bluuebunny/crossref_metadata_2025 huggingface dataset:
Physical and genetic interactions between Alx4 and Cart1
Authors: Qu Shimian , Tucker S. Craig , Zhao Qi , deCrombrugghe Benoit , Wisdom Ron
Published: April 2021

Alx4 and Cart1 are closely related members of the family of transcription factors that contain the paired-type homeodomain. In contrast to other types of homeodomains, the paired-type homeodomain has been shown to mediate high-affinity sequence-specific DNA binding to palindromic elements as either homodimers or as heterodimers with other family members. Alx4 and Cart1 are co-expressed at several sites during development, including the craniofacial mesenchyme, the mesenchymal derivatives of neural crest cells in the first branchial arch and the limb bud mesenchyme. Because of the molecular similarity and overlapping expression pattern, we have analyzed the functional and genetic relationships between Alx4 and Cart1. The two proteins have similar DNA- binding activity in vitro and can form DNA-binding heterodimers; furthermore, they activate transcription of reporter genes that contain high-affinity DNA-binding sites in cell culture in a similar manner. Therefore, at least by these criteria, the two proteins are functionally redundant. Analysis of double mutant animals reveals several genetic interactions. First, mutation of Cart1 exacerbates Alx4- dependent polydactyly in a manner that is dependent on gene dosage. Second, there are complex genetic interactions in the craniofacial region that reveal a role for both genes in the fusion of the nasal cartilages and proper patterning of the mandible, as well as other craniofacial structures. Third, double mutant mice show a split sternum that is not detected in mice with any other genotype. Interpreted in the context of the biochemical characterization, the genetic analysis suggests that Alx4 and Cart1 are indeed functionally redundant, and reveal both unique and redundant functions for these genes in development.
Phloem long-distance transport ofCmNACPmRNA: implications for supracellular regulation in plants
Authors: Ruiz-Medrano Roberto , Xoconostle-Cázares Beatriz , Lucas William J.
Published: April 2021

Direct support for the concept that RNA molecules circulate throughout the plant, via the phloem, is provided through the characterisation of mRNA from phloem sap of mature pumpkin (Cucurbita maxima) leaves and stems. One of these mRNAs, CmNACP, is a member of the NAC domain gene family, some of whose members have been shown to be involved in apical meristem development. In situ RT-PCR analysis revealed the presence of CmNACP RNA in the companion cell-sieve element complex of leaf, stem and root phloem. Longitudinal and transverse sections showed continuity of transcript distribution between meristems and sieve elements of the protophloem, suggesting CmNACP mRNA transport over long distances and accumulation in vegetative, root and floral meristems. In situ hybridization studies conducted on CmNACP confirmed the results obtained using in situ RT-PCR.

Phloem transport of CmNACP mRNA was proved directly by heterograft studies between pumpkin and cucumber plants, in which CmNACP transcripts were shown to accumulate in cucumber scion phloem and apical tissues. Similar experiments were conducted with 7 additional phloem-related transcripts. Collectively, these studies established the existence of a system for the delivery of specific mRNA transcripts from the body of the plant to the shoot apex. These findings provide insight into the presence of a novel mechanism likely used by higher plants to integrate developmental and physiological processes on a whole-plant basis.
discordia mutations specifically misorient asymmetric cell divisions during development of the maize leaf epidermis
Authors: Gallagher Kimberly , Smith Laurie G.
Published: April 2021

In plant cells, cytokinesis depends on a cytoskeletal structure called a phragmoplast, which directs the formation of a new cell wall between daughter nuclei after mitosis. The orientation of cell division depends on guidance of the phragmoplast during cytokinesis to a cortical site marked throughout prophase by another cytoskeletal structure called a preprophase band. Asymmetrically dividing cells become polarized and form asymmetric preprophase bands prior to mitosis; phragmoplasts are subsequently guided to these asymmetric cortical sites to form daughter cells of different shapes and/or sizes. Here we describe two new recessive mutations, discordia1 (dcd1) and discordia2 (dcd2), which disrupt the spatial regulation of cytokinesis during asymmetric cell divisions. Both mutations disrupt four classes of asymmetric cell divisions during the development of the maize leaf epidermis, without affecting the symmetric divisions through which most epidermal cells arise. The effects of dcd mutations on asymmetric cell division can be mimicked by cytochalasin D treatment, and divisions affected by dcd1 are hypersensitive to the effects of cytochalasin D. Analysis of actin and microtubule organization in these mutants showed no effect of either mutation on cell polarity, or on formation and localization of preprophase bands and spindles. In mutant cells, phragmoplasts in asymmetrically dividing cells are structurally normal and are initiated in the correct location, but often fail to move to the position formerly occupied by the preprophase band. We propose that dcd mutations disrupt an actin-dependent process necessary for the guidance of phragmoplasts during cytokinesis in asymmetrically dividing cells.
Transient establishment of anteroposterior polarity in the zebrafish pectoral fin bud in the absence of sonic hedgehog activity
Authors: Neumann Carl J. , Grandel Heiner , Gaffield William , Schulte-Merker Stefan , Nüsslein-Volhard Christiane
Published: April 2021

