bluuebunny/crossref_metadata_2025 huggingface dataset:
Synthesis and release of 9-cis retinoic acid by the urodele wound epidermis
Authors: Viviano Carolyn M. , Horton Claire E. , Maden Malcolm , Brockes Jeremy P.
Published: April 2021

The wound epidermis is a transient secretory epithelium that apposes the mesenchymal blastema of a regenerating urodele limb, and is required for regeneration. Previous studies have shown that the positional identity of the blastema is respecified by retinoic acid (RA; Maden, M. (1982)Nature 295, 672-675), that the blastema contains RA (Scadding, S. R. and Maden, M. (1994) Dev. Biol. 162, 608617), and that an RA-reporter gene introduced into the blastema is differentially activated along the proximodistal axis (Brockes, J. P. (1992)Proc. Natl. Acad. Sci. USA 89, 11386-11390). The newt limb wound epidermis has been explanted with minimal mesenchymal contamination and cultured under conditions where it retains expression and inducibility of marker antigens. We have assayed for the release of retinoids from the wound epidermis by coculture with cells transfected with an RA-responsive reporter gene. The reporter was activated to a level corresponding to stimulation by 0.1–1 nM RA, and this activation was substantially conferred by medium conditioned by the wound epidermis. No significant activation was observed for cells transfected with mutated reporter plasmids and analysed in parallel co-cultures. Wound epidermis from contralateral proximal and distal blastemas were compared for reporter activation, and gave a P/D activation ratio significantly greater than 1.Wound epidermis explants were cultured in the presence of tritiated retinol, and extracts were analysed by HPLC on three different columns. Radioactivity was detected in peaks corresponding to didehydroretinol, 9-cis RA and other unidentified metabolites. Analysis of conditioned media samples, some after pulse chase experiments, detected significant release of retinol, 9-cis RA and other metabolites. Although all-trans RA was detectable, the predominant acidic metabolite was 9-cis RA. These experiments establish the wound epidermis as a source of RA for local cellular interactions in the blastema.
The homeobox-containing gene XANF-1 may control development of the Spemann organizer
Authors: Zaraisky A. G. , Ecochard V. , Kazanskaya O. V. , Lukyanov S. A. , Fesenko I. V. , Duprat A.-M.
Published: April 2021

At the beginning of gastrulation the homeobox-containing gene, XANF-1, is expressed at a low level throughout the animal hemisphere of Xenopus laevis embryos, with a local maximum of expression in the region of the dorsal blastopore lip. By the end of gastrulation expression ceases everywhere except in the most anterior part of the neurectoderm. We have investigated the functions of this gene by microinjecting XANF-1 mRNA in the blastomeres of the 32-cell stage embryo and have observed the following effects. First, microinjections of the mRNA in the animal blastomeres and the blastomeres of the marginal zone elicited massive migration of cells to the interior of the embryo at the early gastrula stage. Second, overexpression of XANF-1 in the ventral marginal zone (VMZ) resulted in the appearance of an additional centre of gastrulation movements and the formation of a secondary axis. In addition we showed that synthetic XANF-1 mRNA was able to cause dorsal-type differentiation in VMZ explants extirpated from the microinjected embryos at the beginning of gastrulation. These results suggest that XANF-1 may control the main functions of cells of the Spemann organizer.
Embryonic development of the Drosophila brain: formation of commissural and descending pathways
Authors: Therianos Stavros , Leuzinger Sandra , Hirth Frank , Goodman Corey S. , Reichert Heinrich
Published: April 2021

The establishment of initial axonal pathways in the embryonic brain of Drosophila melanogaster was investigated at the cellular and molecular level using antibody probes, enhancer detector strains and axonal pathfinding mutants. During embryogenesis, two bilaterally symmetrical cephalic neurogenic regions form, which are initially separated from each other and from the ventral nerve cord. The brain commissure that interconnects the two brain hemispheres is pioneered by axons that project towards the midline in close association with an interhemispheric cellular bridge. The descending longitudinal pathways that interconnect the brain to the ventral nerve cord are prefigured by a chain of longitudinal glial cells and a cellular bridge between brain and subesophageal ganglion; pioneering descending and ascending neurons grow in close association with these structures. The formation of the embryonic commissural and longitudinal pathways is dependent on cells of the CNS midline. Mutations in the commissureless gene, which affects growth cone guidance towards the midline, result in a marked reduction of the brain commissure. Mutations in the single-minded gene and in other spitz group genes, which affect the differentiation of CNS midline cells, result in the absence or aberrant projection of longitudinal pathways. The analysis of axon pathway formation presented here reveals remarkable similarities as well as distinct differences in the embryonic development of the brain and the segmental ganglia, and forms the basis for a comprehensive genetic and molecular genetic dissection of axonal pathfinding processes in the developing brain.
Differential regulation of AP-1 and novel TRE-specific DNA-binding complexes during differentiation of oligodendrocyte-type-2-astrocyte (O-2A) progenitor cells
Authors: Barnett Susan C. , Rosario Marta , Doyle Arthur , Kilbey Anna , Lovatt Archie , Gillespie David A. F.
Published: April 2021

