bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential effects of combined trk receptor mutations on dorsal root ganglion and inner ear sensory neurons
Authors: Minichiello Liliana , Piehl Fredrik , Vazquez Esther , Schimmang Thomas , Hökfelt Tomas , Represa Juan , Klein Rüdiger
Published: April 2021

We have generated double mutant mice deficient in pairs of two different Trk receptors and have analysed the effects on survival and differentiation of dorsal root ganglion (DRG), inner ear cochlear and vestibular sensory neurons. In most combinations of mutant trk alleles, the defects observed in double compared to single mutant mice were additive. However, double homozygous trkA−/−;trkB−/− DRG and trkB−/−;trkC−/− vestibular neurons showed the same degree of survival as single trkA−/− and trkB−/− mice, respectively, suggesting that those neurons required both Trk signaling pathways for survival. In situ hybridisation analysis of DRG neurons of double mutant mice revealed differential expression of excitatory neuropeptides. Whereas calcitonin-gene-related peptide expression correlated with the trkA phenotype, substance P expression was detected in all combinations of double mutant mice. In the inner ear, TrkB- and TrkC-dependent neurons were shown to at least partially depend on each other for survival, most likely indirectly due to abnormal development of their common targets. This effect was not observed in DRGs, where neurons depending on different Trk receptors generally innervate different targets.
Role of the morphogenetic furrow in establishing polarity in the Drosophila eye
Authors: Chanut Françoise , Heberlein Ulrike
Published: April 2021

The Drosophila retina is a crystalline array of 800 ommatidia whose organization and assembly suggest polarization of the retinal epithelium along anteroposterior and dorsoventral axes. The retina develops by a stepwise process following the posterior-to-anterior progression of the morphogenetic furrow across the eye disc. Ectopic expression of hedgehog or local removal of patched function generates ectopic furrows that can progress in any direction across the disc leaving in their wake differentiating fields of ectopic ommatidia. We have studied the effect of these ectopic furrows on the polarity of ommatidial assembly and rotation. We find that the anteroposterior asymmetry of ommatidial assembly parallels the progression of ectopic furrows, regardless of their direction. In addition, ommatidia developing behind ectopic furrows rotate coordinately, forming equators in various regions of the disc. Interestingly, the expression of a marker normally restricted to the equator is induced in ectopic ommatidial fields. Ectopic equators are stable as they persist to adulthood, where they can coexist with the normal equator. Our results suggest that ectopic furrows can impart polarity to the disc epithelium, regarding the direction of both assembly and rotation of ommatidia. We propose that these processes are polarized as a consequence of furrow propagation, while more global determinants of dorsoventral and anteroposterior polarity may act less directly by determining the site of furrow initiation.
Anterior-posterior subdivision and the diversification of the mesoderm in Drosophila
Authors: Borkowski Olga M. Dunin , Brown Nicholas H. , Bate Michael
Published: April 2021

We have used a novel cell marker, in which the twist promoter directs the synthesis of the cell surface protein CD2 (twi-CD2) to examine the development of the mesoderm in the Drosophila embryo after gastrulation and to locate the progenitor cell populations for different meso- dermal derivatives. We find that the early mesoderm in each segment is divided into a more anterior region with relatively low levels of twist and twi-CD2 expression and a more posterior region where twist and twi-CD2 expression are high. This subdivision coincides with regional assignments of cells to form different progenitors: dorsal anterior cells invaginate to form an internal layer from which the visceral mesoderm is derived. Ventral anterior cells form progenitors of mesodermal glial cells. Dorsal posterior cells form heart. Ventral and dorsal posterior cells form somatic muscles. We conclude that the metamerically repeated anterior-posterior subdivision of the mesoderm is an essential element in laying out the pattern of mesodermal progenitor cells and in distinguishing between an internal cell layer which will give rise to the progenitors of visceral muscles and an external layer which will generate the somatic muscles and the heart.
Serrate signals through Notch to establish a Wingless-dependent organizer at the dorsal/ventral compartment boundary of the Drosophila wing
Authors: Diaz-Benjumea F.J. , Cohen S.M.
Published: April 2021

Growth and patterning of the Drosophila wing is controlled by organizing centers located at the anterior-posterior and dorsal-ventral compartment boundaries. Interaction between cells in adjacent compartments establish the organizer. We report here that Serrate and Notch mediate the interaction between dorsal and ventral cells to direct localized expression of Wingless at the D/V boundary. Serrate serves as a spatially localized ligand which directs Wg expression through activation of Notch. Ligand independent activation of Notch is sufficient to direct Wg expression, which in turn mediates the organizing activity of the D/V boundary.
Conserved and divergent expression aspects of the Drosophila segmentation gene hunchback in the short germ band embryo of the flour beetle Tribolium
Authors: Wolff Christian , Sommer Ralf , Schröder Reinhard , Glaser Gerald , Tautz Diethard
Published: April 2021

