bluuebunny/crossref_metadata_2025 huggingface dataset:
Keratinocyte growth factor and its receptor are involved in regulating early lung branching
Authors: Post Martin , Souza Patricia , Liu Jason , Tseu Irene , Wang Jinxia , Kuliszewski Maciej , Tanswell A. Keith
Published: April 2021

Lung branching morphogenesis depends on mesenchymalepithelial tissue interactions. Keratinocyte growth factor (KGF) has been implicated to be a regulator of these tissue interactions. In the present study, we investigated the role of KGF in early rat lung organogenesis. Reverse transcriptase-polymerase chain reaction analysis revealed KGF mRNA expression in the mesenchymal component of the 13-day embryonic lung, while message for KGF receptor (KGFR) was expressed in the epithelium, confirming the paracrine nature of KGF/KGFR axis. Antisense KGF oligonucleotides inhibited DNA synthesis of embryonic lung explants. This inhibitory effect of antisense KGF was partially reversed by the addition of exogenous KGF. Recombinant KGF was mitogenic for 13-day isolated embryonic lung epithelial cells. Medium conditioned by 13-day lung mesenchymal cells also stimulated DNA synthesis of 13-day embryonic lung epithelial cells. This stimulatory effect was partially abrogated by a neutralizing KGF antibody. The number of terminal buds of lung explants cultured in the presence of antisense KGF oligonucleotides was significantly reduced compared to control explants. Exogenous KGF partially abrogated the inhibitory effect of antisense KGF on early lung branching. Sense or scrambled KGF oligonucleotides had no inhibitory effect on lung growth and branching. Addition of neutralizing KGF antibodies to the explants also reduced the degree of branching, while non-immune IgG and neutralizing acidic FGF antibodies had no effect. Explants incubated with antisense oligonucleotides targeted to the initiation site of translation of both the splice variants of the fibroblast growth factor receptor-2 (FGFR2) gene, KGFR and bek, exhibited a similar reduction in lung branching as observed with antisense KGF oligonucleotides. Antisense KGFR-specific oligonucleotides dramatically inhibited lung branching, while exposure of explants to antisense bek-specific oligonucleotides resulted in reduced branching albeit to a lesser degree than that observed with antisense KGFR-specific oligonucleotides. Neither sense nor scrambled KGFR-specific oligonucleotides had any effect on early lung branching. These results suggest that the KGF/KGFR system has a critical role in early lung organogenesis.
Neuronal cell migration in C. elegans: regulation of Hox gene expression and cell position
Authors: Harris Jeanne , Honigberg Lee , Robinson Naomi , Kenyon Cynthia
Published: April 2021

In C. elegans, the Hox gene mab-5, which specifies the fates of cells in the posterior body region, has been shown to direct the migrations of certain cells within its domain of function. mab-5 expression switches on in the neuroblast QL as it migrates into the posterior body region. mab-5 activity is then required for the descendants of QL to migrate to posterior rather than anterior positions. What information activates Hox gene expression during this cell migration? How are these cells subsequently guided to their final positions? We address these questions by describing four genes, egl-20, mig-14, mig-1 and lin-17, that are required to activate expression of mab-5 during migration of the QL neuroblast. We find that two of these genes, egl-20 and mig-14, also act in a mab-5-independent way to determine the final stopping points of the migrating Q descendants. The Q descendants do not migrate toward any obvious physical targets in wild-type or mutant animals. Therefore, these genes appear to be part of a system that positions the migrating Q descendants along the anteroposterior axis.
A conserved region of engrailed, shared among all en-, gsc-, Nk1-, Nk2- and msh-class homeoproteins, mediates active transcriptional repression in vivo
Authors: Smith Sheryl T. , Jaynes James B.
Published: April 2021

The engrailed homeoprotein is a dominantly acting or ‘active’ transcriptional repressor both in cultured cells and in vivo. When retargeted via a homeodomain swap to the endogenous fushi tarazu gene (ftz), it actively represses it, resulting in a ftz mutant phenocopy. We have mapped functional regions of engrailed using this in vivo repression assay. In addition to a region containing an active repression domain identified in cell culture assays (K. Han and J. L. Manley, 1993), we find that two evolutionarily conserved regions contribute to activity. The one of these that does not flank the HD is particularly crucial to repression activity in vivo. We find that this domain is present not only in all engrailed-class homeo-proteins but also in all known members of several other classes, including goosecoid, Nk1, Nk2 and msh. Thus engrailed’s active repression function in vivo is dependent on a highly conserved interaction that was established early in the evolution of the homeobox gene superfamily. We further show using rescue transgenes that the widely conserved in vivo repression domain is required for the normal function of engrailed in the embryo.
Undifferentiated cells in the developing Drosophila eye influence facet assembly and require the Fat facets ubiquitin-specific protease
Authors: Huang Yongzhao , Fischer-Vize Janice A.
Published: April 2021

