At these stages, only a few cells show expression in the ventral ganglion. In the eye imaginal disc, transcript accumulation is most conspicuous in a group of presumptive glia precursor cells posterior to the morphogenetic furrow and in the optic stalk. In the adult brain, expression is prominent in several regions of the optic lobe cortex and along the border between central brain and optic lobes. In the mutation In(1)ombH31, 40 kb of regulatory DNA, downstream from the transcription unit, are removed from the omb gene. In(1)ombH31 is characterized by the lack of a set of giant interneurons from the lobula plate of the adult optic lobes. We find that, already during embryogenesis, there is a drastic difference between wild type and In(1)ombH31 in the level of the omb transcript in the optic lobe primordia. The adult mutant phenotype may thus be caused by omb misexpression during embryonic development.
Altered Epidermal Growth Factor-like sequences provide evidence for a role of Notch as a receptor in cell fate decisions
Authors: Heitzler Pascal , Simpson Pat
Published: April 2021

In Drosophila each neural precursor is chosen from a group of cells through cell interactions mediated by Notch and Delta which may function as receptor and ligand (signal), respectively, in a lateral signalling path-way. The cells of a group are equipotential and express both Notch and Delta. Hyperactive mutant Notch molecules, (Abruptex), probably have an enhanced affinity for the ligand. When adjacent to wild-type cells, cells bearing the Abruptex proteins are unable to produce the signal. It is suggested that in addition to the binding of Notch molecules on one cell to the Delta molecules of opposing cells, the Notch and Delta proteins on the surface of the same cell may interact. Binding between a cell’s own Notch and Delta molecules would alter the availability of these proteins to interact with their counterparts on adjacent cells.
Appearance and evolution of calcium currents and contraction during the early post-fusional stages of rat skeletal muscle cells developing in primary culture
Authors: Cognard Christian , Constantin Bruno , Rivet-Bastide Michèle , Imbert Nathalie , Besse Colette , Raymond Guy
Published: April 2021

Primary cultures from enzymatically dissociated satellite cells of newborn rat skeletal muscles enabled developmental in vitro studies of mechanical and electrical properties during the first steps of myogenesis. The present work focused on the appearance, evolution and roles of two types of calcium currents (ICa,T and ICa,L) and of depolarization-induced contractile activity during the early stages of muscle cell development in primary culture. Prefusional mononucleated cells (myoblasts), young myotubes of 1 day (with less than 10 nuclei) or 2-3 days (more than 9 nuclei) and myoballs from 4-6, 7-9, 10-12 and 13-16 days cultures were patchclamped (whole-cell configuration), and calcium currents and contraction simultaneously recorded. Sodium but not calcium currents could be recorded at the myoblast stage. In young myotubes (1 day), ICa,L was present with high incidence as compared to ICa,T, which was poorly expressed. Contractile responses appeared at the next stage (2-3 days) while the occurrence of ICa,T progressively increased. This developmental evolution of the calcium currents and contraction expression was accompanied by some changes in their characteristics: the ICa,T/ICa,L amplitudes ratio progressively increased and the time-to-peak of contraction progressively decreased with the age of myoballs. Physiological functions for calcium currents in developing muscle are suggested and discussed: ICa,T, which is transiently expressed, could be involved in the pacemaker-like activity while ICa,L could serve as an early contraction triggering mechanism and/or initially to fill and then to maintain the intracellular calcium stores.
Intrinsic and extrinsic factors in the mechanism of neurulation: effect of curvature of the body axis on closure of the posterior neuropore
Authors: Straaten H.W.M. van , Hekking J.W.M. , Consten C. , Copp A.J.
Published: April 2021