The segmentation gene hunchback (hb) plays a central role in determining the anterior-posterior pattern in the Drosophila embryo. We have cloned the homologue of hb from the flour beetle Tribolium and show that, on the basis of its expression pattern, most of its functions seem to be conserved between these two species. Like Drosophila, Tribolium has a maternal hb expression that appears to be under translational control by a factor at the posterior pole of the embryo. The maternal expression is followed by a zygotic expression in the region of the developing head and thoracic segments. During germ band extension, a posterior expression domain appears that is likely to be homologous to the posterior blastoderm expression of hb in Drosophila. These observations suggest that hb may have the same functions in early Drosophila and Tribolium devel-opment, despite the different types of embryogenesis in these two species (long versus short germ development). One differing aspect of hb expression in Tribolium concerns a structure that is not present in Drosophila, namely the serosa. An hb expression domain at the anterior pole precisely demarcates the border between the extra-embryonic serosa and the embryonic field in the Tribolium embryo at an early stage, and hb protein remains expressed in the serosa cells until the end of embryogenesis.
Conservation of Brachyury (T) genes in amphioxus and vertebrates: developmental and evolutionary implications
Authors: Holland Peter W. H. , Koschorz Birgit , Holland Linda Z. , Herrmann Bernhard G.
Published: April 2021

Homologues of the murine Brachyury (T) gene have been cloned from several vertebrates, and are implicated in mesoderm formation and in differentiation of the notochord. In contrast, the roles of the ascidian Brachyury gene may be restricted to presumptive notochord. To understand the evolution of Brachyury genes and their developmental roles, we have searched for homologues in amphioxus, representing the third chordate subphylum and the probable closest relative of the vertebrates. We report the isolation of two amphioxus cDNA clones with clear homology to Brachyury genes, and demonstrate that these derive from separate loci resultant from a recent gene duplication. This finding represents an exception to the emerging consensus of an archetypal prevertebrate genome in amphioxus. The spatial and temporal distribution of Brachyury transcripts during amphioxus development is remarkably similar to vertebrate Brachyury, in presumptive mesoderm, posterior mesoderm and the notochord. Gene expression extends throughout the anteroposterior axis of the notochord, despite the most rostral regions being a more recent specialization; it also persists into larval stages, despite differentiation into contractile tissue. We propose that roles of Brachyury in notochord differentiation are more ancient than roles in mesoderm formation, and that the latter are shared by cephalochordates and all vertebrates.
Regulation of Hoxc-8 during mouse embryonic development: identification and characterization of critical elements involved in early neural tube expression
Authors: Shashikant Cooduvalli S. , Bieberich Charles J. , Belting Heinz-Georg , Wang Jeanne C. H. , Borbély Marc A. , Ruddle Frank H.
Published: April 2021

We have characterized cis-acting elements that direct the early phase of Hoxc-8 expression using reporter gene analysis in transgenic mice. By deletion we show that a 135 bp DNA fragment, located approximately 3 kb upstream of the coding region of Hoxc-8, is capable of directing posterior neural tube expression. This early neural tube (ENT) enhancer consists of four separate elements, designated A, B, C and D, whose nucleotide sequences are similar to binding sites of known transcription factors. Nucleotide substitutions suggest that element A is an essential component of the ENT enhancer. However element A by itself is incapable of directing neural tube expression. This element requires interactions at any two of the other three elements, B, C or D. Thus, the components of the ENT enhancer direct neural tube expression in an interdependent manner. We propose that Hoxc-8 is activated in the neural tube by combinatorial interactions among several proteins acting within a small region. Our transgenic analyses provide a means to identify transcription factors that regulate Hoxc-8 expression during embryogenesis.
Caudalization of neural fate by tissue recombination and bFGF
Authors: Cox Wm. Gregory , Hemmati-Brivanlou Ali
Published: April 2021

In order to study anteroposterior neural patterning in Xenopus embryos, we have developed a novel assay using explants and tissue recombinants of early neural plate. We show, by using region-specific neural markers and lineage tracing, that posterior axial tissue induces midbrain and hindbrain fates from prospective forebrain. The growth factor bFGF mimics the effect of the posterior dorsal explant in that it (i) induces forebrain to express hindbrain markers, (ii) induces prospective hindbrain explants to make spinal cord, but not forebrain and midbrain, and (iii) induces posterior neural fate in ectodermal explants neu-ralized by the dominant negative activin receptor and follistatin without mesoderm induction. The competence of forebrain explants to respond to both posterior axial explants and bFGF is lost by neural groove stages. These findings demonstrate that posterior neural fate can be derived from anterior neural tissue, and identify a novel activity for the growth factor bFGF in neural patterning. Our observations suggest that full anteroposterior neural patterning may be achieved by caudalization of prospec-tive anterior neural fate in the vertebrate embryo.
Rhodopsin plays an essential structural role in Drosophila photoreceptor development
Authors: Kumar Justin P. , Ready Donald F.
Published: April 2021

Null mutations of the Drosophila Rh1 rhodopsin gene, ninaE, result in developmental defects in the photosensitive membranes, the rhabdomeres, of compound eye photoreceptors R1-R6. In normal flies, Rh1 expression begins at about 78% of pupal life. At approximately 90% of pupal life, a specialized catacomb-like membrane architecture develops at the base of normal rhabdomeres. In ninaE null mutants, these catacombs do not form and developing rhabdomere membrane involutes into the cell as curtains of apposed plasma membrane. A filamentous cytoskeletal complex that includes F-actin and the unconventional myosin, NINAC, decorates the cytoplasmic surface of these curtains.
FGF suppresses apoptosis and induces differentiation of fibre cells in the mouse lens
Authors: Chow Robert L. , Roux Graciana Diez , Roghani Monireh , Palmer Michelle A. , Rifkin Daniel B. , Moscatelli David A. , Lang Richard A.
Published: April 2021