AP-1 is an ubiquitous transcription factor which is composed of the Jun and Fos proto-oncogene proteins and is thought to play a role in both cell proliferation and differentiation. We have used an immortal, bipotential oligodendrocyte-type-2 astrocyte progenitor cell line (O-2A/c-myc) which can differentiate into oligodendrocytes or type-2 astrocytes in vitro, to investigate whether AP-1 DNA-binding activity fluctuates during glial cell differentiation. Unexpectedly, DNA-mobility shift assays using a TRE-containing oligonucleotide derived from the promoter of the glial-specific gene, glial fibrillary acidic protein (GFAP/AP-1), revealed that O-2A/c-myc progenitor cells were devoid of conventional AP-1 DNA-binding complexes. O-2A/c-myc cells did however contain several novel GFAP/AP-1-specific DNA-binding complexes, which we have termed APprog. APprog complexes recognise the TRE consensus motif present in the GFAP/AP-1 oligonucleotide together with adjacent 3′ sequences but do not contain c-Jun or any other known Jun-related proteins. When O-2A/c-myc cells underwent terminal differentiation APprog complexes were lost and conventional AP-1 DNA-binding activity became evident, particularly in astrocytes. These changes appear to be closely linked to the differentiation process since they did not occur in a derivative of the O-2A/c-myc cell line that contains an activated v-ras oncogene and which fails to differentiate under appropriate culture conditions. The inverse regulation of conventional AP-1 and APprog complexes within the O-2A lineage suggests that these factors may play a role in the regulation of glial cell differentiation or glial cell-specific gene expression.
dishevelled is a component of the frizzled signaling pathway in Drosophila
Authors: Krasnow Randi E. , Wong Lily L. , Adler Paul N.
Published: April 2021

The tissue polarity genes in Drosophila are required to coordinate cell polarity within the plane of the epidermis. Evidence to date suggests that these genes may encode components of a novel signal transduction pathway. Three of the genes, frizzled (fz), dishevelled (dsh), and prickle (pk) share a similar tissue polarity phenotype, suggesting that they function together in a single process. dsh is also known to function as a mediator of wingless (wg) signaling in a variety of developmental patterning processes in the fly. In this study, we make use of a fz transgene and a hypomorphic fz allele as genetic tools in an attempt to order these genes in a genetic hierarchy. Our results argue that dsh encodes a dosage sensitive component required for fz function and that it likely acts downstream of fz in the generation of tissue polarity. Our findings suggest that dsh may have a general role in signal transduction, perhaps as a component of a receptor complex.
The expression of the Athb-8 homeobox gene is restricted to provascular cells in Arabidopsis thaliana
Authors: Baima Simona , Nobili Fabio , Sessa Giovanna , Lucchetti Sabrina , Ruberti Ida , Morelli Giorgio
Published: April 2021

We have characterized an Arabidopsis homeobox gene coding for a putative DNA binding protein that represents an early marker for vascular development. The full-length cDNA encodes a protein of 833 amino acids that we have designated Athb-8; it contains the conserved DNA binding domain that characterizes the HD-Zip family of transcription factors. RNA analysis showed that the Athb-8 gene is expressed during the vegetative and the reproductive phases of plant growth. A higher steady-state level of the Athb-8 mRNA was found in flowering stem and root. In situ mRNA analysis of Arabidopsis plants demonstrated that Athb-8 expression is restricted to the procambial cells of embryo and developing organs. Moreover, Athb-8-GUS expression was found in single parenchyma cells which are differentiating into tracheary elements in wounded tobacco transgenic plants. Finally, we showed that the auxin, indole-3-acetic acid, which is involved in vascular development and differentiation, modulates the expression of the gene. Taken together, these results suggest that Athb-8 might be a regulator of vascular development in Arabidopsis thaliana.
The limb deformity mutation disrupts the SHH/FGF-4 feedback loop and regulation of 5′ HoxD genes during limb pattern formation
Authors: Haramis Anna G. , Brown Jennifer M. , Zeller Rolf
Published: April 2021

Mutations in the murine limb deformity (ld) gene disrupt differentiation of the Apical Ectodermal Ridge (AER) and patterning of distal limb structures. However, initial outgrowth of the limb bud is not affected, suggesting that early and late functions of the AER are uncoupled. Similarly, activation of the 5′ members of the HoxD gene cluster (Hoxd-11 to Hoxd-13) is not affected in ld mutant posterior limb bud mesenchyme, but the subsequent anteriorization of 5′ HoxD domains is delayed by about 12 hours and is associated with reduced levels of polarising activity. These results indicate that the ld gene products act upstream of 5′ HoxD genes during patterning of the autopod. Expression of the signalling molecule Sonic hedgehog (Shh) in the posterior limb bud mesenchyme is initiated normally, but ceases prematurely indicating a defect in maintenance of Shh by the ld mutant AER. Fur-thermore, no Fgf-4 transcripts are detected in the ld mutant AER, whereas Fgf-8 transcripts remain expressed. However, Shh expression can be rescued by heterospecific grafting of ld mutant posterior mesenchyme under a wild-type chicken AER. These studies show that the AER defect in ld homozygous limb buds causes disruption of the FGF-4/SHH feedback loop and support the proposed essential role for FGF-4 in maintaining Shh expression during limb pattern formation.
Periaxin expression in myelinating Schwann cells: modulation by axon-glial interactions and polarized localization during development
Authors: Scherer Steven S. , Xu Yi-tian , Bannerman Peter G. C. , Sherman Diane L. , Brophy Peter J.
Published: April 2021