Neurulation has been suggested to involve both factors intrinsic and extrinsic to the neuroepithelium. In the curly tail (ct) mutant mouse embryo, final closure of the posterior neuropore is delayed to varying extents resulting in neural tube defects. Evidence was presented recently (Brook et al., 1991, Development 113, 671-678) to suggest that enhanced ventral curvature of the caudal region is responsible for the neurulation defect, which probably originates from an abnormally reduced rate of cell proliferation affecting the hindgut endoderm and notochord, but not the neuroepithelium (Copp et al., 1988, Development 104, 285-295). This axial curvature probably generates a mechanical stress on the posterior neuropore, opposing normal closure. We predicted, therefore, that the ct/ct posterior neuropore should be capable of normal closure if the neuroepithelium is isolated from its adjacent tissues. This prediction was tested by in vitro culture of ct/ct posterior neuropore regions, isolated by a cut caudal to the 5th from last somite. In experimental explants, the neuroepithelium of the posterior neuropore, together with the contiguous portion of the neural tube, were separated mechanically from all adjacent non-neural tissues. The posterior neuropore closed in these explants at a similar rate to isolated posterior neuropore regions of non-mutant embryos. By contrast, control ct/ct explants, in which the caudal region was isolated but the neuroepithelium was left attached to adjacent tissues, showed delayed neurulation. To examine further the idea that axial curvature may be a general mechanism regulating neurulation, we cultured chick embryos on curved substrata in vitro. Slight curvature of the body axis (maximally 1° per mm axial length), of either concave or convex nature, resulted in delay of posterior neuropore closure in the chick embryo. Both incidence and extent of closure delay correlated with the degree of curvature that was imposed. We propose that during normal embryogenesis the rate of neurulation is related to the angle of axial curvature, such that experimental alterations in curvature will have differing effects (either enhancement or delay of closure) depending on the angle of curvature at which neurulation normally occurs in a given species, or at a given level of the body axis.
Involvement of leukemia inhibitory factor and nerve growth factor in the development of dorsal root ganglion neurons
Authors: Murphy Mark , Reid Kate , Brown Melissa A. , Bartlett Perry F.
Published: April 2021

Leukemia inhibitory factor (LIF) was recently shown to stimulate the generation of sensory neurons from the murine neural crest in vitro. Here, we examine the respective activities of LIF and nerve growth factor (NGF) throughout the embryonic development of sensory neurons in dorsal root ganglia (DRG) and neural crest. In cultures of embryonic day 12 (E12) DRG, which contain sensory neuron precursor cells, a combination of both LIF and NGF are required for the differentiation of mature sensory neurons from their neurofilament negative (NF) precursors. The primary differentiation step from NF cell to NF+ immature neuron is promoted by LIF, whereas the survival and further maturation of the newly differentiated neurons depends on NGF. In cultures of sensory neurons isolated at the time of target innervation (E14 and E15 DRG), the survival of the majority of the neurons is dependent on NGF. However, LIF acts as a survival agent for a discrete population of NGF non-responsive neurons. From E16, the number of neurons maintained by LIF increases to >90% by birth. Consistent with the in vitro observations, LIF mRNA could be detected at early developmental stages (E12-E13), within the spinal column and DRG as well as the limbs and, later (after E15), in areas of sensory innervation (skin, limbs, feet and gut). This supports the idea that LIF, as well as NGF, may regulate sensory development in vivo.
Development and organization of glial cells in the peripheral nervous system of Drosophila melanogaster
Authors: Giangrande Angela , Murray Marjorie A. , Palka John
Published: April 2021

We have used enhancer trap lines as markers to recognize glial cells in the wing peripheral nervous system of Drosophila melanogaster. Their characterization has enabled us to define certain features of glial differentiation and organization. In order to ask whether glial cells originate within the disc or whether they migrate to the wing nerves from the central nervous system, we used two approaches. In cultured wing discs from glialspecific lines, peripheral glial precursors are already present within the imaginal tissue during the third larval stage. Glial cells differentiate on a wing nerve even in mutants in which that nerve does not connect to the central nervous system. To assess whether peripheral glial cells originate from ectoderm or from mesoderm, we cultured discs from which the mesodermally derived adepithelial cells had been removed. Our findings indicate that peripheral glial cells originate from ectodermally derived cells. As has already been shown for the embryonic central nervous system, gliogenesis in the periphery is an early event during adult development: glial cells, or their precursors, are already present at stages when neurons are still differentiating. Finally, our results also suggest that peripheral glial cells may not display a stereotyped arrangement.
Two enhancer regions in the mouse En-2 locus direct expression to the mid/hindbrain region and mandibular myoblasts
Authors: Logan C. , Khoo W. K. , Cado D. , Joyner A. L.
Published: April 2021