To determine whether fibroblast growth factor (FGF) has a role in lens development, we have generated transgenic mice expressing a dominant-negative form of the murine FGF receptor-1 (FGFRDN) in the lens. Using the fibre cell- specific αA-crystallin promoter to express the FGFRDN, we have asked whether FGF is required for fibre cell differentiation. The transgenic mice display diminished differentiation of fibre cells as indicated by their reduced elongation. In addition, transgenic lenses have an unusual refractile anomaly that morphological and biochemical data show results from the apoptosis of fibre cells in the central region of the lens. These results show that lens fibre cells are dependent on FGF for their survival and differentiation, and demonstrate that growth factor deprivation in vivo can lead to apoptosis.
An investigation into early placental ontogeny: allantoic attachment to the chorion is selective and developmentally regulated
Authors: Downs Karen M. , Gardner Richard L.
Published: April 2021

Culture of postimplantation conceptuses was used in conjunction with microsurgery to investigate the timing, the mechanism and the developmental regulation of chorioallantoic fusion in the mouse. The timing of fusion was determined in both freshly recovered conceptuses and in those that had been cultured from as early as the mid-streak stage. Attachment of the allantois to the chorion was found to have occurred in most conceptuses by the 6-somite stage, irrespective of whether they had been cultured.

In investigating the mechanism of fusion, we wished to determine whether it depended on directed growth of the allantoic bud or on its differential adhesion to the chorion. Microsurgery was used to transplant allantoic tissue into the exocoelomic cavity of conceptuses from which the resident allantois had been removed. In synchronous grafting experiments, transplanted allantoises typically attached to the chorion despite loss of their connection with the hindgut region of the fetus. Hence selective attachment of the allantois to the chorion clearly cannot depend simply on its directed growth.

While the transplanted allantoic tissue attached to the chorion selectively, it did not attach to it precociously, despite being favourably positioned to do so. These findings argue that the initial attachment of the allantois to the chorion depends on a selective adhesive mechanism that is developmentally regulated. Further grafting experiments in which donor conceptuses were either more or less advanced than hosts revealed that attachment of the allantois to the chorion depends primarily on the stage of the allantois rather than on the stage of the chorion.

Collectively, these findings support the hypothesis that the initial stage of chorioallantoic fusion depends on selective adhesion between regionally differentiated mesodermal surfaces which is governed principally by the stage of development of the allantois.
The Xenopus homologue of Otx2 is a maternal homeobox gene that demarcates and specifies anterior body regions
Authors: Pannese Maria , Polo Cristiana , Andreazzoli Massimiliano , Vignali Robert , Kablar Boris , Barsacchi Giuseppina , Boncinelli Edoardo
Published: April 2021

In this paper we study Xotx2, a Xenopus homeobox gene related to orthodenticle, a gene expressed in the developing head of Drosophila. The murine cognate, Otx2, is first expressed in the entire epiblast of prestreak embryos and later in very anterior regions of late-gastrulae, including the neuroectoderm of presumptive fore- and mid-brain. In Xenopus, RNase protection experiments reveal that Xotx2 is expressed at low levels throughout early development from unfertilized egg to late blastula, when its expression level significantly increases. Whole-mount in situ hybridization shows a localized expression in the dorsal region of the marginal zone at stage 9.5. At stage 10.25 Xotx2 is expressed in dorsal bottle cells and in cells of the dorsal deep zone fated to give rise to prechordal mesendoderm, suggesting a role in the specification of very anterior structures. In stage 10.5 gastrulae, Xotx2 transcripts start to be detectable also in presumptive anterior neuroectoderm, where they persist in subsequent stages. Various treatments of early embryos cause a general reorganization of Xotx2 expression. In particular, retinoic acid treatment essentially abolishes Xotx2 expression in neuroectoderm. Microinjection of Xotx2 mRNA in 1-, 2- and 4-cell stage embryos causes the appearance of secondary cement glands and partial secondary axes in embryos with reduced trunk and tail structures. The presence of the Xotx2 homeodomain is required to produce these effects. In particular, this homeodomain contains a specific lysine residue at position 9 of the recognition helix. Microinjected transcripts of Xotx2 constructs containing a homeodomain where this lysine is substituted by a glutamine or a glutamic acid residue fail to cause these effects.
Induction of the prospective neural crest of Xenopus
Authors: Mayor Roberto , Morgan Richard , Sargent Michael G.
Published: April 2021

