bluuebunny/crossref_metadata_2025 huggingface dataset:
The Drosophila tango gene encodes a bHLH-PAS protein that is orthologous to mammalian Arnt and controls CNS midline and tracheal development
Authors: Sonnenfeld Margaret , Ward Mary , Nystrom Gerald , Mosher Jack , Stahl Stephanie , Crews Stephen
Published: April 2021

The Drosophila single-minded and trachealess bHLH-PAS genes control transcription and development of the CNS midline cell lineage and tracheal tubules, respectively. We show that Single-minded and Trachealess activate transcription by forming dimers with the Drosophila Tango protein that is an orthologue of the mammalian Arnt protein. Both cell culture and in vivo studies show that a DNA enhancer element acts as a binding site for both Single-minded∷Tango and Trachealess∷Tango heterodimers and functions in controlling CNS midline and tracheal transcription. Isolation and analysis of tango mutants reveal CNS midline and tracheal defects, and gene dosage studies demonstrate in vivo interactions between single-minded∷tango and trachealess∷tango. These experiments support the existence of an evolutionarily conserved, functionally diverse bHLH-PAS protein regulatory system.
Two signalling pathways specify localised expression of the Broad-Complex in Drosophila eggshell patterning and morphogenesis
Authors: Deng Wu-Min , Bownes Mary
Published: April 2021

The Drosophila eggshell, which has a pair of chorionic appendages (dorsal appendages) located asymmetrically along both the anterior/posterior and dorsal/ventral axes, provides a good model to study signal instructed morphogenesis. We show that the Broad-Complex, a gene encoding zinc-finger transcription factors, is essential for the morphogenesis of dorsal appendages and is expressed in a bilaterally symmetrical pattern in the lateral-dorsal-anterior follicle cells during late oogenesis. This is induced and specified along the dorsoventral axis by an epidermal growth factor receptor signalling pathway, which includes a localised transforming growth factor-alpha like molecule, Gurken, in the oocyte and the Drosophila EGF receptor homologue, Torpedo, in the surrounding somatic follicle cells. Furthermore, the precisely localised expression of BR-C along the AP axis requires a separate signalling pathway, initiated by a transforming growth factor-beta homologue, Decapentaplegic, in nearby follicle cells. These two signalling pathways, one from the oocyte and the other from the follicle cells, co- ordinately specify patches of follicle cells to express the Broad-Complex in a unique position in respect to both major axes, which in turn directs the differentiation of the dorsal appendages in the correct position on the eggshell.
Steroid regulated programmed cell death during Drosophila metamorphosis
Authors: Jiang Changan , Baehrecke Eric H. , Thummel Carl S.
Published: April 2021

During insect metamorphosis, pulses of the steroid hormone 20-hydroxyecdysone (ecdysone) direct the destruction of obsolete larval tissues and their replacement by tissues and structures that form the adult fly. We show here that larval midgut and salivary gland histolysis are stage-specific steroid-triggered programmed cell death responses. Dying larval midgut and salivary gland cell nuclei become permeable to the vital dye acridine orange and their DNA undergoes fragmentation, indicative of apoptosis. Furthermore, the histolysis of these tissues can be inhibited by ectopic expression of the baculovirus antiapoptotic protein p35, implicating a role for caspases in the death response. Coordinate stage-specific induction of the Drosophila death genes reaper (rpr) and head involution defective (hid) immediately precedes the destruction of the larval midgut and salivary gland. In addition, the diap2 anti-cell death gene is repressed in larval salivary glands as rpr and hid are induced, suggesting that the death of this tissue is under both positive and negative regulation. Finally, diap2 is repressed by ecdysone in cultured salivary glands under the same conditions that induce rpr expression and trigger programmed cell death. These studies indicate that ecdysone directs the death of larval tissues via the precise stage- and tissue-specific regulation of key death effector genes.
Human ZP3 restores fertility in Zp3 null mice without affecting order-specific sperm binding
Authors: Rankin Tracy L. , Tong Zhi-Bin , Castle Philip E. , Lee Eric , Gore-Langton Robert , Nelson Lawrence M. , Dean Jurrien
Published: April 2021

The mammalian zona pellucida surrounding ovulated eggs mediates sperm binding at fertilization, provides a postfertilization block to polyspermy, and facilitates passage of pre-implantation embryos down the oviduct. Although the three zona proteins (ZP1, ZP2, ZP3) are well conserved, mammalian fertilization is relatively specific and human sperm do not bind to the mouse zona pellucida. There are considerable in vitro data that ZP3 acts as a primary sperm adhesion molecule in mice and, by analogy, a similar role has been postulated for human ZP3. Genetically altered mice lacking ZP3 (Zp3tm/tm) do not form a zona pellucida and are infertile. To rescue this phenotype, transgenic mice expressing human ZP3 (67% identical to mouse ZP3) were produced and bred with Zp3tm/tm null mice. The resultant human ZP3 rescue females had chimeric zonae pellucidae composed of mouse ZP1, mouse ZP2 and human ZP3. Human ZP3 expressed in mouse oocytes had an apparent mass (64 kDa) indistinguishable from native human ZP3 and distinct from mouse ZP3 (83 kDa). Despite the presence of human ZP3, human sperm did not bind to the chimeric zona pellucida, and notwithstanding the absence of mouse ZP3, mouse sperm bound to ovulated eggs in vitro and fertility was restored in vivo. These data have implications regarding the molecular basis of mouse and human sperm binding to their respective zonae pellucidae.
Role of caudal in hindgut specification and gastrulation suggests homology between Drosophila amnioproctodeal invagination and vertebrate blastopore
Authors: Wu Linda H. , Lengyel Judith A.
Published: April 2021