An En-2/lacZ gene fusion containing 9.5 kb of En-2 genomic DNA was capable of directing lacZ expression in an En-2-specific manner both temporally and spatially during embryogenesis and in the adult. lacZ expression was confined in the embryo to cells within the mid/hindbrain and mandibular arch regions and in the adult to cells of the molecular and granular layers of the cerebellum, and within the pons and colliculi regions. Interestingly, in the adult, transgene expression patterns within the cerebellum in two lines appeared to mark distinct anterior-posterior compartments. Analysis of the expression pattern of this transgene, in fetal and adult mice lacking a functional En-2 protein, provided evidence that the En-2 gene in mouse is not autoregulated. Deletion analysis of the En-2 genomic region and the use of a heterologous promoter identified two enhancer-containing regions of 1.5 and 1.0 kb in length, 5 of the transcribed sequences, which independently directed expression in the embryo to either the mid/hindbrain region or mandibular myoblasts, respectively. The 1.5 kb fragment contains the most anterior neural enhancer and the 1.0 kb fragment, the earliest myogenic enhancer thus far characterized. These En-2-specific regulatory regions can now be used in a biochemical analysis to identify proteins important in anterior-posterior patterning of the vertebrate CNS and in the specification of muscle identity as well as in a mutational analysis to direct expression of other developmentally important genes to these regions.
Expression of β1 integrin complexes on the surface of unfertilized mouse oocyte
Authors: Tarone Guido , Russo Mario A. , Hirsch Emilio , Odorisio Teresa , Altruda Fiorella , Silengo Lorenzo , Siracusa Gregorio
Published: April 2021

Integrins are a family of cell surface receptors that mediate cell-cell and cell-matrix interactions in a variety of different cellular systems. Here we show that unfertilized mouse oocytes express β1 class integrins both at mRNA and protein levels. Using the reverse transcription polymerase chain reaction and oligonucleotide primers based on the DNA sequence of mouse integrins, the RNA transcripts for the β1, α5 and α6 subunits were detected in unfertilized oocytes. The expression of the mRNAs is paralleled by the expression of the corresponding proteins, in fact, theα 5/ β1 and the α6/ β1 complexes can be immunoprecipitated with specific antibodies from 125I-surface-labeled oocytes. Using subunit-specific antibodies we also demonstrate the presence of the α3/ β1 at the oocyte surface but α1, α2, α4 or αV subunits were not detectable. Since the mouse 3 DNA sequence is not available, we have not tested for the corresponding transcript. Integrin subunits α6 and β1 were differently distributed on the oocyte surface, as visualized by immunofluorescence staining and by immunoelectron microscopy. α6 antigen was mainly confined to the microvillous area of the oocyte surface, while β1 was more homogeneously distributed over the whole oolemma. These data demonstrate for the first time the expression of three β1 integrin complexes in unfertilized mouse oocytes. Such proteins may have a role in sperm-egg interaction or during very early steps of embryogenesis.
A paternally imprinted X chromosome retards the development of the early mouse embryo
Authors: Thornhill Alan R. , Burgoyne Paul S.
Published: April 2021

It has previously been shown that XO mouse fetuses with a paternally derived X chromosome (Xp) are developmentally retarded and consequently smaller than their XX sibs, and that XX fetuses are retarded when compared with their XY sibs. The genetic basis for these early XO-XX and XX-XY differences has not been determined. Here we show that 10.5 day post coitum XO mouse fetuses with a maternal X chromosome, rather than being smaller than their XX sibs, are significantly larger and equivalent in size to their XY sibs. Thus the retardation of XpO fetuses must be due to an effect of their paternally derived X chromosome. The finding that XmO fetuses are larger than XX fetuses and equivalent in size to XY fetuses suggests that the XX-XY difference present at 10.5 days post coitum is largely due to the difference in X chromosome constitution rather than to a Y chromosome effect.
Clonal heterogeneity in the germinal zone of the developing rat telencephalon
Authors: Acklin Susan E. , Kooy Derek van der
Published: April 2021