The Drosophila compound eye develops by a complex series of cell interactions where multiple positive and inhibitory cues guide cells in each facet into their positions and fates. The results of many genetic and molecular experiments have led to the view that facet assembly is directed by cells within developing ommatidial preclusters. Here fat facets mutants and the cloned fat facets gene were used to show that, in order to limit the number of photoreceptors in a facet to eight, undifferentiated cells surrounding assembling facets send an inhibitory signal to extraneous cells within the facet preclusters. Generation of the inhibitory signal requires the ubiquitin-specific protease encoded by the fat facets gene and is thus regulated by ubiquitin-dependent proteolysis.
Targeted disruption of the Wnt2 gene results in placentation defects
Authors: Monkley Susan J. , Delaney Stephen J. , Pennisi David J. , Christiansen Jeffrey H. , Wainwright Brandon J.
Published: April 2021

Wnt genes have been implicated in a range of developmental processes in the mouse including the patterning of the central nervous system and limbs. Reported here for the first time is the expression of Wnt2 in the early heart field of 7.5–8.5 dpc (days post-coitum) mouse embryos, making Wnt2 a potentially useful gene marker for the early stages of heart development. Expression was also detected in the allantois from 8.0 dpc and at later stages in the placenta and umbilicus. Mice deficient in Wnt2, generated by gene targeting, displayed runting and approximately 50% died perinatally. Histological analysis revealed alterations in the size and structure of placentas from these mice from 14.5 dpc. The placental defects were associated primarily with the labyrinthine zone and included oedema and tissue disruption and accumulation of maternal blood in large pools. There was also an apparent decrease in the number of foetal capillaries and an increase in the amount of fibrinoid material in the Wnt2 mutant placentas. These results suggest that Wnt2 is required for the proper vascularisation of the mouse placenta and the placental defects in Wnt2-deficient mice result in a reduction in birthweight and perinatal lethality.
Pax2 contributes to inner ear patterning and optic nerve trajectory
Authors: Torres Miguel , Gómez-Pardo Emilia , Gruss Peter
Published: April 2021

During gestation, the paired box-containing gene Pax2 is expressed in the mid-hindbrain area, developing eye and inner ear. We generated Pax2 null mutant mice, which show the requirement of Pax2 for the establishment of axonal pathways along the optic stalks and ventral diencephalon. In mutant brains, the optic tracts remain totally ipsilateral due to agenesis of the optic chiasma. Furthermore, Pax2 mutants show extension of the pigmented retina into the optic stalks and failure of the optic fissure to close resulting in coloboma. In the inner ear, Pax2 mutants show agenesis of the cochlea and the spiral ganglion, i.e., the parts of the organ responsible for auditory function and in whose primordium Pax2 is expressed. Our results identify Pax2 as a major regulator of patterning during organogenesis of the eye and inner ear and indicate its function in morphogenetic events required for closure of the optic fissure and neural tube.
The regeneration of the cephalic neural crest, a problem revisited: the regenerating cells originate from the contralateral or from the anterior and posterior neural fold
Authors: Couly Gérard , Grapin-Botton Anne , Coltey Pierre , Douarin Nicole M. Le
Published: April 2021

The mesencephalic and rhombencephalic levels of origin of the hypobranchial skeleton (lower jaw and hyoid bone) within the neural fold have been determined at the 5-somite stage with a resolution corresponding to each single rhombomere, by means of the quail-chick chimera technique. Expression of certain Hox genes (Hoxa-2, Hoxa-3 and Hoxb-4) was recorded in the branchial arches of chick and quail embryos at embryonic days 3 (E3) and E4. This was a pre-requisite for studying the regeneration capacities of the neural crest, after the dorsal neural tube was resected at the mesencephalic and rhombencephalic level. We found first that excisions at the 5-somite stage extending from the mid-mesencephalon down to r8 are followed by the regeneration of neural crest cells able to compensate for the deficiencies so produced. This confirmed the results of previous authors who made similar excisions at comparable (or older) developmental stages. When a bilateral excision was followed by the unilateral homotopic graft of the dorsal neural tube from a quail embryo, thus mimicking the situation created by a unilateral excision, we found that the migration of the grafted unilateral neural crest (quail-labelled) is bilateral and compensates massively for the missing crest derivatives. The capacity of the intermediate and ventral neural tube to yield neural crest cells was tested by removing the chick rhombencephalic neural tube and replacing it either uni- or bilaterally with a ventral tube coming from a stage-matched quail. No neural crest cells exited from the ventral neural tube but no deficiency in neural crest derivatives was recorded. Crest cells were found to regenerate from the ends of the operated region. This was demonstrated by grafting fragments of quail neural fold at the extremities of the excised territory. Quail neural crest cells were seen migrating longitudinally from both the rostral and caudal ends of the operated region and filling the branchial arches located inbetween. Comparison of the behaviour of neural crest cells in this experimental situation with that showed by their normal fate map revealed that crest cells increase their proliferation rate and change their migratory behaviour without modifying their Hox code.
Structure of the insect head as revealed by the EN protein pattern in developing embryos
Authors: Rogers Bryan T. , Kaufman Thomas C.
Published: April 2021