Sonic hedgehog (Shh) is expressed in the posterior vertebrate limb bud mesenchyme and directs anteroposterior patterning and growth during limb development. Here we report an analysis of the pectoral fin phenotype of zebrafish sonic you mutants, which disrupt the shh gene. We show that Shh is required for the establishment of some aspects of anteroposterior polarity, while other aspects of anteroposterior polarity are established independently of Shh, and only later come to depend on Shh for their maintenance. We also demonstrate that Shh is required for the activation of posterior HoxD genes by retinoic acid. Finally, we show that Shh is required for normal development of the apical ectodermal fold, for growth of the fin bud, and for formation of the fin endoskeleton.
Msx1 antagonizes the myogenic activity of Pax3 in migrating limb muscle precursors
Authors: Bendall Andrew J. , Ding Jixiang , Hu Gezhi , Shen Michael M. , Abate-Shen Cory
Published: April 2021

The migration of myogenic precursors to the vertebrate limb exemplifies a common problem in development – namely, how migratory cells that are committed to a specific lineage postpone terminal differentiation until they reach their destination. Here we show that in chicken embryos, expression of the Msx1 homeobox gene overlaps with Pax3 in migrating limb muscle precursors, which are committed myoblasts that do not express myogenic differentiation genes such as MyoD. We find that ectopic expression of Msx1 in the forelimb and somites of chicken embryos inhibits MyoD expression as well as muscle differentiation. Conversely, ectopic expression of Pax3 activates MyoD expression, while co-ectopic expression of Msx1 and Pax3 neutralizes their effects on MyoD. Moreover, we find that Msx1 represses and Pax3 activates MyoD regulatory elements in cell culture, while in combination, Msx1 and Pax3 oppose each other’s trancriptional actions on MyoD. Finally, we show that the Msx1 protein interacts with Pax3 in vitro, thereby inhibiting DNA binding by Pax3. Thus, we propose that Msx1 antagonizes the myogenic activity of Pax3 in migrating limb muscle precursors via direct protein-protein interaction. Our results implicate functional antagonism through competitive protein-protein interactions as a mechanism for regulating the differentiation state of migrating cells.
Compensatory defects associated with mutations in Hoxa1 restore normal palatogenesis to Hoxa2 mutants
Authors: Barrow Jeffery R. , Capecchi Mario R.
Published: April 2021

The rhombencephalic neural crest play several roles in craniofacial development. They give rise to the cranial sensory ganglia and much of the craniofacial skeleton, and are vital for patterning of the craniofacial muscles. The loss of Hoxa1 or Hoxa2 function affects the development of multiple neural crest-derived structures. To understand how these two genes function together in craniofacial development, an allele was generated that disrupts both of these linked genes. Some of the craniofacial defects observed in the double mutants were additive combinations of those that exist in each of the single mutants, indicating that each gene functions independently in the formation of these structures. Other defects were found only in the double mutants demonstrating overlapping or synergistic functions. We also uncovered multiple defects in the attachments and trajectories of the extrinsic tongue and hyoid muscles in Hoxa2 mutants. Interestingly, the abnormal trajectory of two of these muscles, the styloglossus and the stylohyoideus, blocked the attachment of the hyoglossus to the greater horn of the hyoid, which in turn correlated exactly with the presence of cleft palate in Hoxa2 mutants. We suggest that the hyoglossus, whose function is to depress the lateral edges of the tongue, when unable to make its proper attachment to the greater horn of the hyoid, forces the tongue to adopt an abnormal posture which blocks closure of the palatal shelves. Unexpectedly, in Hoxa1/Hoxa2 double mutants, the penetrance of cleft palate is dramatically reduced. We show that two compensatory defects, associated with the loss of Hoxa1 function, restore normal attachment of the hyoglossus to the greater horn thereby allowing the palatal shelves to lift and fuse above the flattened tongue.
Infertility associated with incomplete spermatogenic arrest and oligozoospermia in Egr4-deficient mice
Authors: Tourtellotte Warren G. , Nagarajan Rakesh , Auyeung Anthony , Mueller Christina , Milbrandt Jeffrey
Published: April 2021