A double-labeling technique, combining retroviral tagging of individual cell lines (one clone per brain hemisphere) with the simultaneous [3H]thymidine-labeling of dividing cells in S phase, was used to study proliferation characteristics of individual precursor cell lines in the germinal zone of the developing rat forebrain. The cortical germinal zone was found to be segregated into three spatially distinct horizontal populations of precursor cell lineages, which differed in cell cycle kinetics, amount of cell death, and synchronous versus asynchronous mode of proliferation. The striatal germinal zone demonstrated a similar heterogeneity in the cell cycle characteristics of proliferating clones, but did not show nearly as distinct a spatial segregation of these different populations. The results demonstrate the clonal heterogeneity among precursor populations in the telencephalon and the differential spatial organization of the cortical and the striatal germinal zones. This germinal zone heterogeneity may predict some of the differences found among cellular phenotypes in the adult forebrain.
A Xenopus homeobox gene defines dorsal-ventral domains in the developing brain
Authors: Saha Margaret S. , Michel Rosana B. , Gulding Kathryn M. , Grainger Robert M.
Published: April 2021

One of the distinguishing features of vertebrate development is the elaboration of the anterior neural plate into forebrain and midbrain, yet little is known about the early tissue interactions that regulate pattern formation in this region or the genes that mediate these interactions. As an initial step toward analyzing the process of regionalization in the anterior-most region of the brain, we have screened an anterior neural cDNA library for homeobox clones and have identified one which we have called XeNK-2 (Xenopus NK-2) because of its homology to the NK-2 family of homeobox genes. From neurula stages, when XeNK-2 is first detectable, through hatching stages, XeNK-2 mRNA is expressed primarily in the anterior region of the brain. By swimming tadpole stages, XeNK-2 expression resolves into a set of bands positioned at the forebrain-midbrain and the midbrain-hindbrain boundaries, after which XeNK2 transcripts are no longer detectable. In addition to localized expression along the anterior-posterior axis, XeNK-2 may also play a role in the process of regionalization along the dorsal-ventral axis of the developing brain. At all stages examined, XeNK-2 mRNA is restricted to a pair of stripes that are bilaterally symmetrical in the ventral-lateral region of the brain. To begin to identify the tissue interactions that are required for the proper spatial and temporal localization of XeNK-2, we have performed a series of explant experiments. Consistent with earlier work showing that the A/P axis is not fixed at mid-gastrula stages, we show that XeNK-2 expression is activated when assayed in gastrula stage explants taken from any region along the entire A/P axis and that the tissue interactions necessary to localize XeNK-2 along the A/P axis are not completed until later neurula stages.
The development of indirect flight muscle innervation in Drosophila melanogaster
Authors: Fernandes Joyce , VijayRaghavan K.
Published: April 2021

We have examined the development of innervation to the indirect flight muscles of Drosophila. During metamorphosis, the larval intersegmental nerve of the mesothorax is remodelled to innervate the dorsal longitudinal muscles and two of the dorsoventral muscles. Another modified larval nerve innervates the remaining dorsoventral muscle. The dorsal longitudinal muscles develop using modified larval muscles as templates while dorsoventral muscles develop without the use of such templates. The development of innervation to the two groups of indirect flight muscles differs in spatial and temporal patterns, which may reflect the different ways in which these muscles develop. The identification of myoblasts associated with thoracic nerves during larval life and the association of migrating myoblasts with nerves during metamorphosis indicate the existence of nerve-muscle interactions during indirect flight muscle development. In addition, the developing pattern of axonal branching suggests a role for the target muscles in respecifying neuromuscular junctions during metamorphosis.
Retinoic acid receptors and cellular retinoid binding proteins III. Their differential transcript distribution during mouse nervous system development
Authors: Ruberte Esther , Friederich Valérie , Chambon Pierre , Morriss-Kay Gillian
Published: April 2021