The earliest sign of the prospective neural crest of Xenopus is the expression of the ectodermal component of Xsna (the Xenopus homologue of snail) in a low arc on the dorsal aspect of stage 11 embryos, which subsequently assumes the horseshoe shape characteristic of the neural folds as the convergence-extension movements shape the neural plate. A related zinc-finger gene called Slug (Xslu) is expressed specifically in this tissue (i.e. the prospective crest) when the convergence extension movements are completed. Sub-sequently, Xslu is found in pre- and post-migratory cranial and trunk neural crest and also in lateral plate mesoderm after stage 17. Both Xslu and Xsna are induced by mesoderm from the dorsal or lateral marginal zone but not from the ventral marginal zone. From stage 10.5, explants of the prospective neural crest, which is underlain with tissue, are able to express Xslu. However expression of Xsna is not apparently specified until stage 12 and further contact with the inducer is required to raise the level of expression to that seen later in development. Xslu is specified at a later time. Embryos injected with noggin mRNA at the 1-cell stage or with plasmids driving noggin expression after the start of zygotic transcription express Xslu in a ring surrounding the embryo on the ventroposte-rior side. We suggest this indicates (a) that noggin interacts with another signal that is present throughout the ventral side of the embryo and (b) that Xslu is unable to express in the neural plate either because of the absence of a co-inducer or by a positive prohibition of expression. The ventral co-inducer, in the presence of overexpressed noggin, seems to generate an anterior/posterior pattern in the ventral part of the embryo comparable to that seen in neural crest of normal embryos. We suggest that the prospective neural crest is induced in normal embryos in the ectoderm that overlies the junction of the domains that express noggin and Xwnt-8. In support of this, we show animal cap explants from blastulae and gastrulae, treated with bFGF and noggin express Xslu but not NCAM although the mesoderm marker Xbra is also expressed. Explants treated with noggin alone express NCAM only. An indication that induction of the neural plate border is regulated independently of the neural plate is obtained from experiments using ultraviolet irradiation in the pre-cleavage period. At certain doses, the cranial crest domains are not separated into lateral masses and there is a reduction in the size of the neural plate.
Serrate expression can functionally replace Delta activity during neuroblast segregation in the Drosophila embryo
Authors: Gu Yi , Hukriede Neil A. , Fleming Robert J.
Published: April 2021

Serrate and Delta encode structurally related proteins in D. melanogaster that bind within a common extracellular region on the NOTCH receptor molecule. We used ectopic expression to determine if SERRATE could mediate in vivo functions parallel or antagonistic to those proposed for the putative NOTCH ligand DELTA. Our results demonstrate that Serrate can replace Delta gene function during embryonic neuroblast segregation and that expression of Serrate leads to a NOTCH-dependent suppression of achaete expression in proneural clusters. Our findings strongly suggest that SERRATE functions as an alternative ligand capable of NOTCH activation.
The T gene is necessary for normal mesodermal morphogenetic cell movements during gastrulation
Authors: Wilson Valerie , Manson Linda , Skarnes William C. , Beddington Rosa S. P.
Published: April 2021

The T (Brachyury) deletion in mouse is responsible for defective primitive streak and notochord morphogenesis, leading to a failure of the axis to elongate properly posterior to the forelimb bud. T/T embryonic stem (ES) cells colonise wild-type embryos, but in chimeras at 10.5 days post coitum (dpc) onwards they are found predominantly in the distal tail, while trunk paraxial and lateral mesoderm are deficient in T/T cells (Wilson, V., Rashbass, P. and Beddington, R. S. P. (1992) Development 117, 1321-1331). To determine the origin of this abnormal tissue distribution, we have isolated T/T and control T/+ ES cell clones which express lacZ constitutively using a gene trap strategy. Visualisation of T/T cell distribution in chimeric embryos throughout gastrulation up to 10.5 dpc shows that a progressive buildup of T/T cells in the primitive streak during gastrulation leads to their incorporation into the tailbud. These observations make it likely that one role of the T gene product is to act during gastrulation to alter cell surface (probably adhesion) properties as cells pass through the primitive streak.

As the chimeric tail elongates at 10.5 dpc, abnormal morphology in the most distal portion becomes apparent. Comparison of T expression in the developing tailbud with the sites of accumulation of T/T cells in chimeras shows that T/T cells collect in sites where T would normally be expressed. T expression becomes internalised in the tailbud following posterior neuropore closure while, in abnormal chimeric tails, T/T cells remain on the surface of the distal tail. We conclude that prevention of posterior neuropore closure by the wedge of T/T cells remaining in the primitive streak after gastrulation is one source of the abnormal tail phenotypes observed.

Accumulation of T/T cells in the node and anterior streak during gastrulation results in the preferential incorporation of T/T cells into the ventral portion of the neural tube and axial mesoderm. The latter forms compact blocks which are often fused with the ventral neural tube, reminiscent of the notochordal defects seen in intact mutants. Such fusions may be attributed to cell-autonomous changes in cell adhesion, possibly related to those observed at earlier stages in the primitive streak.
Zygotic caudal regulation by hunchback and its role in abdominal segment formation of the Drosophila embryo
Authors: Schulz Cordula , Tautz Diethard
Published: April 2021

caudal (cad) is a maternally and zygotically expressed gene in Drosophila whereby the two phases of expression can functionally replace each other. The zygotic expression forms an abdominal and a posterior domain, whereby only the posterior domain has so far been studied with respect to its regulation and function. We show here that the abdominal cad domain is regulated by the hunchback (hb) gradient through repression at high concentrations and activation at low concentrations of HB protein. To study the function of the abdominal cad domain in the absence of redundant interactions, we have utilized an experimental system in which the embryo lacks the normal bicoid (bcd) and hb expression. An artificial hb gradient is then intro-duced into such embryos, which results in an induction of an ectopic zygotic cad domain in the more anterior region. Employing this system, we show that the cad domain functions by activating the expression of the abdominal gap genes knirps (kni) and giant (gt). We conclude that cad is the so far missing region-specific activator of abdominal segmentation genes.
Digit tip regeneration correlates with regions of Msx1 (Hox 7) expression in fetal and newborn mice
Authors: Reginelli Angela D. , Wang Yao-Qi , Sassoon David , Muneoka Ken
Published: April 2021