Male fertility is complex and depends upon endocrine/paracrine regulatory mechanisms and morphogenetic processes occurring during testicular development, spermatogenesis (mitosis and meiosis) and spermiogenesis (spermatid maturation). Egr4 (NGFI-C, pAT133), a member of the Egr family of zinc-finger transcription factors, is thought to be involved in cellular growth and differentiation, but its specific function has been previously unknown. We derived Egr4 null mice through targeted mutagenesis and found that they were phenotypically normal with the exception that males, but not females, were infertile. Egr4 is expressed at low levels within male germ cells during meiosis and is critical for germ cell maturation during the early-mid pachytene stage. While most Egr4 null male germ cells undergo apoptosis during early-mid pachytene, some are capable of maturing beyond an apparent Egr4-dependent developmental restriction point. Consequently, a limited degree of spermiogenesis occurs but this is accompanied by markedly abnormal spermatozoon morphology and severe oligozoospermia. Egr4 appears to regulate critical genes involved in early stages of meiosis and has a singularly important role in male murine fertility. These data raise the possibility that Egr4 may contribute to some forms of human idiopathic male infertility.
Cardiac looping and the vertebrate left-right axis: antagonism of left-sided Vg1 activity by a right-sided ALK2-dependent BMP pathway
Authors: Ramsdell Ann F. , Yost H. Joseph
Published: April 2021

The rightward looping of the primary heart tube is dependent upon upstream patterning events that establish the vertebrate left-right axis. In Xenopus, a left-sided Vg1 signaling pathway has been implicated in instructing cells to adopt a ‘left-sided identity’; however, it is not known whether ‘right-sided identity’ is acquired by a default pathway or by antagonism of Vg1 signaling. Here, we propose that an antagonistic, BMP/ALK2/Smad-mediated signaling pathway is active on the right side of the Xenopus embryo. Truncated ALK2 receptor expression on the right side of the blastula elicits heart reversals and altered nodal expression. Consistent with these findings, constitutively active ALK2 (CA-ALK2) receptor expression on the left side of the blastula also elicits heart reversals and altered nodal expression. Coexpression of CA-ALK2 with mature Vg1 ligand results in predominantly left-sided nodal expression patterns and normal heart looping, demonstrating that the ALK2 pathway can ‘rescue’ left-right reversals that otherwise occur following right-sided misexpression of mature Vg1 ligand alone. Results with chimeric precursor proteins indicate that the mature domain of BMP ligands can mimic the ability of the ALK2 signaling pathway to antagonize the Vg1 pathway. Consistent with the observed antagonism between BMP and Vg1 ligands, left-sided ectopic expression of Xolloid results in heart reversals. Moreover, ectopic expression of Smad1 or Smad7 identified two downstream modulators of the BMP/ALK2 signaling pathway that also can regulate cardiac orientation. Collectively, these results define a BMP/ALK2-mediated pathway on the right side of the Xenopus embryo and, moreover, suggest that left-right patterning preceding cardiac morphogenesis involves the activation of two distinct and antagonistic, left- and right-sided TGFβ-related signaling pathways.
Spire contains actin binding domains and is related to ascidian posterior end mark-5
Authors: Wellington Andrea , Emmons Steve , James Brian , Calley John , Grover Melissa , Tolias Peter , Manseau Lynn
Published: April 2021

Spire is a maternal effect locus that affects both the dorsal- ventral and anterior-posterior axes of the Drosophila egg and embryo. It is required for localization of determinants within the developing oocyte to the posterior pole and to the dorsal anterior corner. During mid-oogenesis, spire mutants display premature microtubule-dependent cytoplasmic streaming, a phenotype that can be mimicked by pharmacological disruption of the actin cytoskeleton with cytochalasin D. Spire has been cloned by transposon tagging and is related to posterior end mark-5, a gene from sea squirts that encodes a posteriorly localized mRNA. Spire mRNA is not, however, localized to the posterior pole. SPIRE also contains two domains with similarity to the actin monomer-binding WH2 domain, and we demonstrate that SPIRE binds to actin in the interaction trap system and in vitro. In addition, SPIRE interacts with the rho family GTPases RHOA, RAC1 and CDC42 in the interaction trap system. Thus, our evidence supports the model that SPIRE links rho family signaling to the actin cytoskeleton.
Control of DAF-7 TGF-β expression and neuronal process development by a receptor tyrosine kinase KIN-8 in Caenorhabditis elegans
Authors: Koga Makoto , Take-uchi Masaya , Tameishi Tatsuji , Ohshima Yasumi
Published: April 2021

KIN-8 in C. elegans is highly homologous to human ROR- 1 and 2 receptor tyrosine kinases of unknown functions. These kinases belong to a new subfamily related to the Trk subfamily. A kin-8 promoter::gfp fusion gene was expressed in ASI and many other neurons as well as in pharyngeal and head muscles. A kin-8 deletion mutant was isolated and showed constitutive dauer larva formation (Daf-c) phenotype: about half of the F1 progeny became dauer larvae when they were cultivated on an old lawn of E. coli as food. Among the cells expressing kin-8::gfp, only ASI sensory neurons are known to express DAF-7 TGF-β, a key molecule preventing dauer larva formation. In the kin-8 deletion mutant, expression of daf-7::gfp in ASI was greatly reduced, dye-filling in ASI was specifically lost and ASI sensory processes did not completely extend into the amphid pore. The Daf-c phenotype was suppressed by daf- 7 cDNA expression or a daf-3 null mutation. ASI-directed expression of kin-8 cDNA under the daf-7 promoter or expression by a heat shock promoter rescued the dye-filling defect, but not the Daf-c phenotype, of the kin-8 mutant. These results show that the kin-8 mutation causes the Daf- c phenotype through reduction of the daf-7 gene expression and that KIN-8 function is cell-autonomous for the dye- filling in ASI. KIN-8 is required for the process development of ASI, and also involved in promotion of daf- 7 expression through a physiological or developmental function.
Cyclin A and B functions in the early Drosophila embryo
Authors: Stiffler Lisa A. , Ji Jun-Yuan , Trautmann Susanne , Trusty Cory , Schubiger Gerold
Published: April 2021