During early embryogenesis in Drosophila, caudal mRNA is distributed as a gradient with its highest level at the posterior of the embryo. This suggests that the Caudal homeodomain transcription factor might play a role in establishing the posterior domains of the embryo that undergo gastrulation and give rise to the posterior gut. By generating embryos lacking both the maternal and zygotic mRNA contribution, we show that caudal is essential for invagination of the hindgut primordium and for further specification and development of the hindgut. These effects are achieved by the function of caudal in activating different target genes, namely folded gastrulation, which is required for invagination of the posterior gut primordium, and fork head and wingless, which are required to promote development of the internalized hindgut primordium. caudal is not sufficient for hindgut gastrulation and development, however, as it does not play a significant role in activating expression of the genes tailless, huckebein, brachyenteron and bowel. We argue that caudal and other genes expressed at the posterior of the Drosophila embryo (fork head, brachyenteron and wingless) constitute a conserved constellation of genes that plays a required role in gastrulation and gut development.
Identification of an evolutionarily conserved 110 base-pair cis-acting regulatory sequence that governs Wnt-1 expression in the murine neural plate
Authors: Rowitch David H. , Echelard Yann , Danielian Paul S. , Gellner Klaus , Brenner Sydney , McMahon Andrew P.
Published: April 2021

The generation of anterior-posterior polarity in the vertebrate brain requires the establishment of regional domains of gene expression at early somite stages. Wnt-1 encodes a signal that is expressed in the developing midbrain and is essential for midbrain and anterior hindbrain development. Previous work identified a 5.5 kilobase region located downstream of the Wnt-1 coding sequence which is necessary and sufficient for Wnt-1 expression in vivo. Using a transgenic mouse reporter assay, we have now identified a 110 base pair regulatory sequence within the 5.5 kilobase enhancer, which is sufficient for expression of a lacZ reporter in the approximate Wnt-1 pattern at neural plate stages. Multimers of this element driving Wnt-1 expression can partially rescue the midbrain-hindbrain phenotype of Wnt-1−/− embryos. The possibility that this region represents an evolutionarily conserved regulatory module is suggested by the identification of a highly homologous region located downstream of the wnt-1 gene in the pufferfish (Fugu rubripes). These sequences are capable of appropriate temporal and spatial activation of a reporter gene in the embryonic mouse midbrain; although, later aspects of the Wnt-1 expression pattern are absent. Genetic evidence has implicated Pax transcription factors in the regulation of Wnt-1. Although Pax-2 binds to the 110 base pair murine regulatory element in vitro, the location of the binding sites could not be precisely established and mutation of two putative low affinity sites did not abolish activation of a Wnt-1 reporter transgene in vivo. Thus, it is unlikely that Pax proteins regulate Wnt-1 by direct interactions with this cis-acting regulatory region. Our analysis of the 110 base pair minimal regulatory element suggests that Wnt-1 regulation is complex, involving different regulatory interactions for activation and the later maintenance of transgene expression in the dorsal midbrain and ventral diencephalon, and at the midbrain-hindbrain junction.
Epithelial-mesenchymal signaling during the regionalization of the chick gut
Authors: Roberts Drucilla J. , Smith Devyn M. , Goff Deborah J. , Tabin Clifford J.
Published: April 2021

The development of the vertebrate gut requires signaling between the endoderm and mesoderm for establishing its normal anteroposterior (AP) axis and for tissue-specific differentiation. Factors implicated in positional specification of the AP regions of the gut include endodermally expressed Sonic hedgehog (Shh), mesodermally expressed Bmp4 and members of the Hox gene family. We have investigated the roles of these factors during AP regional specification of the chick embryonic gut. Early in gut development, the endoderm sends inductive signals to the mesoderm. Shh has been implicated as one of these signals. We find a differential response to exposure of the inductive influence of Shh along the AP axis of the gut. Virally mediated misexpression of Shh results in ectopic upregulation of its receptor Ptc and a cellular proliferation throughout the gut mesoderm. Although ectopic Shh can induce Bmp4 in the mesoderm of the midgut and hindgut, Bmp4 is not induced in the stomach region of the foregut. The stomach region has a thicker layer of mesoderm than the rest of the gut suggesting that the normal function of Bmp4 could be to limit mesodermal growth in the non-stomach regions of the gut. Ectopic Bmp4 expression in the stomach results in a reduction of the mesodermal component consistent with this hypothesis. In addition to the regional restriction on Bmp4 induction, Shh can only induce Hoxd-13 in the mesoderm of the hindgut. These findings suggest that a prepattern exists in the primitive gut mesoderm prior to expression of Shh in the endoderm. The gut mesoderm is subsequently responsible for inducing region-specific differentiation of its overlying endoderm. We tested the role of Hoxd-13, normally restricted in its mesodermal expression to the most posterior region of the hindgut (cloaca), in controlling adjacent endodermal differentiation. When virally mediated Hoxd-13 is misexpressed in the primitive midgut mesoderm, there is a transformation of the endoderm to the morphology and mucin content of the hindgut. Thus, the positionally restricted expression of a Hox gene in the gut mesoderm influences the inductive signaling that leads to regionally specific differentiation of gut endoderm.
The development of apical embryonic pattern in Arabidopsis
Authors: Long Jeff A. , Barton M. Kathryn
Published: April 2021