The structure of the insect head has long been a topic of enjoyable yet endless debate among entomologists. More recently geneticists and molecular biologists trying to better understand the structure of the head of the Dipteran Drosophila melanogaster have joined the discourse extrapolating from what they have learned about Drosophila to insects in general. Here we present the results of an investigation into the structure of the insect head as revealed by the distribution of engrailed related protein (Engrailed) in the insect orders Diptera, Siphonaptera, Orthoptera and Hemiptera. The results of this comparative embryology in conjunction with genetic experiments on Drosophila melanogaster lead us to conclude: (1) The insect head is composed of six Engrailed accumulating segments, four postoral and two preoral. The potential seventh and eighth segments (clypeus or labrum) do not accumulate Engrailed. (2) The structure known as the dorsal ridge is not specific to the Diptera but is homologous to structures found in other insect orders. (3) A part of this structure is a single segment-like entity composed of labial and maxillary segment derivatives which produce the most anterior cuticle capable of taking a dorsal fate. The segments anterior to the maxillary segment produce only ventral structures. (4) As in Drosophila, the process of segmentation of the insect head is fundamentally different from the process of segmentation in the trunk. (5) The pattern of Engrailed accumulation and its presumed role in the specification and development of head segments appears to be highly conserved while its role in other pattern formation events and tissue-specific expression is variable. An overview of the pattern of Engrailed accumulation in developing insect embryos provides a basis for discussion of the generality of the parasegment and the evolution of Engrailed patterns.
A relationship between apoptosis and flow during programmed capillary regression is revealed by vital analysis
Authors: Meeson Annette , Palmer Michelle , Calfon Marcella , Lang Richard
Published: April 2021

Previous analyses of developmentally programmed capillary regression suggested two distinct causes of vascular endothelial cell (VEC) death. The first appeared to be macrophage-dependent (Lang, R. A. and Bishop, M.

J. (1993) Cell 74, 453-462) while the second was proposed to result from cessation of blood flow (Lang, R. A., Lustig, M., Francois, F., Sellinger, M. and Plesken, H. (1994). Development 120, 3395-3403). Combined, these analyses suggested a model in which initial, macrophage-mediated endothelial cell apoptosis blocked blood flow within a capillary segment and, as a consequence, caused apoptosis of all remaining cells in the affected segment.

In the current study, we have tested this model using a new method that combines vital and histological analyses as a means of determining the fate of whole capillary segments and individual cells in vivo. This technique revealed that one of the first events in regression was the apoptosis of a single VEC in otherwise normal, flowing capillary segments (initiating apoptosis). These isolated, dying VECs projected into and restricted the capillary lumen, imposing either a temporary or permanent block to blood flow. Following cessation of flow, synchronous apoptosis of VECs occurred (secondary apoptosis). In addition, a quantitative analysis revealed a reciprocal relationship between plasma flow and VEC apoptosis. These observations are consistent with a model for capillary regression in which macrophages induce apoptosis in a limited number of VECs and, as a consequence of a block to blood flow, also cause apoptosis in those remaining.
valentino: a zebrafish gene required for normal hindbrain segmentation
Authors: Moens Cecilia B. , Yan Yi-Lin , Appel Bruce , Force Allan G. , Kimmel Charles B.
Published: April 2021

Mutational analysis can serve both to identify new genes essential for patterning embryonic development and to determine their functions. Here we describe the identifica-tion and phenotypic characterization of alleles of valentino, which we recovered in a genetic screen that sought to identify mutations in the zebrafish that disrupt region-specific gene expression patterns in the embryonic brain. valentino is required for normal hindbrain segmentation and the hindbrain of valentino mutant embryos is shortened by the length of one rhombomere. We demon-strate that valentino is required cell-autonomously in the development of rhombomeres 5 and 6, and propose that valentino functions in the subdivision and expansion of a common precursor region in the presumptive hindbrain into the definitive rhombomeres 5 and 6. These results provide genetic evidence for a two-segment periodicity in the hindbrain and suggest that this periodicity arises sequentially, through the specification and later subdivi-sion of a two-rhombomere unit, or ‘protosegment’.
Xenopus VegT RNA is localized to the vegetal cortex during oogenesis and encodes a novel T-box transcription factor involved in mesodermal patterning
Authors: Zhang Jian , King Mary Lou
Published: April 2021

An RNA localized to the vegetal cortex of Xenopus oocytes encodes a novel T-box protein (VegT) capable of inducing either dorsal or posterior ventral mesoderm at different times in development. VegT is a nuclear protein and its C-terminal domain can activate transcription in a yeast reporter assay, observations consistent with VegT functioning as a transcription factor. Zygotic expression is dynamic along the dorsoventral axis, with transcripts first expressed in the dorsal marginal zone. By the end of gas-trulation, VegT is expressed exclusively in posterior ventral and lateral mesoderm and is excluded from the notochord. Later expression is confined to a subset of Rohon-Beard cells, a type of primary sensory neuron. In animal cap assays, VegT is capable of converting prospective ectoderm into ventral lateral mesoderm. Such ectopic expression of VegT induces its own expression as well as that of Xwnt-8 in caps, suggesting that a Wnt pathway may be involved. Mis-expression of VegT in dorsal animal blastomeres fated to contribute to brain suppresses head formation. Our results suggest that VegT is a localized transcription factor, which operates sequentially in several developmental pathways during embryogenesis, including dorsoventral and posterior patterning of mesoderm.
The Xenopus T-box gene, Antipodean, encodes a vegetally localised maternal mRNA and can trigger mesoderm formation
Authors: Stennard Fiona , Carnac Gilles , Gurdon J. B.
Published: April 2021