In eukaryotes, mitotic cyclins localize differently in the cell and regulate different aspects of the cell cycle. We investigated the relationship between subcellular localization of cyclins A and B and their functions in syncytial preblastoderm Drosophila embryos. During early embryonic cycles, cyclin A was always concentrated in the nucleus and present at a low level in the cytoplasm. Cyclin B was predominantly cytoplasmic, and localized within nuclei only during late prophase. Also, cyclin B colocalized with metaphase but not anaphase spindle microtubules. We changed maternal gene doses of cyclins A and B to test their functions in preblastoderm embryos. We observed that increasing doses of cyclin B increased cyclin B-Cdk1 activity, which correlated with shorter microtubules and slower microtubule-dependent nuclear movements. This provides in vivo evidence that cyclin B-Cdk1 regulates microtubule dynamics. In addition, the overall duration of the early nuclear cycles was affected by cyclin A but not cyclin B levels. Taken together, our observations support the hypothesis that cyclin B regulates cytoskeletal changes while cyclin A regulates the nuclear cycles. Varying the relative levels of cyclins A and B uncoupled the cytoskeletal and nuclear events, so we speculate that a balance of cyclins is necessary for proper coordination during these embryonic cycles.
The BMP antagonist Gremlin regulates outgrowth, chondrogenesis and programmed cell death in the developing limb
Authors: Merino R. , Rodriguez-Leon J. , Macias D. , Gañan Y. , Economides A. N. , Hurle J. M
Published: April 2021

In this study, we have analyzed the expression and function of Gremlin in the developing avian limb. Gremlin is a member of the DAN family of BMP antagonists highly conserved through evolution able to bind and block BMP2, BMP4 and BMP7. At early stages of development, gremlin is expressed in the dorsal and ventral mesoderm in a pattern complementary to that of bmp2, bmp4 and bmp7. The maintenance of gremlin expression at these stages is under the control of the AER, ZPA, and BMPs. Exogenous administration of recombinant Gremlin indicates that this protein is involved in the control of limb outgrowth. This function appears to be mediated by the neutralization of BMP function to maintain an active AER, to restrict the extension of the areas of programmed cell death and to confine chondrogenesis to the central core mesenchyme of the bud. At the stages of digit formation, gremlin is expressed in the proximal boundary of the interdigital mesoderm of the chick autopod. The anti-apoptotic influence of exogenous Gremlin, which results in the formation of soft tissue syndactyly in the chick, together with the expression of gremlin in the duck interdigital webs, indicates that Gremlin regulates the regression of the interdigital tissue. At later stages of limb development, gremlin is expressed in association with the differentiating skeletal pieces, muscles and the feather buds. The different expression of Gremlin in relation with other BMP antagonists present in the limb bud, such as Noggin, Chordin and Follistatin indicates that the functions of BMPs are regulated specifically by the different BMP antagonists, acting in a complementary fashion rather than being redundant signals.
The role of Pax6 in restricting cell migration between developing cortex and basal ganglia
Authors: Chapouton Prisca , Gärtner Annette , Götz Magdalena
Published: April 2021

It is not clear to what extent restricted cell migration contributes to patterning of the developing telencephalon, since both restricted and widespread cell migration have been observed. Here, we have analysed dorso-ventral cell migration in the telencephalon of Pax6 mutant mice (Small Eye). The transcription factor Pax6 is expressed in the dorsal telencephalon, the cerebral cortex. Focal injections of adenoviral vectors containing the green fluorescent protein were used to follow and quantify cell movements between two adjacent regions in the developing telencephalon, the cerebral cortex and the ganglionic eminence (the prospective basal ganglia). The analysis in wild-type mice confirmed that the cortico-striatal boundary acts as a semipermeable filter and allows a proportion of cells from the ganglionic eminence to invade the cortex, but not vice versa. Ventro-dorsal cell migration was strongly enhanced in the Pax6 mutant. An essential function of Pax6 in the regionalisation of the telencephalon is then to limit the invasion of the cortex by cells originating in the ganglionic eminence. Cortical cells, however, remain confined to the cortex in the Pax6 mutant. Thus, dorsal and ventral cells are restricted to their respective territories by distinct mechanisms.
Fgfr1 and Fgfr2 have distinct differentiation- and proliferation-related roles in the developing mouse skull vault
Authors: Iseki S. , Wilkie A. O. M. , Morriss-Kay G. M.
Published: April 2021