Periaxin is a newly described protein that is expressed exclusively by myelinating Schwann cells. In developing nerves, periaxin is first detected as Schwann cells ensheathe axons, prior to the appearance of the proteins that characterize the myelin sheath. Periaxin is initially concentrated in the adaxonal membrane (apposing the axon) but, during development, as myelin sheaths mature, periaxin becomes predominately localized at the abaxonal Schwann cell membrane (apposing the basal lamina). In permanently axotomized adult nerves, periaxin is lost from the abaxonal and adaxonal membranes, becomes associated with degenerating myelin sheaths and is phagocytosed by macrophages. In crushed nerves, in which axons regenerate and are remyelinated, periaxin is first detected in the adaxonal membrane as Schwann cells ensheathe regenerating axons, but again prior to the appearance of other myelin proteins. Periaxin mRNA and protein levels change in parallel with those of other myelin-related genes after permanent axotomy and crush. These data demonstrate that periaxin is expressed by myelinating Schwann cells in a dynamic, developmentally regulated manner. The shift in localization of periaxin in the Schwann cell after completion of the spiralization phase of myelination suggests that periaxin participates in membrane-protein interactions that are required to stabilize the mature myelin sheath.
Interchangeability of Caenorhabditis elegans DSL proteins and intrinsic signalling activity of their extracellular domains in vivo
Authors: Fitzgerald Kevin , Greenwald Iva
Published: April 2021

Ligands of the Delta/Serrate/lag-2 (DSL) family and their receptors, members of the lin-12/Notch family, mediate cell-cell interactions that specify cell fate in invertebrates and vertebrates. In C. elegans, two DSL genes, lag-2 and apx-1, influence different cell fate decisions during development. Here we show that APX-1 can fully substitute for LAG-2 when expressed under the control of lag-2 regulatory sequences. In addition, we demonstrate that truncated forms lacking the transmembrane and intracellular domains of both LAG-2 and APX-1 can also substitute for endogenous lag-2 activity. Moreover, we provide evidence that these truncated forms are secreted and able to activate LIN-12 and GLP-1 ectopically. Finally, we show that expression of a secreted DSL domain alone may enhance endogenous LAG-2 signalling. Our data suggest ways that activated forms of DSL ligands in other systems may be created.
Anti-Dorsalizing Morphogenetic Protein is a novel TGF-β homolog expressed in the Spemann organizer
Authors: Moos Malcolm , Wang Shouwen , Krinks Marie
Published: April 2021

We have identified a novel growth factor in Xenopus, which is most closely related to human Bone Morphogenetic Protein-3. Its expression peaks during gastrulation, most prominently in the Spemann organizer, and persists in the posterior neural floor plate and prechordal plate during neurulation. Injection of the corresponding mRNA into dorsal blastomeres results in dose-dependent suppression of dorsal and anterior structures, even in the presence of lithium chloride. Overexpression of the gene downregulates the dorsalizing factors noggin, goosecoid and follistatin, as well as the dorsal markers NCAM, muscle actin and MyoD; conversely, ventral markers are induced. We therefore designate this gene product Anti-Dorsalizing Morphogenetic Protein (ADMP). Though development of dorsoanterior structures is suppressed when exogenous ADMP is injected, the gene is induced by lithium chloride treatment or activin, both of which are known to produce the opposite effect. Thus, the expression of ADMP resembles that of several dorsalizing signals, but its product exerts dorsal-suppressing activity. This suggests that ADMP may moderate organizer-associated dorsalizing influences. These findings are also consistent with the recently advanced proposal of dorsally expressed inhibitory activin-like signals.
Segmental patterning of heart precursors in Drosophila
Authors: Lawrence Peter A. , Bodmer Rolf , Vincent Jean-Paul
Published: April 2021

SUMMARY

The mesoderm of Drosophila embryos is segmented; for instance there are segmentally arranged clusters of cells (some of which are heart precursors) that express even-skipped. Expression of even-skipped depends on Wingless, a secreted molecule. In principle, Wingless could act directly in the mesoderm or it could induce the pattern after crossing from ectoderm to mesoderm. Using mosaic embryos, we show that Wingless produced in the mesoderm is sufficient for even-skipped expression. This proves that induction is not essential. However, induction can occur: when patches of wingless mutant mesoderm are overlaid by wild-type ectoderm, they do express even-skipped. We therefore believe that Wingless from both the ectoderm and mesoderm may contribute to patterning the mesoderm. Using the UAS/Gal4 system, we made embryos in which the Wingless protein is uniformly expressed. This is sufficient to rescue the repeated clusters of even-skipped expressing cells, although they are enlarged. We conclude that the mesoderm is segmented in some way not dependent on the distribution of Wingless, suggesting a more permissive and less instructive role for the protein in this instance.
The Caenorhabditis elegans lin-12 gene mediates induction of ventral uterine specialization by the anchor cell
Authors: Newman Anna P. , White John G. , Sternberg Paul W.
Published: April 2021