We have used differential display to identify genes inducible by activin and isolated a novel member of the T-box gene family that includes the Xenopus genes Xbrachyury and Eomesodermin. Here we show that this novel gene is unique within the T-box family because it is maternally expressed at a high level. Furthermore, it belongs to a rare class of maternal mRNAs in Xenopus that are localised to the vegetal hemisphere of the egg and we have therefore named it Antipodean. We show here that low amounts of Antipodean injected into ectoderm (animal cap cells) strongly induce pan mesodermal genes such as Xbrachyury and ventral mesodermal genes such as Xwnt-8. Overexpression of Antipodean generates mesoderm of ventral character, and induces muscle only weakly. This property is consistent with the observed late zygotic Antipodean mRNA expression in the posterior paraxial mesoderm and ventral blastopore, and its exclusion from the most dorsal mesodermal structure, the notochord. Antipodean is induced by several molecules of the TGF-β class, but in contrast to Xbrachyury, not by bFGF. This result suggests that the expression of these T-box genes may be under the control of different regulatory pathways. Finally, we demonstrate that Antipodean and Eomesoder-min induce each other and both are able to induce Xbrachyury. The early zygotic expression of Antipodean is not induced by Xbrachyury, though later it is to some extent. Considering its maternal content, Antipodean could initiate a cascade of T-box gene activations. The expression of these genes may, in turn, sustain each other’s expression to define and maintain the mesoderm identity in Xenopus.
Prenatal and postnatal requirements of NT-3 for sympathetic neuroblast survival and innervation of specific targets
Authors: ElShamy Wael M. , Linnarsson Sten , Lee Kuo-Fen , Jaenisch Rudolf , Ernfors Patrik
Published: April 2021

Postnatal homozygous neurotrophin-3 mutant mice display a loss of about half the sympathetic superior cervical ganglion (SCG) neurons (Ernfors, P., Lee, K.-F., Kucera, J. and Jaenisch, R. (1994a) Cell 77, 503-512; Farinas, I., Jones, K. R., Backus, C., Wang, X. Y. and Reichardt, L. F. (1994) Nature 369, 658-661). We found that this loss is caused by excessive apoptosis of sympathetic neuroblasts leading to a failure to generate a normal number of neurons during neurogenesis. NT-3 was also found to be required postnatally. In Nt-3−/− mice, sympathetic fibers failed to invade pineal gland and external ear postnatally; whereas other targets of the external and internal carotid nerves, including the submandibular gland and the iris, displayed a normal complement of sympathetic innervation. Sympathetic fibers of mice carrying one functional copy of the Nt-3 gene (Nt-3+/− mice) invaded the pineal gland, but failed to branch and form a ground plexus. Cultured neonatal sympathetic neurons responded to NT-3 by neurite outgrowth and mRNA upregulation of the NT-3 receptor, trkC. Exogenously administered NT-3 promoted sympathetic growth and rescued the sympathetic target deficit of the mutant mice. We conclude that NT-3 is required for the survival of sympathetic neuroblasts during neurogenesis and for sympathetic innervation and branching in specific targets after birth.
Pax-3 is necessary for migration but not differentiation of limb muscle precursors in the mouse
Authors: Daston George , Lamar Elise , Olivier Marc , Goulding Martyn
Published: April 2021

The limb muscles of vertebrates are derived from precursor cells that migrate from the lateral edge of the dermomyotome into the limb bud. Previous studies have shown that the paired domain-containing transcription factor Pax-3 is expressed in the limb in cells that are precursors for limb muscles (Williams, B. and Ordahl, C. P. (1994) Development 120, 785–796). In splotch (Pax-3–) embryos, the limb muscles fail to develop and cells expressing Pax-3 are no longer found in the limb. In this paper we have analyzed the role of Pax-3 in the migration and subsequent differentiation of limb muscle precursors. By labeling somites adjacent to the prospective forelimb with the lipophilic dye DiI, we have shown that cells derived from these somites do not migrate into the limbs of splotch mice. The failure of limb muscle precursors to invade the limb in splotch mice is associated with the absence of c-met expression in premigratory cells, together with a change in the morphology of the ventral dermomyotome. In addition, we have shown the lateral half of somites derived from day E9.25 splotch embryos can undergo muscle differentiation when grafted into the limb bud of stage 20 chick host embryos. Our results indicate that Pax-3 regulates the migration of limb muscle precursors into the limb and is not required for cells in the lateral somite to differentiate into muscle.
Expression of type 1 inositol 1,4,5-trisphosphate receptor during axogenesis and synaptic contact in the central and peripheral nervous system of developing rat
Authors: Dent Myrna A. R. , Raisman Geoff , Lai F. Anthony
Published: April 2021