Fibroblast growth factor receptors (FGFRs) play major roles in skeletogenesis, and activating mutations of the human FGFR1, FGFR2 and FGFR3 genes cause premature fusion of the skull bones (craniosynostosis). We have investigated the patterns of expression of Fgfr1, Fgfr2 and Fgfr3 in the fetal mouse head, with specific reference to their relationship to cell proliferation and differentiation in the frontal and parietal bones and in the coronal suture. Fgfr2 is expressed only in proliferating osteoprogenitor cells; the onset of differentiation is preceded by down-regulation of Fgfr2 and up-regulation of Fgfr1. Following up-regulation of the differentiation marker osteopontin, Fgfr1, osteonectin and alkaline phosphatase are down-regulated, suggesting that they are involved in the osteogenic differentiation process but not in maintaining the differentiated state. Fgfr3 is expressed in the cranial cartilage, including a plate of cartilage underlying the coronal suture, as well as in osteogenic cells, suggesting a dual role in skull development. Subcutaneous insertion of FGF2-soaked beads onto the coronal suture on E15 resulted in up-regulation of osteopontin and Fgfr1 in the sutural mesenchyme, down-regulation of Fgfr2, and inhibition of cell proliferation. This pattern was observed at 6 and 24 hours after bead insertion, corresponding to the timing and duration of FGF2 diffusion from the beads. We suggest (a) that a gradient of FGF ligand, from high levels in the differentiated region to low levels in the environment of the osteogenic stem cells, modulates differential expression of Fgfr1 and Fgfr2, and (b) that signalling through FGFR2 regulates stem cell proliferation whereas signalling through FGFR1 regulates osteogenic differentiation.
Progression of the morphogenetic furrow in the Drosophila eye: the roles of Hedgehog, Decapentaplegic and the Raf pathway
Authors: Greenwood Simon , Struhl Gary
Published: April 2021

During Drosophila eye development, Hedgehog (Hh) protein secreted by maturing photoreceptors directs a wave of differentiation that sweeps anteriorly across the retinal primordium. The crest of this wave is marked by the morphogenetic furrow, a visible indentation that demarcates the boundary between developing photoreceptors located posteriorly and undifferentiated cells located anteriorly. Here, we present evidence that Hh controls progression of the furrow by inducing the expression of two downstream signals. The first signal, Decapentaplegic (Dpp), acts at long range on undifferentiated cells anterior to the furrow, causing them to enter a ‘pre-proneural’ state marked by upregulated expression of the transcription factor Hairy. Acquisition of the pre-proneural state appears essential for all prospective retinal cells to enter the proneural pathway and differentiate as photoreceptors. The second signal, presently unknown, acts at short range and is transduced via activation of the Serine-Threonine kinase Raf. Activation of Raf is both necessary and sufficient to cause pre-proneural cells to become proneural, a transition marked by downregulation of Hairy and upregulation of the proneural activator, Atonal (Ato), which initiates differentiation of the R8 photoreceptor. The R8 photoreceptor then organizes the recruitment of the remaining photoreceptors (R1-R7) through additional rounds of Raf activation in neighboring pre-proneural cells. Finally, we show that Dpp signaling is not essential for establishing either the pre-proneural or proneural states, or for progression of the furrow. Instead, Dpp signaling appears to increase the rate of furrow progression by accelerating the transition to the pre-proneural state. In the abnormal situation in which Dpp signaling is blocked, Hh signaling can induce undifferentiated cells to become pre-proneural but does so less efficiently than Dpp, resulting in a retarded rate of furrow progression and the formation of a rudimentary eye.
The specification and growth factor inducibility of the pronephric glomus in Xenopus laevis
Authors: Brennan, Hannah C. , Nijjar Sarbjit , Jones Elizabeth A.
Published: April 2021

We report a study on the specification of the glomus, the filtration device of the amphibian pronephric kidney, using an explant culturing strategy in Xenopus laevis. Explants of presumptive pronephric mesoderm were dissected from embryos of mid-gastrula to swimming tadpole stages. These explants were cultured within ectodermal wraps and analysed by RT-PCR for the presence of the Wilm’s Tumour-1 gene, xWT1, a marker specific for the glomus at the stages analysed, together with other mesodermal markers. We show that the glomus is specified at stage 12.5, the same stage at which pronephric tubules are specified. We have previously shown that pronephric duct is specified somewhat later, at stage 14.