We have studied the transcript distribution of the retinoic acid receptors (RARs) and the cytoplasmic retinoid binding proteins during embryonic development of the mouse nervous system. Of the three retinoic acid receptors, only RAR-γ was not expressed in developing neural structures. RAR-β and RAR-α both showed rostral limits of expression in the medulla oblongata equivalent to their patterns of expression in the neuroepithelium of the early hindbrain neural tube. Within their expression domains in the spinal cord and brain, RAR-α was ubiquitously expressed, whereas RAR-β transcripts showed very specific patterns of expression, suggesting that this receptor is involved in mediating retinoic acid-induced gene expression in relation to the development of specific neural structures or pathways.

The cytoplasmic binding proteins, cellular retinoic acid binding proteins type I and II (CRABP I and CRABP II) and cellular retinol binding protein type I (CRBP I), were widely distributed in developing neural structures. Their differential spatiotemporal patterns of expression suggest that fine regional control of availability of retinoic acid (RA) to the nuclear receptors plays an important role in organization and differentiation of the nervous system. For instance, expression of CRABP I in the migrating cells that give rise to the olivary and pontine nuclei, which develop abnormally in conditions of retinoid excess, is consistent with observations from a variety of other systems indicating that CRABP I limits the access of RA to the nuclear receptors in normal physiological conditions. Similarly, expression of CRBP I in the choroid plexuses, which develop abnormally in conditions of vitamin A deficiency, is consistent with observations indicating that this binding protein mediates the synthesis of RA in tissues requiring high levels of RA for their normal developmental programme. RAR-β and CRABP II, which are both RA-inducible, were coexpressed with CRBP I in the choroid plexus and in many other sites, perhaps reflecting the fact that all three genes are RAinducible. The function of CRABP II is not well understood; its domains of expression showed overlaps with both CRABP I and CRBP I.
Epithelial cell proliferation and islet neogenesis in IFN-g transgenic mice
Authors: Gu Danling , Sarvetnick Nora
Published: April 2021

We have identified a model system for the study of pancreatic islet development and regeneration in transgenic mice bearing the interferon-gamma (IFN-g) gene expressed in the pancreatic islets. Previous studies showed that the locally produced IFN-g causes lymphocyte infiltration and islet cell destruction. Here we demonstrate that new islet cells are formed continuously from duct cells as evidenced by (1) the dramatic proliferation of duct cells, (2) the appearance of primitive cells and (3) their subsequent differentiation to endocrine cells. The IFN-g induced islet neogenesis is similar to embryonic islet morphogenesis and offers a model system for studying factors modulating islet development. Additionally, the duct cells occasionally transdifferentiate to gastrointestinal-like cell types and hepatocytes. These results underscore the lymphokine’s ability to initiate a complex ‘transdifferentiation’ pathway, providing a window for understanding lineage interrelationships within a terminally differentiated structure.
Differential expression of multiple fork head related genes during gastrulation and axial pattern formation in the mouse embryo
Authors: Sasaki Hiroshi , Hogan Brigid L. M.
Published: April 2021