Release of intracellular Ca2+ is triggered by the second messenger inositol 1,4,5-trisphosphate, which binds to the inositol 1,4,5-trisphosphate receptor and gates the opening of an intrinsic calcium channel in the endoplasmic reticulum. In order to understand the importance of this mechanism in development, we have examined the distribution of the type 1 inositol 1,4,5-trisphosphate receptor during development, in some areas of the rat brain and spinal cord and in peripheral neurons, using in situ hybridization and immunohistochemistry. In brain, we find that type 1 inositol 1,4,5-trisphosphate receptor is expressed in neurons from very early in development; low levels of expression are first detected after the neurons have migrated to their final positions, when they start to differentiate and begin axonal growth. Increasing levels of expression are observed later in development, during the time of synaptogenesis and dendritic contact. Glial cells do not express type 1 inositol 1,4,5-trisphosphate receptor, except for a transient period of expression, probably by oligodendrocytes, in developing fibre tracts during the onset of myelination. In contrast with the brain, both grey and white matter of the spinal cord express type 1 inositol 1,4,5-trisphosphate receptor throughout development, and it remains present in the adult spinal cord. We also show, for the first time, that type 1 inositol 1,4,5-trisphosphate receptor is expressed in the peripheral nervous system. Strong labelling was observed in the dorsal root ganglia and during development this expression seems to coincide with the onset of axogenesis. These results suggest that type 1 inositol 1,4,5-trisphosphate receptor may be involved in the regulatory mechanism controlling Ca2+ levels in neurons during the periods of cell differentiation, axogenesis and synaptogenesis.
Analysis of cell movement during the culmination phase of Dictyostelium development
Authors: Dormann Dirk , Siegert Florian , Weijer Cornelis J.
Published: April 2021

Co-ordinated cell movement of tens of thousands of cells and periodic signals characterise the multicellular development of the cellular slime mould Dictyostelium discoideum. We investigated cell movement by analysing timelapse video recordings made during the slug stage and the culmination phase of Dictyostelium development. Slugs viewed from the side showed an even, straight forward movement with the tip slightly raised in the air. Slugs, that had migrated for a prolonged period of time either culminated or showed a behaviour best described as abortive culmination. Culmination is initiated by a local aggregation of anterior-like cells at the base of the slug at the prestalkprespore boundary, where they form a stationary mass of cells. Prespore cells continue to move forward over this stationary pile and, as a result, are lifted into the air. The stationary group of anterior-like cells thereby end up to the back of the slug. At this point the slug either falls back on the agar surface or continues culmination. If the slug continues to migrate these cells regain motility, move forward to the prespore-prestalk boundary and form a new pile again. In the case of culmination the neutral red stained cells in the pile move to the back of the slug and form a second signalling centre beside the tip. Both centres are characterised by vigorous rotational cell movement. The cells belonging to the basal centre will form the basal disc and the lower cup in the fruiting body. The upper cup will be formed by the prestalk cells rotating most vigorously at the prestalk-prespore boundary. The remaining neutral red stained anterior-like cells in the prespore zone sort either to the upper or lower organising centre in the fruiting body.
Late effects of retinoic acid on neural crest and aspects of rhombomere identity
Authors: Gale Emily , Prince Victoria , Lumsden Andrew , Clarke Jon , Holder Nigel , Maden Malcolm
Published: April 2021

We exposed st.10 chicks to retinoic acid (RA), both globally, and locally to individual rhombomeres, to look at its role in specification of various aspects of hindbrain derived morphology. Previous studies have looked at RA exposure at earlier stages, during axial specification. Stage 10 is the time of morphological segmentation of the hindbrain and is just prior to neural crest migration. Rhombomere 4 localised RA injections result in specific alterations of pathways some crest cells that normally migrate to sites of differentiation of neurogenic derivatives. The r4 crest cells that give rise to mesenchymal derivatives are unaffected. In addition, r4 gene expression is also partially altered by RA; within 6 hours of r4 exposure to RA, ectopic expression of Krox-20 is seen in r4 and Hoxb-1 expression is lost while Hoxa-2 expression continues normally. When we examined these RA-treated animals later in development, they showed an anterior displacement of the facial ganglion in addition to a mis-direction of the extensions of its distal axons and a dramatic decrease in the number of contralateral vestibuloacoustic neurons normally seen in r4. Only this r4-specific neuronal type is affected in r4; the motor neuron projections seem normal in experimental animals. The specificity of this result, combined with the loss of Hoxb-1 expression in r4 and the work by Krumlauf and co-workers showing gain of contralateral neurons co-localised with ectopic Hoxb-1 expression, indicates a role for Hoxb-1 and RA in the specification of this cell type in normal development. These results suggest that RA, at st.10, is able to affect some aspects of segment identity while leaving others unchanged.
Mos is required for MAP kinase activation and is involved in microtubule organization during meiotic maturation in the mouse
Authors: Verlhac Marie-Hélène , Kubiak Jacek Z. , Weber Michèle , Géraud Gérard , Colledge William H. , Evans Martin J. , Maro Bernard
Published: April 2021