Furthermore, we have analysed the growth factor inducibility of the glomus in the presence or absence of retinoic acid (RA) by RT-PCR. We define for the first time the conditions under which these growth factors induce glomus tissue in animal cap tissue. Activin together with high concentrations of RA can induce glomus tissue from animal cap ectoderm. Unlike the pronephric tubules, the glomus can also be induced by FGF and RA.
Cell death and cell protection genes determine the fate of pistils in maize
Authors: Calderon-Urrea Alejandro , Dellaporta Stephen L.
Published: April 2021

The formation of unisexual flowers in maize requires the selective elimination and sexual maturation of floral organs in an initially bisexual floral meristem. Elimination of pistil primordia occurs in the primary and secondary florets of the tassel spikelets, and in the secondary florets of ear spikelets. Ill-fated pistil cells undergo a cell death process associated with nuclear degeneration in a specific spatial- temporal pattern that begins in the subepidermis, eventually aborting the entire organ. The sex determination genes tasselseed1 and tasselseed2 are required for death of pistil cells. tasselseed1 is required for the accumulation of TASSELSEED2 mRNA in pistil cells. All pistil primordia express TASSELSEED2 RNA but functional pistils found in ear spikelets are protected from cell death by the action of the silkless1 gene. silkless1 blocks tasselseed-induced cell death in the pistil primordia of primary ear florets. A model is proposed for the control of pistil fate by the action of the ts1-ts2-sk1 pathway.
BMP signaling plays a role in visceral endoderm differentiation and cavitation in the early mouse embryo
Authors: Coucouvanis Electra , Martin Gail R.
Published: April 2021

At E4.0 the inner cell mass of the mouse blastocyst consists of a core of embryonic ectoderm cells surrounded by an outer layer of primitive (extraembryonic) endoderm, which subsequently gives rise to both visceral endoderm and parietal endoderm. Shortly after blastocyst implantation, the solid mass of ectoderm cells is converted by a process known as cavitation into a pseudostratified columnar epithelium surrounding a central cavity. We have previously used two cell lines, which form embryoid bodies that do (PSA1) or do not (S2) cavitate, as an in vitro model system for studying the mechanism of cavitation in the early embryo. We provided evidence that cavitation is the result of both programmed cell death and selective cell survival, and that the process depends on signals from visceral endoderm (Coucouvanis, E. and Martin, G. R. (1995) Cell 83, 279-287). Here we show that Bmp2 and Bmp4 are expressed in PSA1 embryoid bodies and embryos at the stages when visceral endoderm differentiation and cavitation are occurring, and that blocking BMP signaling via expression of a transgene encoding a dominant negative mutant form of BMP receptor IB inhibits expression of the visceral endoderm marker, Hnf4, and prevents cavitation in PSA1 embryoid bodies. Furthermore, we show that addition of BMP protein to cultures of S2 embryoid bodies induces expression of Hnf4 and other visceral endoderm markers and also cavitation. Taken together, these data indicate that BMP signaling is both capable of promoting, and required for differentiation of, visceral endoderm and cavitation of embryoid bodies. Based on these and other data, we propose a model for the role of BMP signaling during peri-implantation stages of mouse embryo development.
Cerebral cortical specification by early potential restriction of progenitor cells and later phenotype control of postmitotic neurons
Authors: Arimatsu Yasuyoshi , Ishida Mami , Takiguchi-Hayashi Keiko , Uratani Yoshihiko
Published: April 2021

Neurons expressing latexin, a carboxypeptidase A inhibitor, are restricted to lateral areas in the cerebral cortex of adult and early postnatal rats. To address the precise timing of cortical regional specification at the cellular level, we monitored latexin expression in developing cortical cells under specific conditions in vitro. Individual cortical cells were labeled with 5-bromo-2′-deoxyuridine in vivo, dissociated and exposed to a defined new environment in a monolayer or a reaggregated-cell culture system. While a substantial fraction of early progenitor cells derived from the lateral cerebral wall became latexin-expressing neurons in both systems, far fewer progenitors from dorsal cortex did so under the same environmental conditions, indicating early establishment of cortical regional specification at the progenitor cell level. Furthermore, it was shown that the probability for postmitotic cells within lateral cortex to become latexin-expressing neurons was influenced by temporally regulated regional environmental signals. These findings suggest that developing cortical cells are progressively specified for a regional molecular phenotype during both their proliferative and postmitotic periods.
Control of auxin-regulated root development by the Arabidopsis thaliana SHY2/IAA3 gene
Authors: Tian Qing , Reed Jason W.
Published: April 2021