We report that during mouse fetal development transcripts of Msx1 and Msx2 become progressively restricted to cells that will form more distal digit structures; the Msx2 expression domain is always more distal than Msx1. At birth both Msx1 and Msx2 are expressed in cells of the nail bed and hair follicle. We have found that the regenerative ability of mouse digit tips is restricted to levels in which the amputation plane is within the region of Msx1, but not Msx2, expression in early fetal digits and to levels where both Msx1 and Msx2 are expressed in late fetal and neonatal digits. Fetal digit tip regeneration is rapid and completed by birth, whereas neonatal digit tip regeneration requires 4 weeks and is sometimes imperfect. In both fetal and neonatal digits, we find that both Msx1 and Msx2 are expressed during regeneration, but not during wound healing associated with proximal amputations where no regenerative response is observed. These data support the hypothesis that the expression of Msx genes are important for digit cells to initiate and participate in a regenerative response.
Capacitation of mouse spermatozoa: I. Correlation between the capacitation state and protein tyrosine phosphorylation
Authors: Visconti Pablo E. , Bailey Janice L. , Moore Grace D. , Pan Dieyun , Olds-Clarke Patricia , Kopf Gregory S.
Published: April 2021

The molecular basis of mammalian sperm capacitation, defined functionally as those processes that confer on the sperm the acquisition of fertilization-competence either in vivo in the female reproductive tract or in vitro, is poorly understood. We demonstrate here that capacitation of caudal epididymal mouse sperm in vitro is accompanied by a time-dependent increase in the protein tyrosine phosphorylation of a subset of proteins of Mr 40,000–120,000. Incubation of sperm in media devoid of bovine serum albumin, CaCl2 or NaHCO3, components which individually are required for capacitation, prevent the sperm from undergoing capacitation as assessed by the ability of the cells to acquire the pattern B chlortetracycline fluorescence, to undergo the zona pellucida-induced acrosome reaction and, in some cases, to fertilize metaphase IIarrested eggs in vitro. In each of these cases the protein tyrosine phosphorylation of the subset of capacitation-associated proteins does not occur. Protein tyrosine phosphorylation of these particular proteins, as well as sperm capacitation, can be recovered in media devoid of each of these three constituents (bovine serum albumin, CaCl2 or NaHCO3) by adding back the appropriate component in a concentration-dependent manner. The requirement of NaHCO3 for these phosphorylations is not due to an alkalinization of intracellular sperm pH or to an increase in media pH. Caput epididymal sperm, which lack the ability to undergo capacitation in vitro, do not display this capacitation-dependent subset of tyrosine phosphorylated proteins in complete media even after extended incubation periods, and do not fertilize metaphase II-arrested eggs in vitro. These data suggest that protein tyrosine phosphorylation in sperm may represent an important regulatory pathway that may ultimately modulate events associated with capacitation.
Capacitation of mouse spermatozoa: II. Protein tyrosine phosphorylation and capacitation are regulated by a cAMP-dependent pathway
Authors: Visconti Pablo E. , Moore Grace D. , Bailey Janice L. , Leclerc Pierre , Connors Stephanie A. , Pan Dieyun , Olds-Clarke Patricia , Kopf Gregory S.
Published: April 2021

In the accompanying report (Visconti, P.E., Bailey, J.L., Moore, G.D., Pan, D., Olds-Clarke, P. and Kopf, G.S. (1995) Development, 121, 1129–1137) we demonstrated that the tyrosine phosphorylation of a subset of mouse sperm proteins of Mr 40,000–120,000 was correlated with the capacitation state of the sperm. The mechanism by which protein tyrosine phosphorylation is regulated in sperm during this process is the subject of this report. Cauda epididymal sperm, when incubated in media devoid of NaHCO3, CaCl2 or bovine serum albumin do not display the capacitation-associated increases in protein tyrosine phosphorylation of this subset of proteins. This NaHCO3, CaCl2 or bovine serum albumin requirement for protein tyrosine phosphorylation can be completely overcome by the addition of biologically active, but not inactive, cAMP analogues. Addition of the active cAMP analogues to sperm incubated in media devoid of NaHCO3, CaCl2 or bovine serum albumin overcomes the inability of these media to support capacitation, as assessed by the ability of the cells to acquire the pattern B chlortetracycline fluorescence, to undergo the zona pellucida-induced acrosome reaction and, in some cases, to fertilize metaphase II-arrested eggs in vitro. The effects of the cAMP analogues to enhance protein tyrosine phosphorylation and to promote capacitation appears to be at the level of the cAMP-dependent protein kinase (PKA), since two specific inhibitors of this enzyme (H-89 and Rp-cAMPS) block the capacitationdependent increases in protein tyrosine phosphorylation in sperm incubated in media supporting capacitation. Capacitation, as assessed by the aforementioned endpoints, also appears to be inhibited by H-89 in a concentrationdependent manner. These results provide further evidence for the interrelationship between protein tyrosine phosphorylation and the appearance of the capacitated state in mouse sperm. They also demonstrate that both protein tyrosine phosphorylation and capacitation appear to be regulated by cAMP/PKA. Up-regulation of protein tyrosine phosphorylation by cAMP/PKA in sperm is, to our knowledge, the first demonstration of such an interrelationship between tyrosine kinase/phosphatase and PKA signaling pathways.
Analysis of the mechanism(s) of metaphase I arrest in maturing mouse oocytes
Authors: Hampl Aleš , Eppig John J.
Published: April 2021