The anchor cell (AC) of the Caenorhabditis elegans gonad has a critical role in the development of a functional egglaying system, which is accomplished through cell-cell interactions. Lateral inhibitory lin-12-mediated signaling among two bipotential cells causes one to adopt the ventral uterine precursor (VU) cell fate while the other becomes the AC. The AC then induces formation of vulval tissue. We find that the AC also induces a particular ventral uterine intermediate precursor fate (π) by a mechanism that is genetically and temporally distinct from vulval induction. This process requires lin-12, but unlike previously described lin-12-mediated decisions, signaling is unidirectional, is between dissimilar cells and does not involve lateral inhibition. The π fates are necessary for egg laying and appear to produce a distinct specialized cell type. Thus, patterning of the ventral uterus by the AC is crucial to the development of a functional egg-laying system.
EGF, TGF-α and EGFR expression in human preimplantation embryos
Authors: Chia Choy May , Winston Robert M. L. , Handyside Alan H.
Published: April 2021

Epidermal growth factor (EGF) and transforming growth factor alpha (TGF-α) through their common receptor, epidermal growth factor receptor (EGFR) are known to enhance mitogenesis, development and implantation in several species. In the mouse, co-culture of grouped embryos in microdrops increases the cell number and pro-portion developing to the blastocyst stage. A similar effect is observed with culture of single embryos in medium sup-plemented with EGF or TGF-α highlighting their embry-otrophic effects.

To study the role of EGF, TGF-α and EGFR in early human development, two methods applicable for analysis of expression at the single embryo level have been employed. In the first method, reverse transcription-poly-merase chain reaction has been used to examine the presence of transcripts. Following reverse transcription, strategically designed nested primers, optimised for speci-ficity, were used for amplification from the cDNA equiva-lent of a single embryo. The products were then verified by restriction enzyme digestion and sequence analysis. In the second method, immunocytochemistry has been used to co-localise the expressed proteins. Individual embryos were paraffin embedded and serial sectioned, allowing adjacent sections to be examined with different antibodies and controls. Monoclonal TGF-α and polyclonal EGF and EGFR primary antibodies were used. Staining was performed by peroxidase-conjugated avidin-biotin immunocytochemistry with the appropriate controls. The combination of these two methods can potentially be used for simultaneous analysis of several growth factors and/or their receptors in the same human embryos.

Transcripts for EGF, TGF-α and EGFR were detected in unfertilized oocytes and embryos between 8-cell and blastocyst stages on day 3 to 6 post-insemination. Similarly, at the protein level, all three were detected in unfertilized oocytes and throughout preimplantation development to day 8. At the blastocyst stage, expression was observed in both the trophectoderm and inner cell mass but decreased in more advanced blastocysts except in the polar trophec-toderm and inner cell mass. This pattern of expression contrasts with the murine and bovine species in which TGF-α and EGFR but not EGF are expressed at preim-plantation stages. The concurrent co-expression of these growth factors and their receptor suggests a role for autocrine stimulation in preimplantation development. In the human, reduced protein levels of EGF, TGF-α and EGFR in advanced blastocysts may reflect a switch to dependence on paracrine stimulation. This may explain the inadequacy of simple culture media to maintain the devel-opment of human embryos at later stages and the benefits of co-culture with somatic cells. The continuous expression of EGF, TGF-α and EGFR in the polar trophectoderm may be significant for implantation.
Target-induced neurogenesis in the leech CNS involves efferent projections to the target
Authors: Becker Tom , Berliner Alyson J. , Nitabach Michael N. , Gan Wen-Biao , Macagno Eduardo R.
Published: April 2021

During a critical period in leech embryogenesis, the sex nerves that connect the 5th and 6th midbody ganglia (MG5 and MG6) to the primordium of the male sexual organ carry a spatially localized signal that induces the birth of several hundred neurons specific to these ganglia. We examined particular cellular elements (afferents, efferents, non-neuronal components) within these nerves as potential conveyors of the inductive signal. We show that axons of peripheral sensory neurons in the male genitalia travel along the sex nerves and into MG5 and MG6, but reach the CNS after the critical period has elapsed and cannot, therefore, be involved in the induction. Of the six sex nerves, four contain non-neuronal cells that span the entire distance between the male genitalia and the sex ganglia. However, when male genitalia were transplanted to ectopic locations close to MG6, induction occurred frequently but only in MG6, mediated by ectopic nerves that do not contain these cells. Thus, non-neuronal cells specific to the normal sex nerves are not necessary for induction. In addition, dye injections into the target during the critical period failed to reveal migrating cells in the sex nerves that could convey the inductive signal to the CNS. Finally, we show that 11 pairs of central neurons in each ganglion project to the male organ early during the critical period. In the adult, at least 3 additional pairs of neurons in MG6 also innervate this target. We conclude that the only components of the sex nerves that connect the sex ganglia to the target during the critical period that could be associated with induced central mitogenesis are the axons of central neurons that innervate the male genitalia.
Defective development of the embryonic and extraembryonic circulatory systems in vascular cell adhesion molecule (VCAM-1) deficient mice
Authors: Kwee Lia , Baldwin H. Scott , Shen Hong Min , Stewart Colin L. , Buck Catherine , Buck Clayton A. , Labow Mark A.
Published: April 2021