The apical portion of the Arabidopsis globular stage embryo gives rise to the cotyledons and the shoot apical meristem (SAM). The SHOOT MERISTEMLESS (STM) gene is required for SAM formation during embryogenesis and for SAM function throughout the lifetime of the plant. To more precisely define the development of molecular pattern in the apical portion of the embryo, and the role of the STM gene in the development of this pattern, we have examined AINTEGUMENTA (ANT), UNUSUAL FLORAL ORGANS (UFO) and CLAVATA1 (CLV1) expression in wild-type and stm mutant embryos. The transcripts of these genes mark subdomains within the apical portion of the embryo. Our results indicate that: (1) the molecular organization characteristic of the vegetative SAM is not present in the globular embryo but instead develops gradually during embryogenesis; (2) radial pattern exists in the apical portion of the embryo prior to and independent of STM with STM expression itself responding to radial information; (3) the embryonic SAM consists of central and peripheral subdomains that express different combinations of molecular markers and differ in their ultimate fates; and (4) STM activity is required for UFO expression, STM is required for maintenance but not onset of CLV1 expression and the pattern of ANT expression is independent of STM.
POU transcription factors control expression of CNS stem cell-specific genes
Authors: Josephson R. , Müller T. , Pickel J. , Okabe S. , Reynolds K. , Turner P. A. , Zimmer A. , McKay R. D. G.
Published: April 2021

Multipotential stem cells throughout the developing central nervous system have common properties. Among these is expression of the intermediate filament protein nestin and the brain fatty acid binding protein (B-FABP). To determine if common mechanisms control transcription in CNS stem cells, the regulatory elements of these two genes were mapped in transgenic mice. A 257 basepair enhancer of the rat nestin gene is sufficient for expression throughout the embryonic neuroepithelium. This enhancer contains two sites bound by the class III POU proteins Brn-1, Brn-2, Brn-4, and Tst-1. Only one of the two POU sites is required for CNS expression. An adjacent hormone response element is necessary for expression in the dorsal midbrain and forebrain. The regulatory sites of the B-FABP gene are strikingly similar to those of the nestin gene. A hybrid POU/Pbx binding site is recognized in vitro by Pbx-1, Brn-1 and Brn-2. This site is essential for expression in most of the CNS. In addition, a hormone response element is necessary for forebrain expression. Both the nestin and B-FABP genes therefore depend on POU binding sites for general CNS expression, with hormone response elements additionally required for activity in the anterior CNS. These data indicate that regulation by POU proteins and hormone receptors is a general mechanism for CNS stem cell-specific transcription.
Geminin, a neuralizing molecule that demarcates the future neural plate at the onset of gastrulation
Authors: Kroll Kristen L. , Salic Adrian N. , Evans Louise M. , Kirschner Marc W.
Published: April 2021

In an expression cloning screen in Xenopus embryos, we identified a gene that when overexpressed expanded the neural plate at the expense of adjacent neural crest and epidermis. This gene, which we named geminin, had no sequence similarity to known gene families. We later discovered that geminin’s neuralizing domain was part of a bifunctional protein whose C-terminal coiled-coil domain may play a role in regulating DNA replication. We report here on the neuralizing function of geminin. The localization, effect of misexpression and activity of a dominant negative geminin suggest that the product of this gene has an essential early role in specifying neural cell fate in vertebrates. Maternal geminin mRNA is found throughout the animal hemisphere from oocyte through late blastula. At the early gastrula, however, expression is restricted to a dorsal ectodermal territory that prefigures the neural plate. Misexpression of geminin in gastrula ectoderm suppresses BMP4 expression and converts prospective epidermis into neural tissue. In ectodermal explants, geminin induces expression of the early proneural gene neurogenin-related 1 although not itself being induced by that gene. Later, embryos expressing geminin have an expanded dorsal neural territory and ventral ectoderm is converted to neurons. A putative dominant negative geminin lacking the neuralizing domain suppresses neural differentiation and, when misexpressed dorsally, produces islands of epidermal gene expression within the neurectodermal territory, effects that are rescued by coexpression of the full-length molecule. Taken together, these data indicate that geminin plays an early role in establishing a neural domain during gastrulation.
Key aspects of the primary sex determination mechanism are conserved across the genus Drosophila
Authors: Erickson James W. , Cline Thomas W.
Published: April 2021

In D. melanogaster, a set of ‘X:A numerator genes’, which includes sisterlessA (sisA), determines sex by controlling the transcription of Sex-lethal (Sxl). We characterized sisA from D. pseudoobscura and D. virilis and studied the timing of sisA and Sxl expression with single cell-cycle resolution in D. virilis, both to guide structure-function studies of sisA and to help understand sex determination evolution. We found that D. virilisSISA shares 58% amino acid identity with its melanogaster ortholog. The identities confirm SISA as an atypical bZIP transcription factor. Although virilisSISA can substitute for melanogasterSISA, the protein is not fully functional in a heterologous context. The putative sisA regulatory sequence CAGGTAG is a potential ‘numerator box,’ since it is shared with the other strong X:A numerator gene, sisB, and its target, SxlPe. Temporal and spatial features of sisA and SxlPe expression are strikingly conserved, including rapid onset and cessation of transcription in somatic nuclei, early cessation of sisA transcription in budding pole cells and persistent high-level sisA expression in yolk nuclei. Expression of sisA and Sxl is as tightly coupled in virilis as it is in melanogaster. Taken together, these data indicate that the same primary sex determination mechanism exists throughout the genus Drosophila.
Specification of cell fate in the sea urchin embryo: summary and some proposed mechanisms
Authors: Davidson Eric H. , Cameron R. Andrew , Ransick Andrew
Published: April 2021