Fully grown mouse oocytes are normally competent to progress from prophase I to metaphase II without interruption. However, growing mouse oocytes initially become only partially competent to undergo meiotic maturation. Meiotic maturation in these oocytes does not progress beyond metaphase I. In contrast to the oocytes of most strains of mice, most oocytes of strain LT/Sv mice become arrested at metaphase I even when they are fully grown. The initiation of oocyte maturation is correlated with an increase in p34cdc2 kinase activity that continues to rise until metaphase I. The transition into anaphase I is normally correlated with a decrease in p34cdc2 kinase activity. This study demonstrated that metaphase I arrest in both partially competent growing oocytes and fully grown LT/Sv oocytes is correlated with a sustained elevation of p34cdc2 kinase activity. In fact, p34cdc2 activity continued to increase during the time when activity normally decreased. In normally maturing oocytes, some, but not all, of the cyclin B, the regulatory protein associated with p34cdc2, became degraded in oocytes that entered anaphase I. In contrast, the amount of cyclin B present in the metaphase I-arrested oocytes continued to increase at the time when it was being degraded in normal oocytes progressing to metaphase II. These results suggest that the progression of meiosis is arrested at metaphase I in both groups of oocytes because of continued p34cdc2 kinase activity sustained, at least in part, by restricted degradation of cyclin B. Finally, metaphase I arrest in these oocytes is sustained by a mechanism that differs from that maintaining metaphase II arrest, since an inhibitor of protein phosphorylation, 6-DMAP, induced a dramatic decline in p34cdc2 kinase activity and the resumption of meiosis in metaphase I-arrested oocytes but not in metaphase IIarrested oocytes. Moreover, without 6-DMAP treatment, cyclin B was more stable in the metaphase I-arrested oocytes than in metaphase II-arrested oocytes.
Developmental expression of mouse stromelysin-3 mRNA
Authors: Lefebvre Olivier , Régnier Catherine , Chenard Marie-Pierre , Wendling Corinne , Chambon Pierre , Basset Paul , Rio Marie-Christine
Published: April 2021

We have used northern blot analysis and in situ hybridization to study the spatial distribution of stromelysin-3 (ST3) expression during mouse embryogenesis. ST3 mRNA was observed in trophoblastic cells at the site of embryonic implantation (7.5–8.5 days) and in a variety of developing embryonic tissues. In these tissues, the highest ST3 expression levels were observed during the development of the external features of limb, tail and snout, and during bone and spinal cord morphogenesis. In limb, tail and snout, ST3 expression was specifically detected in mesenchymal cells lining the basement membrane at the junction of primitive dermis and epidermis, and adjacent to epithelial cells undergoing proliferation and/or apoptosis. In bone, ST3 was expressed in invasive mesenchymal cells and, in the spinal cord in neuroepithelial cells of the floor plate, at the time that this structure is crossed by commissural axons. Altogether, these observations suggest a role for ST3 during embryonic morphogenesis, in tissue remodeling processes associated with cell proliferation, death and/or invasion. Moreover, when compared to urokinase and tissue plasminogen activators, the spatiotemporal pattern of ST3 expression shows some similarities, but was not completely superimposable, suggesting that these genes may cooperate in some developing tissues and have specific functions in others.
Hoxa 11 structure, extensive antisense transcription, and function in male and female fertility
Authors: Hsieh-Li Hsiu Mei , Witte David P. , Weinstein Michael , Branford William , Li Hung , Small Kersten , Potter S. Steven
Published: April 2021

Hoxa 11 is a murine Abdominal-B-type homeobox gene. The structure of this gene is presented, including genomic and cDNA sequence. The cDNA includes the complete open reading frame and based on primer extension results is near full length. Surprisingly, the antisense strand of Hoxa 11 was found to be transcribed. Moreover, these antisense transcripts were processed and polyadenylated. The developmental expression patterns for both sense and antisense transcripts were examined using serial section and wholemount in situ hybridizations. Hoxa 11 transcription patterns were defined in the limbs, kidney and stromal cells surrounding the Mullerian and Wolffian ducts. Of particular interest, in the developing limbs, the sense and antisense transcripts showed complementary expression patterns, with antisense RNAs increasing in abundance in regions where sense RNAs were diminishing in abundance. Furthermore, targeted mutation of Hoxa 11 is shown to result in both male and female sterility. The female mutants produce normal ova, which develop properly postfertilization when transferred to wild-type surrogate mothers. The Hoxa 11 homozygous mutants are shown to provide a defective uterine environment. The mutant males exhibited a malformation of the vas deferens that resembles a partial homeotic transformation to an epididymis. In addition, the mutant testes fail to descend properly into the scrotum and, likely as a result, spermatogenesis is perturbed.
Multiple roles for FGF-3 during cranial neural development in the chicken
Authors: Mahmood Radma , Kiefer Paul , Guthrie Sarah , Dickson Clive , Mason Ivor
Published: April 2021