The plant hormone auxin controls many aspects of development and acts in part by inducing expression of various genes. Arabidopsis thaliana semidominant shy2 (short hypocotyl) mutations cause leaf formation in dark-grown plants, suggesting that SHY2 has an important role in regulating development. Here we show that the SHY2 gene encodes IAA3, a previously known member of the Aux/IAA family of auxin-induced genes. Dominant shy2 mutations cause amino acid changes in domain II, conserved among all members of this family. We isolated loss-of-function shy2 alleles including a putative null mutation. Gain-of-function and loss-of-function shy2 mutations affect auxin-dependent root growth, lateral root formation, and timing of gravitropism, indicating that SHY2/IAA3 regulates multiple auxin responses in roots. The phenotypes suggest that SHY2/IAA3 may activate some auxin responses and repress others. Models invoking tissue-specificity, feedback regulation, or control of auxin transport may explain these results.
The trithorax group gene osa encodes an ARID-domain protein that genetically interacts with the Brahma chromatin-remodeling factor to regulate transcription
Authors: Vázquez Martha , Moore Lisa , Kennison James A.
Published: April 2021

The trithorax group gene brahma (brm) encodes the ATPase subunit of a chromatin-remodeling complex involved in homeotic gene regulation. We report here that brm interacts with another trithorax group gene, osa, to regulate the expression of the Antennapedia P2 promoter. Regulation of Antennapedia by BRM and OSA proteins requires sequences 5′ to the P2 promoter. Loss of maternal osa function causes severe segmentation defects, indicating that the function of osa is not limited to homeotic gene regulation. The OSA protein contains an ARID domain, a DNA-binding domain also present in the yeast SWI1 and Drosophila DRI proteins. We propose that the OSA protein may target the BRM complex to Antennapedia and other regulated genes.
Signaling by the TGF-β homolog decapentaplegic functions reiteratively within the network of genes controlling retinal cell fate determination in Drosophila
Authors: Chen Rui , Halder Georg , Zhang Zhihuan , Mardon Graeme
Published: April 2021

Retinal cell fate determination in Drosophila is controlled by an interactive network of genes, including eyeless, eyes absent, sine oculis and dachshund. We have investigated the role of the TGF-β homolog decapentaplegic in this pathway. We demonstrate that, during eye development, while eyeless transcription does not depend on decapentaplegic activity, the expression of eyes absent, sine oculis and dachshund are greatly reduced in a decapentaplegic mutant background. We also show that decapentaplegic signaling acts synergistically with and at multiple levels of the retinal determination network to induce eyes absent, sine oculis and dachshund expression and ectopic eye formation. These results suggest a mechanism by which a general patterning signal such as Decapentaplegic cooperates reiteratively with tissue-specific factors to determine distinct cell fates during development.
Role of the EGF receptor pathway in growth and patterning of the Drosophila wing through the regulation of vestigial
Authors: Nagaraj Raghavendra , Pickup Amanda T. , Howes Robert , Moses Kevin , Freeman Matthew , Banerjee Utpal
Published: April 2021

Growth and patterning of the Drosophila wing disc depends on the coordinated expression of the key regulatory gene vestigial both in the Dorsal-Ventral (D/V) boundary cells and in the wing pouch. We propose that a short-range signal originating from the core of the D/V boundary cells is responsible for activating EGFR in a zone of organizing cells on the edges of the D/V boundary. Using loss-of-function mutations and ectopic expression studies, we show that EGFR signaling is essential for vestigial transcription in these cells and for making them competent to undergo subsequent vestigial-mediated proliferation within the wing pouch.
HyBra1, a Brachyury homologue, acts during head formation in Hydra
Authors: Technau Ulrich , Bode Hans R.
Published: April 2021

A homologue of the T-box gene, Brachyury, has been isolated from hydra. The gene, termed HyBra1, is expressed in the endoderm and is associated with the formation of the hypostome, the apical part of the head in four different developmental situations. In adults, which are continuously undergoing patterning, HyBra1 is continuously expressed in the hypostome. During budding, hydra’s asexual form of reproduction, the gene is expressed in a small area that will eventually form the hypostome of the developing bud before any morphological sign of budding is apparent. The gene is also expressed very early during head regeneration and is confined to the region that will form the hypostome. During embryogenesis, HyBra1 is expressed shortly before hatching in the region that will form the apical end of the animal, the hypostome. The absence of expression at the apical end of decapitated animals of reg-16, a head formation-deficient mutant, provides additional evidence for a role of HyBra1 during head formation. Further, treatments that alter the head activation gradient have no effect on HyBra1 expression indicating the role of the gene is confined to head formation. Transplantation experiments indicate that the expression occurs before head determination has occurred, but expression does not irreversibly commit tissue to forming a head. A comparison of the function of the Brachyury homologues suggests an evolutionary conservation of a molecular mechanism that has been co-opted for a number of developmental processes throughout evolution.
Inhibition of in vitro enteric neuronal development by endothelin-3: mediation by endothelin B receptors
Authors: Wu Jun J. , Chen Jing-Xian , Rothman Taube P. , Gershon Michael D.
Published: April 2021