Four genes encoding fork-head-domain-containing proteins (FD genes) have been isolated from a mouse 8.5 days post coitum (p.c.) embryo cDNA library. Two are mouse homologues of rat HNF-3β and HNF-3α. The other two are novel and have been named MF-1 and MF-2 (for mesoderm/mesenchyme fork head). Wholemount in situ hybridization of embryos between 6.5 and 9.5 days p. c. shows that each gene has a unique expression pattern. HNF-3β is expressed in the node, notochord, floor plate and gut, while HNF-3α is mainly in the definitive endoderm and gut, but also in the floor plate of the midbrain. These results suggest that HNF3β and HNF-3α, in addition to their known functions as transcriptional activators in adult liver, play a role in body axis formation, neural tube patterning and definitive endoderm formation during gastrulation. MF-1 RNA is present in non-notochordal mesoderm, and in neural-crest-derived head mesenchyme, while MF-2 transcripts are found in the sclerotomes of the somites and in head mesenchyme, including that from neural crest. Studies on gastrulation stage embryos suggest that the early temporal and spatial patterns of HNF-3β? MF1 and HNF-3α correlate with populations of cells undergoing commitment to different developmental fates. A model is proposed linking FD gene expression with gastrulation events in the mouse.
Vital dye labelling of Xenopus laevis trunk neural crest reveals multipotency and novel pathways of migration
Authors: Collazo Andres , Bronner-Fraser Marianne , Fraser Scott E.
Published: April 2021

Although the Xenopus embryo has served as an important model system for both molecular and cellular studies of vertebrate development, comparatively little is known about its neural crest. Here, we take advantage of the ease of manipulation and relative transparency of Xenopus laevis embryos to follow neural crest cell migration and differentiation in living embryos. We use two techniques to study the lineage and migratory patterns of frog neural crest cells: (1) injections of DiI or lysinated rhodamine dextran (LRD) into small populations of neural crest cells to follow movement and (2) injections of LRD into single cells to follow cell lineage. By using non-invasive approaches that allow observations in living embryos and control of the time and position of labelling, we have been able to expand upon the results of previous grafting experiments. Migration and differentiation of the labelled cells were observed over time in individual living embryos, and later in sections to determine precise position and morphology. Derivatives populated by the neural crest are the fins, pigment stripes, spinal ganglia, adrenal medulla, pronephric duct, enteric nuclei and the posterior portion of the dorsal aorta. In the rostral to mid-trunk levels, most neural crest cells migrate along two paths: a dorsal pathway into the fin, followed by presumptive fin cells, and a ventral pathway along the neural tube and notochord, followed by presumptive pigment, sensory ganglion, sympathetic ganglion and adrenal medullary cells. In the caudal trunk, two additional paths were noted. One group of cells moves circumferentially within the fin, in an arc from dorsal to ventral; another progresses ventrally to the anus and subsequently populates the ventral fin. By labelling individual precursor cells, we find that neural tube and neural crest cells often share a common precursor. The majority of clones contain labelled progeny cells in the dorsal fin. The remainder have progeny in multiple derivatives including spinal ganglion cells, pigment cells, enteric cells, fin cells and/or neural tube cells in all combinations, suggesting that many premigratory Xenopus neural crest precursors are multipotent.
wingless expression mediates determination of peripheral nervous system elements in late stages of Drosophila wing disc development
Authors: Phillips R. G. , Whittle J. R. S.
Published: April 2021

We have used conditional wingless genotypes to dissect the role of this gene in late stages of wing disc development. One of these genotypes (wgIL/wg-lacZ) is simultaneously a reporter of wingless transcription and temperature-sensitive for wingless function, and has allowed us to define its pattern of transcription in the absence of wingless activity. The primordia of a subset of the bristles of the notum, which develop in or immediately adjacent to wingless-expressing cells, depend upon wing less activity. The time-course of this contribution and the effect on proneural gene expression together suggest that wingless may regulate the activity of products of the achaete-scute complex in proneural clusters. wing less activity is also required at the presumptive wing margin and is a necessary precondition for the change in proliferation pattern in this region. The involvement of wingless in transducing or mediating positional signals for spatial patterning in imaginal disc development is discussed.
FGF signalling in the early specification of mesoderm in Xenopus
Authors: Amaya Enrique , Stein Pascal A. , Musci Thomas J. , Kirschner Marc W.
Published: April 2021