An early set of blastomere specifications occurs during cleavage in the sea urchin embryo, the result of both conditional and autonomous processes, as proposed in the model for this embryo set forth in 1989. Recent experimental results have greatly illuminated the mechanisms of specification in some early embryonic territories, though others remain obscure. We review the progressive process of specification within given lineage elements, and with reference to the early axial organization of the embryo. Evidence for the conditional specification of the veg2 lineage subelement of the endoderm and other potential interblastomere signaling interactions in the cleavage-stage embryo are summarized. Definitive boundaries between mesoderm and endoderm territories of the vegetal plate, and between endoderm and overlying ectoderm, are not established until later in development. These processes have been clarified by numerous observations on spatial expression of various genes, and by elegant lineage labeling studies. The early specification events depend on regional mobilization of maternal regulatory factors resulting at once in the zygotic expression of genes encoding transcription factors, as well as downstream genes encoding proteins characteristic of the cell types that will much later arise from the progeny of the specified blastomeres. This embryo displays a maximal form of indirect development. The gene regulatory network underlying the embryonic development reflects the relative simplicity of the completed larva and of the processes required for its formation. The requirements for postembryonic adult body plan formation in the larval rudiment include engagement of a new level of genetic regulatory apparatus, exemplified by the Hox gene complex.
cFKBP/SMAP; a novel molecule involved in the regulation of smooth muscle differentiation
Authors: Fukuda Kimiko , Tanigawa Yoko , Fujii Gen , Yasugi Sadao , Hirohashi Setsuo
Published: April 2021

During embryogenesis, smooth muscle cells of the gut differentiate from mesenchymal cells derived from splanchnic mesoderm. We have isolated a gene involved in the differentiation of smooth muscle cells in the gut using differential display between the chicken proventriculus in which the smooth muscle layer develops poorly and the gizzard in which smooth muscles develop abundantly. The protein encoded by this gene showed highest similarity to mouse FK506 binding protein, FKBP65, and from the function of this protein it was designated chicken FKBP/smooth muscle activating protein (cFKBP/SMAP).

cFKBP/SMAP was first expressed in smooth muscle precursor cells of the gut and, after smooth muscles differentiate, expression was restricted to smooth muscle cells. In organ culture of the gizzard, the differentiation of smooth muscle cells was inhibited by the addition of FK506, the inhibitor of FKBPs. Moreover, overexpression of cFKBP/SMAP in lung and gizzard mesenchymal cells induced smooth muscle differentiation. In addition, cFKBP/SMAP-induced smooth muscle differentiation was inhibited by FK506. We postulate therefore that cFKBP/SMAP plays a crucial role in smooth muscle differentiation in the gut and provides a powerful tool to study smooth muscle differentiation mechanisms, which have been poorly analyzed so far.
Genetic interactions and dosage effects of Polycomb group genes in mice
Authors: Bel Sophie , Coré Nathalie , Djabali Malek , Kieboom Karin , Lugt Nathalie Van der , Alkema Mark J. , Lohuizen Maarten Van
Published: April 2021

In Drosophila and mouse, Polycomb group genes are involved in the maintenance of homeotic gene expression patterns throughout development. Here we report the skeletal phenotypes of compound mutants for two Polycomb group genes bmi1 and M33. We show that mice deficient for both bmi1 and M33 present stronger homeotic transformations of the axial skeleton as compared to each single Polycomb group mutant, indicating strong dosage interactions between those two genes. These skeletal transformations are accompanied with an enhanced shift of the anterior limit of expression of several Hox genes in the somitic mesoderm. Our results demonstrate that in mice the Polycomb group genes act in synergy to control the nested expression pattern of some Hox genes in somitic mesodermal tissues during development.
Transcriptional regulation of atonal during development of the Drosophila peripheral nervous system
Authors: Sun Yan , Jan Lily Yeh , Jan Yuh Nung
Published: April 2021

atonal is a proneural gene for the development of Drosophila chordotonal organs and photoreceptor cells. We show here that atonal expression is controlled by modular enhancer elements located 5′ or 3′ to the atonal-coding sequences. During chordotonal organ development, the 3′ enhancer directs expression in proneural clusters; whereas successive modular enhancers located in the 5′ region drive tissue-specific expression in chordotonal organ precursors in the embryo and larval leg, wing and antennal imaginal discs. Similarly, in the eye disc, the 3′ enhancer directs initial expression in a stripe anterior to the morphogenetic furrow. These atonal-expressing cells are then patterned through a Notch-dependent process into initial clusters, representing the earliest patterning event yet identified during eye morphogenesis. A distinct 5′ enhancer drives expression in intermediate groups and R8 cells within and posterior to the morphogenetic furrow. Both enhancers are required for normal atonal function in the eye. The 5′ enhancer, but not the 3′ enhancer, depends on endogenous atonal function, suggesting a switch from regulation directed by other upstream genes to atonal autoregulation during the process of lateral inhibition. The regulatory regions identified in this study can thus account for atonal expression in every tissue and essentially in every stage of its expression during chordotonal organ and photoreceptor development.
The distribution of endogenous retinoic acid in the chick embryo: implications for developmental mechanisms
Authors: Maden Malcolm , Sonneveld Edwin , van der Saag Paul T. , Gale Emily
Published: April 2021