Mos is normally expressed during oocyte meiotic maturation in vertebrates. However, apart from its cytostatic factor (CSF) activity, its precise role during mouse meiosis is still unknown. First, we analyzed its role as a MAP kinase kinase kinase. Mos is synthesized concomitantly with the activation of MAP kinase in mouse oocytes. Moreover, MAP kinase is not activated during meiosis in oocytes from mos–/– mice. This result implies that Mos is necessary for MAP kinase activation in mouse oocytes. Raf-1, another MAP kinase kinase kinase, is already present in immature oocytes, but does not seem to be active when MAP kinase is activated. Moreover, the absence of MAP kinase activation in mos–/– oocytes demonstrates that Raf-1 cannot compensate for the lack of Mos. These results suggest that Raf1 is not involved in MAP kinase activation. Second, we analyzed the organization of the microtubules and chromosomes in oocytes from mos–/– mice. We observed that during the transition between two meiotic M-phases, the microtubules and chromosomes evolve towards an interphase-like state in mos–/– oocytes, while in the control mos+/− oocytes they remain in an M-phase configuration, as in the wild type. Moreover, after spontaneous activation, the majority of mos–/– oocytes are arrested for at least 10 hours in a third meiotic M-phase where they exhibit monopolar half-spindles. These observations present the first evidence, in intact oocytes, of a role for the Mos/…/MAP kinase cascade in the control of microtubule and chromatin organization during meiosis.
Dysgenesis of cephalic neural crest derivatives in Pax7−/− mutant mice
Authors: Mansouri Ahmed , Stoykova Anastasia , Torres Miguel , Gruss Peter
Published: April 2021

Pax7 is a member of the paired box containing gene family. Its expression pattern suggests a function in cephalic neural crest derivatives, skeletal muscle and central nervous system development. To understand the role of Pax7 during mouse embryogenesis, we used the homologous recombination technique in embryonic stem cells and generated Pax7–/– mice. Homozygous animals are born but die shortly after weaning. They exhibit malformations in facial structures involving the maxilla and nose. Our analysis suggests that the observed phenotype is due to a cephalic neural crest defect. No obvious phenotype could be detected in the central nervous system and skeletal muscle. Functional redundancy between Pax7 and Pax3 is discussed.
Allele-specific in situ hybridization (ASISH) analysis: a novel technique which resolves differential allelic usage of H19 within the same cell lineage during human placental development
Authors: Adam Gail I. R. , Cui Hengmi , Miller Stephen J. , Flam Folke , Ohlsson Rolf
Published: April 2021

Precursory studies of H19 transcription during human foetal development have demonstrated maternally derived monoallelic expression. Analyses in extra-embryonic tissues, however, have been more equivocal, with discernible levels of expression of the paternal allele of H19 documented in the first trimester placenta. By refining the in situ hybridization technique we have developed an assay to enable the functional imprinting status of H19 to be determined at the cellular level. This assay involves the use of oligonucleotide DNA probes that are able to discriminate between allelic RNA transcripts containing sequence polymorphisms. Biallelic expression of H19 is confined to a subpopulation of cells of the trophoblast lineage, the extravillous cytotrophoblast, while the mesenchymal stroma cells maintain the imprinted pattern of monoallelic expression of H19 throughout placental development. This data demonstrates that the low level of paternal H19 expression previously detected in normal human placenta is not due to a random loss of functional imprinting, but appears to result from a developmentally regulated cell type-specific activation of the paternal allele. In addition, biallelic expression of H19 does not seem to affect the functional imprinting of the insulin-like growth factor II gene, which is monoallelically expressed at relatively high levels in the extra-villous cytotrophoblasts. These results imply that the allelic usage of these two genes in normal human placental development may not be directly analogous to the situation previously documented in the mouse embryo.
Rhombomere of origin determines autonomous versus environmentally regulated expression of Hoxa3 in the avian embryo
Authors: Saldivar Jose R. , Krull Catherine E. , Krumlauf Robb , Ariza-McNaughton Linda , Bronner-Fraser Marianne
Published: April 2021

We have investigated the pattern and regulation of Hoxa3 expression in the hindbrain and associated neural crest cells in the chick embryo, using whole mount in situ hybridization in conjunction with DiI labeling of neural crest cells and microsurgical manipulations. Hoxa3 is expressed in the neural plate and later in the neural tube with a rostral border of expression corresponding to the boundary between rhombomeres (r) 4 and 5. Initial expression is diffuse and becomes sharp after boundary formation. Hoxa3 exhibits uniform expression within r5 after formation of rhombomeric borders. Cell marking experiments reveal that neural crest cells migrating caudally, but not rostrally, from r5 and caudally from r6 express Hoxa3 in the normal embryo. Results from transposition experiments demonstrate that expression of Hoxa3 in r5 neural crest cells is not strictly cell-autonomous. When r5 is transposed with r4 by rostrocaudal rotation of the rhombomeres, Hoxa3 is expressed in cells migrating lateral to transposed r5 and for a short time, in condensing ganglia, but not by neural crest within the second branchial arch. Since DiI-labeled cells from transposed r5 are present in the second arch, Hoxa3-expressing neural crest cells from r5 appear to down-regulate their Hoxa3 expression in their new environment. In contrast, when r6 is transposed to the position of r4 after boundary formation, Hoxa3 is maintained in both migrating neural crest cells and those positioned within the second branchial arch and associated ganglia. These results suggest that Hoxa3 expression is cellautonomous in r6 and its associated neural crest. Our results suggest that neural crest cells expressing the same Hox gene are not equivalent; they respond differently to environmental signals and exhibit distinct degrees of cell autonomy depending upon their rhombomere of origin.
The Drosophila stonewall gene encodes a putative transcription factor essential for germ cell development
Authors: Clark Kathleen A. , McKearin Dennis M.
Published: April 2021