FGF-3 has been implicated in the development of the hindbrain and otocyst in vertebrate embryos. Since the chicken embryo offers a favourable system in which to study the development of these structures, we have isolated and characterised cDNAs for chicken Fgf-3 and determined its pattern of expression in chick embryos from stage 3 (primitive streak) to stage 25 (early organogenesis). Within the developing cranial neural tube, Fgf-3 exhibits dynamic spatial and temporal expression. During extension of the head process, RNA is detected in the midline of the developing neural plate. In neurulating embryos, transcripts are observed initially in rhombomeres 4 and 5 of the hindbrain and later, in rhombomere 6. During hindbrain development, expression is lost from these rhombomeres, but becomes restricted to rhombomere boundaries, providing an intracellular marker which distinguishes a population of cells within boundary regions. Fgf-3 expression is elevated in ventral and medial boundary regions and is greatly reduced in dorsal parts. Studies of regenerating rhombomere boundaries show that Fgf-3 expression is induced in reforming boundaries when evennumbered rhombomere tissue is grafted next to odd, but not when like is juxtaposed to like. Fgf-3 disappears from boundary regions just prior to the loss of the morphological boundaries suggesting a boundary-associated function.

Other sites of expression have also been identified. At early stages of development Fgf-3 is expressed in the epiblast and mesendoderm of the primitive streak, in mesoderm lateral to the streak and in Hensen’s node. In older embryos transcripts are detected in the endoderm of the pharyngeal pouches, the ectoderm of the second and third pharyngeal arches and the stomodeum. Expression was also detected in the segmental plate and in the posterior half of the three most-recently generated somites.
Terminal diversification of the myocyte lineage generates Purkinje fibers of the cardiac conduction system
Authors: Gourdie Robert G. , Mima Tatsuo , Thompson Robert P. , Mikawa Takashi
Published: April 2021

The rhythmic contraction of the vertebrate heart is dependent on organized propagation of electrical excitation through the cardiac conduction system. Because both muscleand neuron-specific genes are co-expressed in cells forming myocardial conduction tissues, two origins, myogenic and neural, have been suggested for this specialized tissue. Using replication-defective retroviruses, encoding recombinant β-galactosidase (β-gal), we have analyzed cell lineage for Purkinje fibers (i.e., the peripheral elements of the conduction system) in the chick heart. Functioning myocyte progenitors were virally tagged at embryonic day 3 of incubation (E3). Clonal β-gal+ populations of cells, derived from myocytes infected at E3 were examined at 14 (E14) and 18 (E18) days of embryonic incubation. Here, we report that a subset of clonally related myocytes differentiates into conductile Purkinje fibers, invariably in close spatial association with forming coronary arterial blood vessels. These β-gal+ myogenic clones, containing both working myocytes and Purkinje fibers, did not incorporate cells contributing to tissues of the central conduction system (e.g. atrioventricular ring and bundles). In quantitative analyses, we found that whereas the number of β-gal+ myocyte nuclei per clone more than doubled between E14 and E18, the number of β-gal+ Purkinje fiber nuclei remained constant. These data provide evidence that: (1) Purkinje fibers and working myocytes share a common myogenic precursor in the embryonic tubular heart; (2) differentiation of Purkinje fibers may involve earlier commitment to non-proliferation than that of working myocytes and; (3) the peripheral (i.e., the intramural Purkinje fiber network) and central components of the cardiac conduction system are derived from independent parental cells, and that these two components are linked together to establish the sequentially integrated conduction system of the adult heart. We discuss potential mechanisms for induction of Purkinje fibers from embryonic myocytes in association with coronary vasculogenesis.
A cloned, immortal line of murine melanoblasts inducible to differentiate to melanocytes
Authors: Sviderskaya Elena V. , Wakeling William F. , Bennett Dorothy C.
Published: April 2021

Cultures of differentiated melanocytes can readily be grown from the dissociated epidermis of neonatal mice, and immortal cell lines often develop from these. However, the first cells that grow and transiently dominate the cultures, while similar to melanocytes, are unpigmented. These have been shown to be precursors of melanocytes and may be termed melanoblasts. Under our previous standard culture conditions, involving the use of keratinocyte feeder cells, foetal calf serum, the phorbol ester 12-O-tetradecanoyl phorbol acetate (TPA) and cholera toxin, all the melanoblasts spontaneously differentiated to pigmented melanocytes within about 3 weeks. We now describe some factors affecting the proliferation and differentiation of diploid murine melanoblasts in the presence of serum. Murine stem cell factor/steel factor (SCF), basic fibroblast growth factor (bFGF) and murine leukaemia inhibitory factor/differentiation-inhibiting activity (LIF/DIA) all increased melanoblast numbers. SCF and LIF also slightly inhibited melanoblast differentiation, while cholera toxin and TPA promoted differentiation. Using some of these findings, and by regular replacement of keratinocyte or fibroblastoid feeder cells, we have established a clonal line of immortal murine melanoblasts, ‘melb-a’. These cells express tyrosinase-related protein-2 but not, in general, tyrosinase. They can be induced to differentiate irreversibly to functional melanocytes (also proliferative and immortal) by plating in the absence of feeder cells. Thus a new immortal melanocyte line, ‘melan-a2’, has also been produced.
Analysis of cubitus interruptus regulation in Drosophila embryos and imaginal disks
Authors: Schwartz Carol , Locke John , Nishida Craig , Kornberg Thomas B.
Published: April 2021