The aim of these experiments was to determine the endogenous distribution of retinoic acid (RA) across a wide range of embryonic stages in the chick embryo. By high pressure liquid chromatography, it was revealed that didehydroRA is the most prevalent retinoic acid in the chick embryo and that the tissues of the stage 24 embryo differed widely in their total RA content (didehydroRA + all-trans-RA). Some tissues such as the heart had very little RA and some such as the neural tube had very high levels, the total variation between these two being 29-fold. We showed that these tissues also synthesised RA and released it into the medium, thus validating the use of the F9 reporter cell system for further analyses of younger staged embryos. With these F9 cells, we showed that, at stage 4, the posterior end of the embryo had barely detectably higher levels of RA than the anterior end, but that a significant level of RA generation was detected as soon as somitogenesis began. Then a sharp on/off boundary of RA was present at the level of the first somite. We could find no evidence for a posterior-to-anterior gradient of RA. Throughout further development, various consistent observations were made: the developing brain did not generate RA, but the spinal part of the neural tube generated it at very high levels so there must be a sharp on/off boundary in the region of the hindbrain/spinal cord junction; the mesenchyme surrounding the hindbrain generated RA whereas the hindbrain itself did not; there was a variation in RA levels from the midline outwards with the highest levels of RA in the spinal neural tube followed by lower levels in the somites followed by lower levels in the lateral plate; the posterior half of the limb bud generated higher levels than the anterior half. With these observations, we were able to draw maps of endogenous RA throughout these early stages of chick embryogenesis and the developmental implications of these results are discussed.
Differential activation of Myf5 and MyoD by different Wnts in explants of mouse paraxial mesoderm and the later activation of myogenesis in the absence of Myf5
Authors: Tajbakhsh S. , Borello U. , Vivarelli E. , Kelly R. , Papkoff J. , Duprez D. , Buckingham M. , Cossu G.
Published: April 2021

Activation of myogenesis in newly formed somites is dependent upon signals derived from neighboring tissues, namely axial structures (neural tube and notochord) and dorsal ectoderm. In explants of paraxial mesoderm from mouse embryos, axial structures preferentially activate myogenesis through a Myf5-dependent pathway and dorsal ectoderm preferentially through a MyoD-dependent pathway.

Here we report that cells expressing Wnt1 will preferentially activate Myf5 while cells expressing Wnt7a will preferentially activate MyoD. Wnt1 is expressed in the dorsal neural tube and Wnt7a in dorsal ectoderm in the early embryo, therefore both can potentially act in vivo to activate Myf5 and MyoD, respectively. Wnt4, Wnt5a and Wnt6 exert an intermediate effect activating both Myf5 and MyoD equivalently in paraxial mesoderm. Sonic Hedgehog synergises with both Wnt1 and Wnt7a in explants from E8.5 paraxial mesoderm but not in explants from E9.5 embryos.

Signaling through different myogenic pathways may explain the rescue of muscle formation in Myf5 null embryos, which do not form an early myotome but later develop both epaxial and hypaxial musculature. Explants of unsegmented paraxial mesoderm contain myogenic precursors capable of expressing MyoD in response to signaling from a neural tube isolated from E10.5 embryos, the developmental stage when MyoD is present throughout the embryo. Myogenic cells cannot activate MyoD in response to signaling from a less mature neural tube.

Together these data suggest that different Wnt molecules can activate myogenesis through different pathways such that commitment of myogenic precursors is precisely regulated in space and time to achieve the correct pattern of skeletal muscle development.
Involvement of the Sonic Hedgehog, patched 1 and bmp2 genes in patterning of the zebrafish dermal fin rays
Authors: Laforest Lynda , Brown Christopher W. , Poleo Germán , Géraudie Jacqueline , Tada Masazumi , Ekker Marc , Akimenko Marie-Andrée
Published: April 2021

The signaling molecule encoded by Sonic hedgehog (shh) participates in the patterning of several embryonic structures including limbs. During early fin development in zebrafish, a subset of cells in the posterior margin of pectoral fin buds express shh. We have shown that regulation of shh in pectoral fin buds is consistent with a role in mediating the activity of a structure analogous to the zone of polarizing activity (ZPA) (Akimenko and Ekker (1995)Dev. Biol. 170, 243 – 247). During growth of the bony rays of both paired and unpaired fins, and during fin regeneration, there does not seem to be a region equivalent to the ZPA and one would predict that shh would play a different role, if any, during these processes specific to fish fins. We have examined the expression of shh in the developing fins of 4-week old larvae and in regenerating fins of adults. A subset of cells in the basal layer of the epidermis in close proximity to the newly formed dermal bone structures of the fin rays, the lepidotrichia, express shh, and ptc1 which is thought to encode the receptor of the SHH signal. The expression domain of ptc1 is broader than that of shh and adjacent blastemal cells releasing the dermal bone matrix also express ptc1. Further observations indicate that the bmp2 gene, in addition to being expressed in the same cells of the basal layer of the epidermis as shh, is also expressed in a subset of the ptc1-expressing cells of the blastema. Amputations of caudal fins immediately after the first branching point of the lepidotrichia, and global administration of all-trans-retinoic acid, two procedures known to cause fusion of adjacent rays, result in a transient decrease in the expression of shh, ptc1 and bmp2. The effects of retinoic acid on shh expression occur within minutes after the onset of treatment suggesting direct regulation of shh by retinoic acid. These observations suggest a role for shh, ptc1 and bmp2 in patterning of the dermoskeleton of developing and regenerating teleost fins.
Avian neural crest-derived neurogenic precursors undergo apoptosis on the lateral migration pathway
Authors: Wakamatsu Yoshio , Mochii Makoto , Vogel Kristine S. , Weston James A.
Published: April 2021