VCAM-1 is a cytokine-inducible cell surface protein capable of mediating adhesion to leukocytes expressing α4 integrins. Mice deficient in VCAM-1 expression were produced by targeted homologous recombination in ES cells. VCAM-1-deficient embryos were not viable and exhibited either of two distinct phenotypes. Approximately half of the embryos died before embryonic day 11.5 and exhibited a severe defect in placental development in which the allantois failed to fuse with the chorion. The remaining VCAM-1-deficient embryos survived to embryonic day 11.5-12.5 and displayed several abnormalities in their developing hearts including a reduction of the compact layer of the ventricular myocardium and intraventricular septum. The hearts also contained significant amounts of blood in the pericardial space and lacked an epicardium. α4 and VCAM-1 were found to be expressed in wild-type embryos in a reciprocal fashion in the chorion and allantois and in the epicardium and the underlying myocardium, although VCAM-1 was expressed in the intraventricular septum in the absence of adjacent α4-expressing cells. These data suggest important roles for VCAM-1 and α4 in the development of the placenta and the heart.
Mice deficient in cellular retinoic acid binding protein II (CRABPII) or in both CRABPI and CRABPII are essentially normal
Authors: Lampron Carmen , Rochette-Egly Cécile , Gorry Philippe , Dollé Pascal , Mark Manuel , Lufkin Thomas , LeMeur Marianne , Chambon Pierre
Published: April 2021

We have disrupted the CRABPII gene using homologous recombination in embryonic stem cells, and shown that this disruption results in a null mutation. CRABPII null mutant mice are essentially indistinguishable from wild-type mice as judged by their normal development, fertility, life span and general behaviour, with the exception of a minor limb malformation. Moreover, CRABPI−/−/CRABPII−/− double mutant mice also appear to be essentially normal, and both CRABPII−/− single mutant and CRABPI−/−/CRABPII−/− double mutant embryos are not more sensitive than wild-type embryos to retinoic acid excess treatment in utero. Thus, CRABPI and CRABPII are dispensable both during mouse development and adult life. Our present results demonstrate that CRABPs are not critically involved in the retinoic acid signaling pathway, and that none of the functions previously proposed for CRABPs are important enough to account for their evolutionary conservation.
Cell adhesion events mediated by α4 integrins are essential in placental and cardiac development
Authors: Yang Joy T. , Rayburn Helen , Hynes Richard O.
Published: April 2021

α4 integrins are cell surface receptors that mediate cell-extracellular matrix (ECM) and cell-cell adhesions by interacting with fibronectin (FN) and vascular cell adhesion molecule 1 (VCAM-1), respectively. We have generated a null mutation in the gene for the α4 integrin subunit. Homozygous null embryos express no α4 integrins and show two unexpected defects, both of which lead to embryonic lethality. The first defect is failure of fusion of the allantois with the chorion during placentation. The second is in the development of the epicardium and coronary vessels leading to cardiac hemorrhrage. Both processes clearly involve α4 integrin interactions that were previously unsuspected. α4 integrin and VCAM-1 are expressed at the sites of these interactions. These results raise the possibility of abortifacients targeting α4 integrins, and raise serious questions about potential side effects of drugs currently being designed to block α4 integrin functions in inflammation.
Identification of a germ-cell-specific transcriptional repressor in the promoter of Tctex-1
Authors: O’Neill Michael J. , Artzt Karen
Published: April 2021

The Tctex-1 gene family maps to the t complex of the mouse and consists of four copies on chromosome 17 in both wild-type and t-haplotypes. Tctex-1 mRNA is eightfold overexpressed in male and female germ cells in t-haplotype compound heterozygotes (tx/ty). In order to determine the cause of this aberrant expression and the role of this gene family in spermatogenesis and oogenesis it was subjected to extensive molecular analysis. We find that Tctex-1 protein is present in sperm tails and oocytes and that it is present at equal levels in wild-type and t-haplotype testis. Surprisingly, the excess message in t-haplotypes is not translated. Sequence analysis of the gene family reveals that one copy in t-haplotypes has a mutated start codon. This same copy is deleted for a protein-binding motif in its promoter. This motif, GIM (Germ cell Inhibitory Motif) has strong homology to the Xenopus AP-2-binding site but does not appear to be a binding site for mammalian AP-2. A factor(s) present in testis and ovary, but absent in other mouse tissues binds specifically to this site. Transfection assays using Tctex-1 promoter constructs suggest that GIM functions as a transcriptional repressor. The possible role of Tctex-1 in t complex transmission ratio distortion and sterility is discussed.
The biogenesis of the coiled body during early mouse development
Authors: Ferreira João , Carmo-Fonseca Maria
Published: April 2021

The coiled body is an ubiquitous nuclear organelle that contains essential components of the pre-mRNA splicing machinery as well as the nucleolar protein fibrillarin. Here we have studied the biogenesis of the coiled body in early mouse embryos. The results show that coiled bodies form and concentrate splicing snRNPs as early as in the maternal and paternal pronuclei of 1-cell embryos. This argues that the coiled body is likely to play a basic role in the nucleus of mammalian cells. In order to correlate the appearance of coiled bodies with the onset of transcriptional activity, embryos were incubated with brominated UTP and the incorporated nucleotide was visualized by fluorescence microscopy. In agreement with previous studies, transcriptional activity was first observed during the 2-cell stage. Thus, coiled bodies form before activation of embryonic gene expression.