The cubitus interruptus (ci) gene of Drosophila is expressed in all anterior compartment cells in both embryos and imaginal disks where it encodes a putative zinc-finger protein related to the vertebrate Gli and C. elegans Tra-1 proteins. Using ci/lacZ fusions, we located regulatory sequences responsible for the normal pattern of ci expression, and obtained evidence that separate elements regulate its expression in embryos and imaginal disks. Mutants that delete a portion of this regulatory region express ci ectopically in the posterior compartments of their wing imaginal disks and have wings with malformed posterior compartments. Similar deletions of ci/lacZ fusion constructs also result in ectopic posterior compartment expression. Evidence that the engrailed protein normally represses ci in posterior compartments includes the expansion of ci expression into posterior compartment cells that lack engrailed function, diminution of ci expression upon overexpression of engrailed protein in anterior compartment cells, and the ability of engrailed protein to bind to the ci regulatory region in vivo and in vitro. We suggest that engrailed protein directly represses ci expression in posterior compartment cells.
Differentiating neurons activate transcription of the brain lipid-binding protein gene in radial glia through a novel regulatory element
Authors: Feng Lei , Heintz Nathaniel
Published: April 2021

Formation and maintenance of a radial glial scaffold is fundamental for development of the vertebrate central nervous system. In mammals, radial glia arise in the neuroepithelium immediately prior to differentiation and migration of neurons away from the ventricular zones, and they are maintained until neuronal migration subsides. We have previously shown that expression of the brain lipid-binding protein (BLBP) in radial glia throughout the developing CNS is strictly correlated with the differentiation and migration of neurons upon these cells, and that BLBP function is required to maintain differentiation of primary cerebellar glial cells in vitro (Feng, L., Hatten, M. E. and Heintz, N. (1994). Neuron 12, 895-908). In this study, we demonstrate that BLBP transcription in vivo involves multiple regulatory elements, and that the dynamic temporal and spatial pattern of BLBP expression in radial and Bergmann glial cells throughout the developing CNS is programmed by a single radial glial cell-specific element (RGE). Furthermore, we demonstrate that BLBP expression in primary cerebellar glial cells requires coculture with differentiating neurons, and that this induction is regulated by the radial glia-specific element. The fact that transcription of BLBP in response to neurons in vitro and its dynamic regulation in radial glia through-out the CNS in vivo are both controlled by the RGE provides the first direct evidence supporting a role for differentiating neurons in the epigenetic regulation of radial glial cell function in vivo.
Control of neuronal pathway selection by the Drosophila LIM homeodomain gene apterous
Authors: Lundgren Scott E. , Callahan Christopher A. , Thor Stefan , Thomas John B.
Published: April 2021

The Drosophila apterous gene encodes a LIM homeo-domain protein expressed embryonically in a small subset of differentiating neurons. To establish the identity of these neurons and to study the role of apterous in their development, we made apterous promoter fusions to an axon-targeted reporter gene. We found that all apterous-expressing neurons are interneurons that choose a single pathway within the developing central nervous system. In apterous mutants, these neurons choose incorrect pathways and fail to fasciculate with one another. Our results indicate that apterous functions to control neuronal pathway selection and suggest that other vertebrate and invertebrate members of the LIM homeodomain class of proteins may serve similar functions.
Regulation of the infraabdominal regions of the bithorax complex of Drosophila by gap genes
Authors: Casares Fernando , Sánchez-Herrero Ernesto
Published: April 2021

The expression of the abdominal-A and Abdominal-B genes of the bithorax complex of Drosophila is controlled by cis-regulatory infraabdominal regions. The activation of these regions along the anteroposterior axis of the embryo determines where abdominal-A and Abdominal-B are transcribed. There is spatially restricted transcription of the infraabdominal regions (infraabdominal transcripts) that may reflect this specific activation. We show that the gap genes hunchback, Krüppel, tailless and knirps control abdominal-A and Abdominal-B expression early in development. The restriction of abdominal-A and Abdominal-B transcription is preceded by (and requires) the spatially localized activation of regulatory regions, which can be detected by the distribution of infraabdominal transcripts. The activation of these regions (except the infraabdominal-8 one) could require no specific gap gene. Instead, a general mechanism of activation, combined with repression by gap genes in the anteroposterior axis, seems to be responsible for delimiting infraabdominal active domains. The gradients of the hunchback and Krüppel products seem to be key elements in this restricted activation.
A sex-specific number of germ cells in embryonic gonads of Drosophila
Authors: Poirié Marylène , Niederer Eva , Steinmann-Zwicky Monica
Published: April 2021

Male first instar larvae possess more germ cells in their gonads than female larvae of the same stage. To determine the earliest time point of sexual dimorphism in germ cell number, we have counted the germ cells of sexed embryos at different developmental stages. We found no difference in germ cell number of male and female embryos at the blastoderm and early gastrulation stage, or when germ cells are about to exit the midgut pocket. We find, however, that males have significantly more germ cells than females as soon as the germ cells are near the places where the gonads are formed and in all later stages. Our results show that germ cells are subject to a sex-specific control mechanism that regulates the number of germ cells already in embryos.