Neural crest cells of vertebrate embryos disperse on distinct pathways and produce different derivatives in specific embryonic locations. In the trunk of avian embryos, crest-derived cells that initially migrate on the lateral pathway, between epidermal ectoderm and somite, produce melanocytes but no neuronal derivatives. Although we found that melanocyte precursors are specified before they disperse on the lateral pathway, we also observed that a few crest-derived neuronal cells are briefly present on the same pathway. Here, we show that neuronal cells are removed by an episode of apoptosis. These observations suggest that localized environmental factor(s) affect the distribution of fate-restricted crest derivatives and function as a ‘proof-reading mechanism’ to remove ‘ectopic’ crest-derived cells.
A potential role for the OTX2 homeoprotein in creating early ‘highways’ for axon extension in the rostral brain
Authors: Nguyen Ba-Charvet Kim Tuyen , Boxberg Ysander von , Guazzi Stefania , Boncinelli Edoardo , Godement Pierre
Published: April 2021

Brain pattern formation starts with a subdivision of the neuroepithelium through site-specific expression of regulatory genes and, subsequently, the boundaries between presumptive neuromeres may provide a scaffold for early formation of axon tracts. In the mouse forebrain, the transcription factor OTX2 is strongly expressed at several such boundaries. Combining dye tracing and staining for OTX2 protein, we show that a number of early fibre tracts develop within stripes of OTX2 expression. To analyse a putative influence of OTX2 on the expression of molecules involved in neurite growth, we generated several clones of NIH3T3 cells stably expressing OTX2 protein at varying levels. As shown by immunoblotting, Otx2 transfection affects the expression of a variety of cell and substratum adhesion molecules, rendering the cells a favourable substratum in neurite outgrowth assays. Among the molecules upregulated with increasing levels of OTX2 are NCAM, tenascin-C and DSD-1-PG, which also in situ colocalize with zones of OTX2 expression at boundaries. These data suggest that Otx2 might be involved in defining local substrata for axon extension in the forebrain.
Evidence for a frizzled-mediated wnt pathway required for zebrafish dorsal mesoderm formation
Authors: Nasevicius Aidas , Hyatt Tana , Kim Hyon , Guttman Julie , Walsh Erin , Sumanas Saulius , Wang Yanshu , Ekker Stephen C.
Published: April 2021

We have used zebrafish as a model system for the study of vertebrate dorsoventral patterning. We isolated a maternally expressed and dorsal organizer localized member of the frizzled family of wnt receptors. Wild-type and dominant, loss-of-function molecules in misexpression studies demonstrate frizzled function is necessary and sufficient for dorsal mesoderm specification. frizzled activity is antagonized by the action of GSK-3, and we show GSK-3 is also required for zebrafish dorsal mesoderm formation. frizzled cooperatively interacts with the maternally encoded zebrafish wnt8 protein in dorsal mesodermal fate determination. This frizzled -mediated wnt pathway for dorsal mesoderm specification provides the first evidence for the requirement of a wnt-like signal in vertebrate axis determination.
The sacral neural crest contributes neurons and glia to the post-umbilical gut: spatiotemporal analysis of the development of the enteric nervous system
Authors: Burns Alan J. , Douarin Nicole M. Le
Published: April 2021

The majority of the enteric nervous system is derived from vagal neural crest cells (NCC), which migrate to the developing gut, proliferate, form plexuses and differentiate into neurons and glia. However, for some time, controversy has existed as to whether cells from the sacral region of the neural crest also contribute to the enteric nervous system. The aim of this study was to investigate the spatiotemporal migration of vagal and sacral NCC within the developing gut and to determine whether the sacral neural crest contributes neurons and glia to the ENS. We utilised quail-chick chimeric grafting in conjunction with antibody labelling to identify graft-derived cells, neurons and glia. We found that vagal NCC migrated ventrally within the embryo and accumulated in the caudal branchial arches before entering the pharyngeal region and colonising the entire length of the gut in a proximodistal direction. During migration, vagal crest cells followed different pathways depending on the region of the gut being colonised. In the pre-umbilical intestine, NCC were evenly distributed throughout the splanchnopleural mesenchyme while, in the post-umbilical intestine, they occurred adjacent to the serosal epithelium. Behind this migration front, NCC became organised into the presumptive Auerbach’s and Meissner’s plexuses situated on either side of the developing circular muscle layer. The colorectum was found to be colonised in a complex manner. Vagal NCC initially migrated within the submucosa, internal to the circular muscle layer, before migrating outwards, adjacent to blood vessels, towards the myenteric plexus region. In contrast, sacral NCC, which also formed the entire nerve of Remak, were primarily located in the presumptive myenteric plexus region and subsequently migrated inwards towards the submucosal ganglia. Although present throughout the post-umbilical gut, sacral NCC were most numerous in the distal colorectum where they constituted up to 17% of enteric neurons, as identified by double antibody labelling using the quail-cell-specific marker, QCPN and the neuron-specific marker, ANNA-1. Sacral NCC were also immunopositive for the glial-specific antibody, GFAP, thus demonstrating that this region of the neural crest contributes neurons and glia to the enteric nervous system.
Transducing positional information to the Hox genes: critical interaction of cdx gene products with position-sensitive regulatory elements
Authors: Charité Jeroen , de Graaff Wim , Consten Dimitri , Reijnen Mark J. , Korving Jeroen , Deschamps Jacqueline
Published: April 2021