The appearance of coiled bodies in 1-cell embryos was preceded by the formation of morphologically distinct structures that also contain coilin and which we therefore refer to as pre-coiled bodies. At the electron microscopic level pre-coiled bodies have a compact fibrillar structure, whereas coiled bodies resemble a tangle of coiled threads. Although both pre-coiled bodies and coiled bodies contain the nucleolar protein fibrillarin, the assembly of coiled bodies is separated both in time and in space from ribosome synthesis. Our results suggest that the embryonic ‘nucleolus-like body’ is a structural scaffold that nucleates independently the formation of the coiled body and the assembly of the machinery responsible for ribosome biosynthesis.
Repression of Pax-2 by WT1 during normal kidney development
Authors: Ryan Gabriella , Steele-Perkins Victoria , Morris Jennifer F. , Rauscher Frank J. , Dressler Gregory R.
Published: April 2021

The developmental, regulatory gene Pax-2 is activated during early kidney morphogenesis and repressed in mature renal epithelium. Persistent Pax-2 expression is also observed in a variety of kidney tumors. Yet, little is known about the signals regulating this transient expression pattern in the developing kidney. We have examined the spatial and temporal expression patterns of Pax-2 and the Wilms’ tumor suppresser protein WT1 with specific antibodies in developing mouse kidneys. A marked increase in WT1 protein levels coincided precisely with down-regulation of the Pax-2 gene in the individual precursor cells of the visceral glomerular epithelium, suggesting a direct effect of the WT1 repressor protein on Pax-2 regulatory elements. To examine whether WT1 could directly repress Pax-2 transcription, binding of WT1 to three high affinity sites in the 5’ untranslated Pax-2 leader sequence was demonstrated by DNAseI footprinting analysis. Furthermore, co-transfection assays using CAT reporter constructs under the control of Pax-2 regulatory sequences demonstrated WT1-dependent transcriptional repression. These three WT1 binding sites were also able to repress transcription, in a WT1-dependent manner, when inserted between a heterologous promoter and the reporter gene. The data indicate that Pax-2 is a likely target gene for WT1 and suggest a direct link, at the level of transcriptional regulation, between a developmental control gene, active in undifferentiated and proliferating cells, and a known tumor suppresser gene.
Differential regulation of transcription factor gene expression and phenotypic markers in developing sympathetic neurons
Authors: Groves Andrew K. , George Kathleen M. , Tissier-Seta Jean-Phillipe , Engel James Douglas , Brunet Jean-François , Anderson David J.
Published: April 2021

We have examined the regulation of transcription factor gene expression and phenotypic markers in developing chick sympathetic neurons. Sympathetic progenitor cells first express the bHLH transcriptional regulator Cash-1 (a chicken achaete-scute homologue), followed by coordinate expression of Phox2, a paired homeodomain protein, and GATA-2, a zinc finger protein. SCG10, a pan-neuronal membrane protein, is first detected one stage later, followed by the catecholaminergic neurotransmitter enzyme tyrosine hydroxylase (TH). We have used these markers to ask two questions: (1) is their expression dependent upon inductive signals derived from the notochord or floor plate?; (2) does their sequential expression reflect a single linear pathway or multiple parallel pathways? Notochord ablation experiments indicate that the floor plate is essential for induction of GATA-2, Phox2 and TH, but not for that of Cash-1 and SCG10. Taken together these data suggest that the development of sympathetic neurons involves multiple transcriptional regulatory cascades: one, dependent upon notochord or floor plate-derived signals and involving Phox2 and GATA-2, is assigned to the expression of the neurotransmitter phenotype; the other, independent of such signals and involving Cash-1, is assigned to the expression of pan-neuronal properties. The parallel specification of different components of the terminal neuronal phenotype is likely to be a general feature of neuronal development.
Avian neural crest cells can migrate in the dorsolateral path only if they are specified as melanocytes
Authors: Erickson Carol A. , Goins Tina L.
Published: April 2021

Neural crest cells are conventionally believed to migrate arbitrarily into various pathways and to differentiate according to the environmental cues that they encounter. We present data consistent with the notion that melanocytes are directed, by virtue of their phenotype, into the dorsolateral path, whereas other neural crest derivatives are excluded.

In the avian embryo, trunk neural crest cells that migrate ventrally differentiate largely into neurons and glial cells of the peripheral nervous system. Neural crest cells that migrate into the dorsolateral path become melanocytes, the pigment cells of the skin. Neural crest cells destined for the dorsolateral path are delayed in their migration until at least 24 hours after migration commences ventrally. Previous studies have suggested that invasion into the dorsolateral path is dependent upon a change in the migratory environment. A complementary possibility is that as neural crest cells differentiate into melanocytes they acquire the ability to take this pathway. When quail neural crest cells that have been grown in culture for 12 hours are labeled with Fluoro-gold and then grafted into the early migratory pathway at the thoracic level, they migrate only ventrally and are coincident with the host neural crest. When fully differentiated melanocytes (96 hours old) are back-grafted under identical conditions, however, they enter the dorsolateral path and invade the ectoderm at least one day prior to the host neural crest. Likewise, neural crest cells that have been cultured for at least 20 hours and are enriched in melanoblasts immediately migrate in the dorsolateral path, in addition to the ventral path, when back-grafted into the thoracic level. A population of neural crest cells depleted of melanoblasts – crest cells derived from the branchial arches – are not able to invade the dorsolateral path, suggesting that only pigment cells or their precursors are able to take this migratory route. These results suggest that as neural crest cells differentiate into melanocytes they can exploit the dorsolateral path immediately.