The differentiation of Drosophila germ cells is a useful model for studying mechanisms of cell specification. We report the identification of a gene, stonewall, that is required for germ cell development. Mutations in stonewall block proper oocyte differentiation and frequently cause the presumptive oocyte to develop as a nurse cell. Eventually, germ cells degenerate apoptotically. Stonewall is a germ cell nuclear protein; Stonewall has a DNA binding domain that shows similarities to the Myb and Adf-1 transcription factors and has other features that suggest that it is a transcription activating factor. We suggest that Stonewall transcriptional regulation is essential in cystocytes for maturation into specialized nurse cells and oocyte.
The Drosophila tissue polarity gene inturned acts cell autonomously and encodes a novel protein
Authors: Park Woo Jin , Liu Jingchun , Sharp Edward J. , Adler Paul N.
Published: April 2021

Mutations in the inturned (in) gene result in abnormal wing hair polarity and in many wing cells forming two or more hairs instead of the normal single hair. We have generated genetic mosaics in a number of different experiments and find that the in gene is required in all regions of the wing and that it functions in a cell autonomous fashion. We report the molecular cloning of the in gene, the molecular mapping of in mutations and the isolation and sequencing of an in cDNA clone. The in gene encodes a novel protein whose sequence suggests it will be membrane bound. The ability of an in cDNA, the expression of which is driven by the basal activity of the hsp70 promoter to rescue an in mutation suggests that patterned expression of in is unlikely to play a role in the function of this gene.
Sex-specific control of Sex-lethal is a conserved mechanism for sex determination in the genus Drosophila
Authors: Bopp Daniel , Calhoun Gretchen , Horabin Jamila I. , Samuels Mark , Schedl Paul
Published: April 2021

In D. melanogaster the binary switch gene Sex-lethal (Sxl) plays a pivotal role in somatic sex determination – when the Sxl gene is on the female pathway is followed, while the male pathway is followed when the gene is off. In the present study we have asked whether the Sxl gene is present in other species of the genus Drosophila and whether it is subject to a similar sex-specific on-off regulation. Sxl proteins were found in all of the drosophilids examined, and they display a sex-specific pattern of expression. Furthermore, characterization of the Sxl gene in the distant drosophilan relative, D. virilis, reveals that the structure and sequence organization of the gene has been well conserved and that, like melanogaster, alternative RNA processing is responsible for its sex-specific expression. Hence, this posttranscriptional on-off regulatory mechanism probably existed before the separation of the drosophilan and sophophoran subgenera and it seems likely that Sxl functions as a sex determination switch gene in most species in the Drosophila genus. Although alternative splicing appears to be responsible for the on-off regulation of the Sxl gene in D. virilis, this species is unusual in that Sxl proteins are present not only in females but also in males. The D. virilis female and male proteins appear to be identical over most of their length except for the aminoterminal approx. 25 aa which are encoded by the differentially spliced exons. In transcriptionally active polytene chromosomes, the male and female proteins bind to the same cytogenetic loci, including the sites corresponding to the D. virilis Sxl and tra genes. Hence, though the male proteins are able to interact with appropriate target premRNAs, they are apparently incapable of altering the splicing pattern of these pre-mRNAs.
PDX-1 is required for pancreatic outgrowth and differentiation of the rostral duodenum
Authors: Offield Martin F. , Jetton Tom L. , Labosky Patricia A. , Ray Michael , Stein Roland W. , Magnuson Mark A. , Hogan Brigid L. M. , Wright Christopher V. E.
Published: April 2021

It has been proposed that the Xenopus homeobox gene, XlHbox8, is involved in endodermal differentiation during pancreatic and duodenal development (Wright, C. V. E., Schnegelsberg, P. and De Robertis, E. M. (1988). Development 105, 787–794). To test this hypothesis directly, gene targeting was used to make two different null mutations in the mouse XlHbox8 homolog, pdx-1. In the first, the second pdx-1 exon, including the homeobox, was replaced by a neomycin resistance cassette. In the second, a lacZ reporter was fused in-frame with the N terminus of PDX-1, replacing most of the homeodomain. Neonatal pdx-1–/– mice are apancreatic, in confirmation of previous reports (Jonsson, J., Carlsson, L., Edlund, T. and Edlund, H. (1994). Nature 371, 606-609). However, the pancreatic buds do form in homozygous mutants, and the dorsal bud undergoes limited proliferation and outgrowth to form a small, irregularly branched, ductular tree. This outgrowth does not contain insulin or amylase-positive cells, but glucagon-expressing cells are found. The rostral duodenum shows a local absence of the normal columnar epithelial lining, villi, and Brunner’s glands, which are replaced by a GLUT2-positive cuboidal epithelium resembling the bile duct lining. Just distal of the abnormal epithelium, the numbers of enteroendocrine cells in the villi are greatly reduced. The PDX-1/ β-galactosidase fusion allele is expressed in pancreatic and duodenal cells in the absence of functional PDX-1, with expression continuing into perinatal stages with similar boundaries and expression levels. These results offer additional insight into the role of pdx-1 in the determination and differentiation of the posterior foregut, particularly regarding the proliferation and differentiation of the pancreatic progenitors.
Modular cis-regulatory organization of Endo16, a gut-specific gene of the sea urchin embryo
Authors: Yuh Chiou-Hwa , Davidson Eric H.
Published: April 2021