Studies of pattern formation in the vertebrate central nervous system indicate that anteroposterior positional information is generated in the embryo by signalling gradients of an as yet unknown nature. We searched for transcription factors that transduce this information to the Hox genes. Based on the assumption that the activity levels of such factors might vary with position along the anteroposterior axis, we devised an in vivo assay to detect responsiveness of cis-acting sequences to such differentially active factors. We used this assay to analyze a Hoxb8 regulatory element, and detected the most pronounced response in a short stretch of DNA containing a cluster of potential CDX binding sites. We show that differentially expressed DNA binding proteins are present in gastrulating embryos that bind to these sites in vitro, that cdx gene products are among these, and that binding site mutations that abolish binding of these proteins completely destroy the ability of the regulatory element to drive regionally restricted expression in the embryo. Finally, we show that ectopic expression of cdx gene products anteriorizes expression of reporter transgenes driven by this regulatory element, as well as that of the endogenous Hoxb8 gene, in a manner that is consistent with them being essential transducers of positional information. These data suggest that, in contrast to Drosophila Caudal, vertebrate cdx gene products transduce positional information directly to the Hox genes, acting through CDX binding sites in their enhancers. This may represent the ancestral mode of action of caudal homologues, which are involved in anteroposterior patterning in organisms with widely divergent body plans and modes of development.
The role of Alx-4 in the establishment of anteroposterior polarity during vertebrate limb development
Authors: Takahashi Motohisa , Tamura Koji , Büscher Dirk , Masuya Hiroshi , Yonei-Tamura Sayuri , Matsumoto Ken , Naitoh-Matsuo Mayumi , Takeuchi Jun , Ogura Keiko , Shiroishi Toshihiko , Ogura Toshihiko , Izpisúa Belmonte Juan Carlos
Published: April 2021

We have determined that Strong’s Luxoid (lstJ) mice have a 16 bp deletion in the homeobox region of the Alx-4 gene. This deletion, which leads to a frame shift and a truncation of the Alx-4 protein, could cause the polydactyly phenotype observed in lstJ mice. We have cloned the chick homologue of Alx-4 and investigated its expression during limb outgrowth. Chick Alx-4 displays an expression pattern complementary to that of shh, a mediator of polarizing activity in the limb bud. Local application of Sonic hedgehog (Shh) and Fibroblast Growth Factor (FGF), in addition to ectodermal apical ridge removal experiments suggest the existence of a negative feedback loop between Alx-4 and Shh during limb outgrowth. Analysis of polydactylous mutants indicate that the interaction between Alx-4 and Shh is independent of Gli3, a negative regulator of Shh in the limb. Our data suggest the existence of a negative feedback loop between Alx-4 and Shh during vertebrate limb outgrowth.
The Polycomb-group gene eed is required for normal morphogenetic movements during gastrulation in the mouse embryo
Authors: Faust Cynthia , Lawson Kirstie A. , Schork Nicholas J. , Thiel Bonnie , Magnuson Terry
Published: April 2021

We have characterized an induced mutation, called embryonic ectoderm development or eed, that disrupts A-P patterning of the mouse embryo during gastrulation. Positional cloning of this gene revealed it to be the highly conserved homologue of the Drosophila gene extra sex combs, which is required for maintenance of long-term transcriptional repression of homeotic gene expression. Mouse embryos homozygous for loss-of-function alleles of eed initiate gastrulation but display abnormal mesoderm production. Very little embryonic mesoderm is produced; in contrast, extraembryonic mesoderm is relatively abundant. These observations, along with mRNA in situ hybridization analyses, suggested a defect in the anterior primitive streak, from which much of the embryonic mesoderm of the wild-type embryo is derived. To analyse this defect, we initiated clonal analysis of the pre-streak epiblast in eed mutant embryos, using the lineage tracer horseradish peroxidase (HRP). The results of these studies indicate that epiblast cells ingress through the anterior streak, but the newly formed mesoderm does not migrate anteriorly and is mislocalized to the extraembryonic compartment. Abnormal localization of mesoderm to the extraembryonic region did not appear to be due to a restriction and alteration of distal epiblast cell fate, since the majority of clones produced from regions fated to ingress through the anterior streak were mixed, displaying descendants in both embryonic and extraembryonic derivatives. eed mutant embryos also fail to display proper epiblast expansion, particularly with respect to the A-P axis. Based on patterns of clonal spread and calculated clone doubling times for the epiblast, this does not appear to be due to decreased epiblast growth. Rather, epiblast, which is normally fated to make a substantial contribution to the axial midline, appears to make mesoderm preferentially. The data are discussed in terms of global morphogenetic movements in the mouse gastrula and a disruption of signalling activity in the anterior primitive streak.
Intraaortic hemopoietic cells are derived from endothelial cells during ontogeny
Authors: Jaffredo Thierry , Gautier Rodolphe , Eichmann Anne , Dieterlen-Lièvre Françoise
Published: April 2021