Even when 12-hour crest cells are grafted into stage 19-21 embryos at an axial level where host crest are invading the dorsolateral path, these young neural crest cells do not migrate dorsolaterally. Conversely, melanoblasts or melanocytes grafted under the same circumstances are found in the ectoderm. These latter results suggest that during normal development neural crest cells must be specified, if not already beginning to differentiate, as melanocytes in order to take this path. Thus melanocyte precursors are one subpopulation of the neural crest that has been documented to exploit a specific pathway on the basis of their phenotype.
Disruption of intermediate filament organization leads to structural defects at the intersomite junction in Xenopus myotomal muscle
Authors: Cary Robert B. , Klymkowsky Michael W.
Published: April 2021

In mature striated muscle, intermediate filaments (IFs) are associated with the periphery of Z-discs and sites of myofibril-membrane attachment. Previously T. Schultheiss, Z. X. Lin, H. Ishikawa, I. Zamir, C. J. Stoeckert and H. Holtzer (1991) J. Cell Biol. 114, 953)reported that the disruption of IF organization in cultured chick myotubes had no detectable effect on muscle cell structure. Cultured muscle is not, however, under the mechanical loads characteristic of muscle in situ. The dorsal myotomal muscle (DMM) of the Xenopus tadpole provides an accessible model system in which to study the effects of mutant IF proteins on an intact, functional muscle. DNAs encoding truncated forms of Xenopus vimentin or desmin were injected into fertilized Xenopus eggs. Embryos were allowed to develop to the tadpole stage and then examined by confocal or electron microscopy. DMM cells containing the truncated IF polypeptides displayed disorganized IF systems. While the alignment of Z-lines appeared unaffected, cells accumulating mutant IF polypeptides displayed abnormal organization at the intersomite junction. Myocyte termini are normally characterized by deep invaginations of the sarcolemma. In myocytes expressing mutated IF polypeptides, these membrane invaginations were reduced or completely absent. Furthermore, the attachment of myofibrils to the junctional membrane was often aberrant or completely disrupted. These results suggest that in active muscle IFs play an important role in the organization and/or stabilization of myofibril-membrane attachment sites.
An analysis of the response to gut induction in the C. elegans embryo
Authors: Goldstein Bob
Published: April 2021

Establishment of the gut founder cell (E) in C. elegans involves an interaction between the P2 and the EMS cell at the four cell stage. Here I show that the fate of only one daughter of EMS, the E cell, is affected by this induction. In the absence of the P2-EMS interaction, both E and its sister cell, MS, produce pharyngeal muscle cells and body wall muscle cells, much as MS normally does.

By cell manipulations and inhibitor studies, I show first that EMS loses the competence to respond before it divides even once, but P2 presents an inducing signal for at least three cell cycles. Second, induction on one side of the EMS cell usually blocks the other side from responding to a second P2-derived signal. Third, microfilaments and microtubules may be required near the time of the interaction for subsequent gut differentiation. Lastly, cell manipulations in pie-1 mutant embryos, in which the P2 cell is transformed to an EMS-like fate and produces a gut cell lineage, revealed that gut fate is segregated to one of P2’s daughters cell-autonomously. The results contrast with previous results from similar experiments on the response to other inductions, and suggest that this induction may generate cell diversity by a different mechanism.
Evolutionary-conserved enhancers direct region-specific expression of the murine Hoxa-1 and Hoxa-2 loci in both mice and Drosophila
Authors: Frasch Manfred , Chen Xiaowei , Lufkin Thomas
Published: April 2021

The HOM-C/Hox complexes are an evolutionary related family of genes that have been shown to direct regionspecific development of the animal body plan. We examined in transgenic mice the DNA regulatory elements that determine the temporal and spatially restricted expression of two of the earliest and most anteriorly expressed murine genes, Hoxa-1 and Hoxa-2, which are homologues of the labial and proboscipedia genes of Drosophila. In both mouse and Drosophila, these genes have been shown to play a critical role in head development. We identified three independent enhancers which direct distinct portions of the Hoxa-1 and Hoxa-2 expression domains during early murine embryogenesis. Two enhancers mediate hindbrainspecific expression, being active in either rhombomere 2, the most anterior rhombomere expressing Hoxa-2, or in rhombomere 4, a region where Hoxa-1 and Hoxa-2 have been shown to exert critical developmental roles. The third enhancer is essential for the most extensive expression domain of Hoxa-1 and contains a retinoic acid response element. Point mutations within the retinoic acid response element abolish expression in neuroepithelium caudal to rhombomere 4, supporting a natural role for endogenous retinoids in patterning of the hindbrain and spinal cord. Analysis of the murine Hoxa-2 rhombomere 2-specific enhancer in Drosophila embryos revealed a distinct expression domain within the arthropod head segments, which parallels the expression domain of the Hoxa-2 homologue proboscipedia. These results suggest an evolutionary conservation between HOM-C/Hox family members, which includes a conservation of certain DNA regulatory elements and possible regulatory cascades.
Targeted disruption of the low-affinity leukemia inhibitory factor receptor gene causes placental, skeletal, neural and metabolic defects and results in perinatal death
Authors: Ware Carol B. , Horowitz Mark C. , Renshaw Blair R. , Hunt Joan S. , Liggitt Denny , Koblar Simon A. , Gliniak Brian C. , McKenna Hilary J. , Papayannopoulou Thalia , Thoma Bettina , Cheng Linzhao , Donovan Peter J. , Peschon Jacques J. , Bartlett Perry F. , Willis Cynthia R. , Wright Barbara D. , Carpenter Melissa K. , Davison Barry L. , Gearing David P.
Published: April 2021