The Endo16 gene of Strongylocentrotus purpuratus is expressed at the blastula stage of embryogenesis throughout the vegetal plate, at the gastrula stage in the whole of the archenteron and in postgastrular stages only in the midgut. We showed earlier that a 2300 bp upstream sequence suffices to faithfully recreate this pattern of expression when fused to a CAT reporter gene. Here we define the functional organization of this cis-regulatory domain, which includes over thirty high specificity binding sites, serviced by at least thirteen different putative transcription factors, in addition to >20 sites for a factor commonly found in the regulatory sequences of other sea urchin genes as well (SpGCF1). The Endo16 cis-regulatory domain consists of several different functional elements, or modules, each containing one or two unique DNA-binding factor target sites, plus sites for factors binding in other modules as well. Modular regulatory function was defined in experiments in which regions of the cis-regulatory DNA containing specific clusters of sites were tested in isolation, combined with one another, or by selective deletion, and the effects on expression of the CAT reporter were determined by whole-mount in situ hybridization or CAT enzyme activity measurements. The most proximal module (A) is mainly responsible for early embryonic expression, and module A alone suffices to locate expression in the vegetal plate and archenteron. The adjacent module (B) is responsible for a steep postgastrular rise in expression, when the gene is transcribed only in the midgut and, prior to this, module B alone also suffices to promote expression in the vegetal plate and archenteron. The most distal module, G, acts as a booster for either A or B modules. However, no combination of A, B and G modules generates vegetal plate or gut expression exclusively. Ectopic expression of A-, B- and G-CAT fusion constructs occurs in the adjacent (veg1-derived) ectoderm and in skeletogenic mesenchyme cells. For expression to be confined to endoderm requires negative regulatory functions mediated by modules E, F and DC. Modules E and F each repress ectopic expression specifically in veg1 ectoderm. Module DC represses ectopic expression specifically in skeletogenic mesenchyme. Expression of some Endo16 constructs is dramatically increased by treatment with LiCl, which expands the territory in which the endogenous Endo16 gene is expressed at the expense of veg1 ectoderm. The same modules that act to repress ectopic expression in untreated embryos are required for enhanced expression of constructs after LiCl treatment. Furthermore, both the negative spatial control functions and response to LiCl require the presence of module A. The total regulatory requirements of the Endo16 gene during embryogenesis can be expressed in terms of the positive and negative functions of the individual modules and the interactions between modules that are identified in this study.
The roles of hedgehog, wingless and lines in patterning the dorsal epidermis in Drosophila
Authors: Peter Bokor , DiNardo Stephen
Published: April 2021

Rows of cells that flank the parasegment boundary make up a signaling center within the epidermis of the Drosophila embryo. Signals emanating from these cells, encoded by hedgehog (hh) and wingless (wg), are shown to be required for all segment pattern dorsally. Wg activity is required for the differentiation of one cell type, constituting half the parasegment. The gene lines appears to act in parallel to the Wg pathway in the elaboration of this cell type. Hh activity is responsible for three other cell types in the parasegment. Some cell types are specified as Hh activity and interfere with the function of patched, analogous to patterning of imaginal discs. However, some pattern is independent of the antagonism of patched by Hh, and relies instead on novel interactions with lines. Lastly, we provide evidence that decapentaplegic does not mediate patterning by Hh in the dorsal epidermis.
Peptide YY expression is an early event in colonic endocrine cell differentiation: evidence from normal and transgenic mice
Authors: Upchurch Brent H. , Fung Brenda P. , Rindi Guido , Ronco Anne , Leiter Andrew B.
Published: April 2021

The hormone peptide YY is produced by endocrine cells in the pancreas, ileum and colon. We have previously shown that peptide YY is coexpressed in all four islet cell types in the murine pancreas when they first appear, suggesting a common peptide YY-producing progenitor. In the colon, peptide YY has been frequently identified in glucagon- expressing L-type endocrine cells. Characterization of colonic endocrine tumors in transgenic mice expressing simian virus 40 large T antigen under the control of the peptide YY gene 5′ flanking region revealed tumor cells producing not only peptide YY and glucagon, but also neurotensin, cholecystokinin, substance P, serotonin, secretin, and gastrin. This suggested that multiple enteroendocrine lineages were related to peptide YY-producing cells. Sub- sequent examination of the ontogeny of colonic endocrine differentiation in nontransgenic mice revealed that peptide YY was the first hormone to appear during development, at embryonic day 15.5. Between embryonic days 16.5 and 18.5, cells expressing glucagon, cholecystokinin, substance P, serotonin, secretin, neurotensin, gastrin and somato- statin first appeared and peptide YY was coexpressed in each cell type at this time. Peptide YY coexpression continued in a significant fraction of most enteroendocrine cell types throughout fetal and postnatal development and into adulthood, with the exception of serotonin-producing cells. This latter population of cells expanded dramatically after birth with rare coexpression of peptide YY. These studies indicate that expression of peptide YY is an early event in colonic endocrine differentiation and support the existence of a common progenitor for all endocrine cells in the colon.