We have investigated the developmental relationship of the hemopoietic and endothelial lineages in the floor of the chicken aorta, a site of hemopoietic progenitor emergence in the embryo proper. We show that, prior to the onset of hemopoiesis, the aortic endothelium uniformly expresses the endothelium-specific membrane receptor VEGF-R2. The onset of hemopoiesis can be determined by detecting the common leukocyte antigen CD45. VEGF-R2 and CD45 are expressed in complementary fashion, namely the hemopoietic cluster-bearing floor of the aorta is CD45+/VEGF-R2−, while the rest of the aortic endothelium is CD45−/VEGF-R2+.

To determine if the hemopoietic clusters are derived from endothelial cells, we tagged the E2 endothelial tree from the inside with low-density lipoproteins (LDL) coupled to DiI. 24 hours later, hemopoietic clusters were labelled by LDL. Since no CD45+ cells were inserted among endothelial cells at the time of vascular labelling, hemopoietic clusters must be concluded to derive from precursors with an endothelial phenotype.
XBF-1, a winged helix transcription factor with dual activity, has a role in positioning neurogenesis in Xenopus competent ectoderm
Authors: Bourguignon Catherine , Li Jian , Papalopulu Nancy
Published: April 2021

Neuronal differentiation in the vertebrate nervous system is temporally and spatially controlled by mechanisms which are largely unknown. Here we investigate the role of XBF-1, an anterior neural plate-specific winged helix transcription factor, in controlling the pattern of neurogenesis in Xenopus ectoderm. We show that, in the anterior neural plate of normal embryos, prospective neurogenesis is positioned at the anterior boundary of the XBF-1 expression domain. By misexpressing XBF-1 in the posterior neural plate we show that a high dose of XBF-1 has a dual effect; it suppresses endogenous neuronal differentiation in high expressing cells and induces ectopic neuronal differentiation in adjacent cells. In contrast, a low dose of XBF-1 does not suppress but instead, expands the domain of neuronal differentiation in the lateral and ventral sides of the embryo. XBF-1 regulates the expression of XSox3, X-ngnr-1, X-Myt-1 and X-Delta-1 suggesting that it acts early in the cascade leading to neuronal differentiation. A fusion of XBF-1 to a strong repressor domain (EnR) mimics most of the XBF-1 effects suggesting that the wild type XBF-1 is a transcriptional repressor. However, fusion of XBF-1 to a strong activation domain (E1A) specifically suppresses neuronal differentiation suggesting that XBF-1 may also work as a transcriptional activator. Based on these findings, we propose that XBF-1 is involved in positioning neuronal differentiation by virtue of its concentration dependent, dual activity, as a suppressor and an activator of neurogenesis.
In vivo evidence that Patched and Smoothened constitute distinct binding and transducing components of a Hedgehog receptor complex
Authors: Chen Yu , Struhl Gary
Published: April 2021

During Drosophila development, cells belonging to the posterior compartment of each segment organize growth and patterning by secreting Hedgehog (Hh), a protein which induces a thin strip of adjacent cells in the anterior compartment to express the morphogens Decapentaplegic (Dpp) and Wingless (Wg). Hedgehog is bound and transduced by a receptor complex that includes Smoothened (Smo), a member of the Frizzled (Fz) family of seven-pass transmembrane receptors, as well as the multiple-pass transmembrane protein Patched (Ptc). Ptc is required for the binding of Hh to the complex as well as for the Hh-dependent activation of Smo within the complex. Here, we identify a likely null allele of the smo gene and use it to determine whether Hh is bound by Ptc alone, or by Smo in concert with Ptc. We find that cells devoid of Smo can sequester Hh, but that their ability to do so depends, as in wild-type cells, on the expression of high levels of Ptc protein. These results suggest that Ptc normally binds Hh without any help from Smo and hence favor a mechanism of signal transduction in which Hh binds specifically to Ptc and induces a conformational change leading to the release of latent Smo activity.
Functional dominance among Hox genes: repression dominates activation in the regulation of dpp
Authors: Capovilla Maria , Botas Juan
Published: April 2021

Here we investigate the mechanisms by which Hox genes compete for the control of positional identity. Functional dominance is often observed where different Hox genes are co-expressed, and frequently the more posteriorly expressed Hox gene is the one that prevails, a phenomenon known as posterior prevalence. We use dpp674, a visceral mesoderm-specific enhancer of decapentaplegic (dpp), to investigate functional dominance among Hox genes molecularly. We find that posterior prevalence does not adequately describe the regulation of dpp by Hox genes. Instead, we find that abdominal-A (abd-A) dominates over the more posterior Abdominal-B (Abd-B) and the more anterior Ultrabithorax (Ubx). In the context of the dpp674 enhancer, abd-A functions as a repressor whereas Ubx and Abd-B function as activators. Thus, these results suggest that other cases of Hox competition and functional dominance may also be understood in terms of competition for target gene regulation in which repression dominates over activation.