We have examined the role of FGF signalling in the development of muscle and notochord and in the expression of early mesodermal markers in Xenopus embryos. Disruption of the FGF signalling pathway by expression of a dominant negative construct of the FGF receptor (XFD) generally results in gastrulation defects that are later evident in the formation of the trunk and tail, though head structures are formed nearly normally. These defects are reflected in the loss of notochord and muscle. Even in embryos that show mild defects and gastrulate properly, muscle formation is impaired, suggesting that morphogenesis and tissue differentiation each depend on FGF. The XFD protein inhibits the expression of the immediate early gene brachyury throughout the marginal zone, including the dorsal side; it does not, however, inhibit the dorsal lip marker goosecoid, which is expressed in the first involuting mesoderm at the dorsal side that will underlie the head. The XFD protein also inhibits Xpo expression, an immediate early marker of ventral and lateral mesoderm. These results suggest that FGF is involved in the earliest events of most mesoderm induction that occur before gastrulation and that the early dorsal mesoderm is already composed of two cell populations that differ in their requirements for FGF.
flk-1, an flt-related receptor tyrosine kinase is an early marker for endothelial cell precursors
Authors: Yamaguchi Terry P. , Dumont Daniel J. , Conlon Ronald A. , Breitman Martin L. , Rossant Janet
Published: April 2021

We have used RT-PCR to screen pluripotent murine embryonic stem cells to identify receptor tyrosine kinases (RTKs) potentially involved in the determination or differentiation of cell lineages during early mouse development. Fourteen different tyrosine kinase sequences were identified. The expression patterns of four RTKs have been examined and all are expressed in the mouse embryo during, or shortly after, gastrulation. We report here the detailed expression pattern of one such RTK, the flt-related gene flk-1. In situ hybridization analysis of the late primitive streak stage embryo revealed that flk-1 was expressed in the proximallateral embryonic mesoderm; tissue fated to become heart. By headfold stages, staining was confined to the endocardial cells of the heart primordia as well as to the blood islands of the visceral yolk sac and the developing allantois. Patchy, speckled staining was detected in the endothelium of all the major embryonic and extraembryonic blood vessels as they formed. During early organogenesis, expression was detected in the blood vessels of highly vascularized tissues such as the brain, liver, lungs and placenta. Since flk-1 was expressed in early mesodermal cells prior to any morphological evidence for endothelial cell differentiation (vasculogenesis), as well as in cells that form blood vessels from preexisting ones (angiogenesis), it appears to be a very early marker of endothelial cell precursors. We have previously reported that another novel RTK, designated tek, was expressed in differentiating endothelial cells. We show here that flk-1 transcripts are expressed one full embryonic day earlier than the first tek transcripts. The expression of these two RTKs appear to correlate with the specification and early differentiation of the endothelial cell lineage respectively, and therefore may play important roles in the establishment of this lineage.
Control of segmental asymmetry in Drosophila embryos
Authors: Manoukian Armen S. , Krause Henry M.
Published: April 2021

During Drosophila development, an important aspect of body patterning is the division of the embryo into repeating morphological units referred to as parasegments. The parasegmental domains are first defined at the blastoderm stage by alternating stripes of transcripts encoded by the pair-rule genes fushi tarazu (ftz) and even-skipped (eve) and later by stripes encoded by the segment polarity genes engrailed (en) and wingless. Here, we show that the runt gene (run) is required to generate asymmetries within these parasegmental domains. Using a heat-shock-inducible run transgene, we found that ectopic run expression leads to rapid repression of eve stripes and a somewhat delayed expansion of ftz stripes. Unexpectedly, we also found that ectopic run was a rapid and potent repressor of odd-numbered en stripes. Two remarkably different segmental phenotypes were generated as a consequence of these effects. In solving the mechanisms underlying these phenotypes, we discovered that the positioning of en stripes is largely determined by the actions of negative regulators. Our data indicate that run is required to limit the domains of en expression in the odd-numbered parasegments, while the odd-skipped gene is required to limit the domains of en expression in the even-numbered parasegments. Activation of en at the anterior margins of both sets of parasegments requires the repression of run and odd by the product of the eve gene. The spatial restriction of gene expression via negative and double negative pathways such as these is likely to be a common theme during development.