The low-affinity receptor for leukemia inhibitory factor (LIFR)* interacts with gp130 to induce an intracellular signal cascade. The LIFR-gp130 heterodimer is implicated in the function of diverse systems. Normal placentation is disrupted in LIFR mutant animals, which leads to poor intrauterine nutrition but allows fetuses to continue to term. Fetal bone volume is reduced greater than three-fold and the number of osteoclasts is increased six-fold, resulting in severe osteopenia of perinatal bone. Astrocyte numbers are reduced in the spinal cord and brain stem. Late gestation fetal livers contain relatively high stores of glycogen, indicating a metabolic disorder. Hematologic and primordial germ cell compartments appear normal. Pleiotropic defects in the mutant animals preclude survival beyond the day of birth.
CSF-1 and mouse preimplantation development in vitro
Authors: Bhatnagar Piyush , Papaioannou Virginia E. , Biggers John D.
Published: April 2021

The effects of macrophage colony stimulating factor on the development of the zygote to the blastocyst stage of an outbred strain of mouse have been studied in KSOM, an improved medium that supports a high rate of in vitro development. Macrophage colony stimulating factor accelerates the formation of the blastocyst cavity by day 4 (96 hours post-hCG). It also increases overall embryonic cell number through a differential increase in the number of trophoblast cells, with no significant effect on the number of inner cell mass cells. By day 5 of culture (120 hours posthCG), colony stimulating factor-treated embryos have about 20 more trophoblast cells than control embryos, an increase of about 30 percent of the total number of cells in a control blastocyst. The maximum response of embryos was obtained at a concentration around 540 U ml−1 colony stimulating factor (=918 Stanley units ml−1), and the cytokine can produce the same effects even if it is present in the medium for only part of the culture period. This in vitro stimulation of preimplantation development with macrophage colony stimulating factor is compatible with continued normal fetal development in vivo.
Muscle sensory neurons require neurotrophin-3 from peripheral tissues during the period of normal cell death
Authors: Oakley R. A. , Garner A. S. , Large T. H. , Frank E.
Published: April 2021

To determine if muscle sensory neurons require neurotrophin-3 (NT3) during the period of normal cell death, we used an NT3-specific antiserum to deplete NT3 from peripheral tissues during this period in chick embryos. DiI staining of dorsal roots indicated that limb injections of anti-NT3 reduced the spinal projection of muscle spindle afferents. In contrast, injection of the antiserum into the spinal cord had no demonstrable effect, indicating that the reduced projection following limb injection was due to peripheral blockade of NT3 signaling. Counts of neurons retrogradely labeled from muscle and cutaneous nerves showed that peripheral blockade of NT3 selectively reduced the survival of muscle sensory neurons without affecting the survival of cutaneous sensory neurons or motoneurons. In situ hybridization with trkC probes indicated that, during the period of cell death, most large diameter muscle sensory neurons express trkC transcripts, whereas few cutaneous neurons express this receptor for NT3. We conclude that large diameter muscle afferents, including spindle afferents, require NT3 from peripheral tissues to survive the normal period of sensory neuron death in vivo.
Axonal guidance in the chicken retina
Authors: Stier Heike , Schlosshauer Burkhard
Published: April 2021

During retina development, ganglion cells extend their axons exclusively into the innermost tissue layer, but not into outer retina layers.

In order to elucidate guiding mechanisms for axons, tissue strips of embryonic chicken retinae were explanted onto retinal cryosections (cryoculture). Ganglion cell axons originating from the explant grew preferentially on the innermost retina layer of cryosections, whereas outer tissue layers were avoided, very much as in vivo.

Stereotropism, interaction with laminin of the basal lamina and axonal fasciculation did not significantly affect oriented axonal outgrowth, so that stereotropism as a guidance mechanism could be excluded. Ganglion cell axons were not directed by physical barriers, e.g. microstructured silicon oxide chips. Similarly, UV induced protein inactivation revealed that laminin present in the inner retina did not provide a guidance cue. Even in the absence of ganglion cell axons in retinal cryosections due to prior optic nerve transection in ovo, the growth preference for the innermost retina layer was maintained in cryocultures.

However, oriented elongation of axons along the innermost retina layer was lost when radial glial endfeet were selectively eliminated in retinal cryosections. In addition, glial endfeet provided an excellent growth substratum when pure preparations of endfeet were employed in explant cultures. The preference for glial endfeet positioned at the inner retina surface was accompanied by the avoidance of outer retina layers, most likely because of inhibitory components in this region. This assumption is corroborated by the finding that addition of exogenous growth-promoting laminin to cryosections did not abolish the inhibition. Laminin on glass surfaces provided an excellent substratum. Axonal outgrowth was also seriously hampered on specifically purified cells of the outer retina. Most notable, however, in cryocultures aberrant innervation of outer retina layers could be induced by prior heat or protease treatment of cryosections, which pointed to proteins as potential inhibitory components.

In summary the data substantiate the hypothesis that within the retina, ganglion cell axons are guided by a dual mechanism based on a permissive and an inhibitory zone. Radial glia is likely to be instructive in this process.