The terminal colon is aganglionic in mice lacking endothelin-3 or its receptor, endothelin B. To analyze the effects of endothelin-3/endothelin B on the differentiation of enteric neurons, E11-13 mouse gut was dissociated, and positive and negative immunoselection with antibodies to p75NTR were used to isolate neural crest- and non-crest-derived cells. mRNA encoding endothelin B was present in both the crest- and non-crest-derived cells, but that encoding preproendothelin-3 was detected only in the non-crest-derived population. The crest- and non-crest-derived cells were exposed in vitro to endothelin-3, IRL 1620 (an endothelin B agonist), and/or BQ 788 (an endothelin B antagonist). Neurons and glia developed only in cultures of crest-derived cells, and did so even when endothelin-3 was absent and BQ 788 was present. Endothelin-3 inhibited neuronal development, an effect that was mimicked by IRL 1620 and blocked by BQ 788. Endothelin-3 failed to stimulate the incorporation of [3H]thymidine or bromodeoxyuridine. Smooth muscle development in non-crest-derived cell cultures was promoted by endothelin-3 and inhibited by BQ 788. In contrast, transcription of laminin α1, a smooth muscle-derived promoter of neuronal development, was inhibited by endothelin-3, but promoted by BQ 788. Neurons did not develop in explants of the terminal bowel of E12 ls/ls (endothelin-3-deficient) mice, but could be induced to do so by endothelin-3 if a source of neural precursors was present. We suggest that endothelin-3/endothelin B normally prevents the premature differentiation of crest-derived precursors migrating to and within the fetal bowel, enabling the precursor population to persist long enough to finish colonizing the bowel.
Requirement of a novel gene, Xin, in cardiac morphogenesis
Authors: Wang Da-Zhi , Reiter Rebecca S. , Lin Jenny Li-Chun , Wang Qin , Williams Haley S. , Krob Sonja L. , Schultheiss Thomas M. , Evans Sylvia , Lin Jim Jung-Ching
Published: April 2021

A novel gene, Xin, from chick (cXin) and mouse (mXin) embryonic hearts, may be required for cardiac morphogenesis and looping. Both cloned cDNAs have a single open reading frame, encoding proteins with 2,562 and 1,677 amino acids for cXin and mXin, respectively. The derived amino acid sequences share 46% similarity. The overall domain structures of the predicted cXin and mXin proteins, including proline-rich regions, 16 amino acid repeats, DNA-binding domains, SH3-binding motifs and nuclear localization signals, are highly conserved. Northern blot analyses detect a single message of 8.9 and 5.8 kilo base (kb) from both cardiac and skeletal muscle of chick and mouse, respectively. In situ hybridization reveals that the cXin gene is specifically expressed in cardiac progenitor cells of chick embryos as early as stage 8, prior to heart tube formation. cXin continues to be expressed in the myocardium of developing hearts. By stage 15, cXin expression is also detected in the myotomes of developing somites. Immunofluorescence microscopy reveals that the mXin protein is colocalized with N-cadherin and connexin-43 in the intercalated discs of adult mouse hearts.

Incubation of stage 6 chick embryos with cXin antisense oligonucleotides results in abnormal cardiac morphogenesis and an alteration of cardiac looping. The myocardium of the affected hearts becomes thickened and tends to form multiple invaginations into the heart cavity. This abnormal cellular process may account in part for the abnormal looping. cXin expression can be induced by bone morphogenetic protein (BMP) in explants of anterior medial mesoendoderm from stage 6 chick embryos, a tissue that is normally non-cardiogenic. This induction occurs following the BMP-mediated induction of two cardiac-restricted transcription factors, Nkx2.5 and MEF2C. Furthermore, either MEF2C or Nkx2.5 can transactivate a luciferase reporter driven by the mXin promoter in mouse fibroblasts. These results suggest that Xin may participate in a BMP-Nkx2.5-MEF2C pathway to control cardiac morphogenesis and looping.
Characterization of an amphioxus paired box gene,AmphiPax2/5/8: developmental expression patterns in optic support cells, nephridium, thyroid-like structures and pharyngeal gill slits, but not in the midbrain-hindbrain boundary region
Authors: Kozmik Zbynek , Holland Nicholas D. , Kalousova Anna , Paces Jan , Schubert Michael , Holland Linda Z.
Published: April 2021

On the basis of developmental gene expression, the vertebrate central nervous system comprises: a forebrain plus anterior midbrain, a midbrain-hindbrain boundary region (MHB) having organizer properties, and a rhombospinal domain. The vertebrate MHB is characterized by position, by organizer properties and by being the early site of action of Wnt1 and engrailed genes, and of genes of the Pax2/5/8 subfamily. Wada and others (Wada, H., Saiga, H., Satoh, N. and Holland, P. W. H. (1998) Development 125, 1113-1122) suggested that ascidian tunicates have a vertebrate-like MHB on the basis of ascidian Pax258 expression there. In another invertebrate chordate, amphioxus, comparable gene expression evidence for a vertebrate-like MHB is lacking. We, therefore, isolated and characterized AmphiPax2/5/8, the sole member of this subfamily in amphioxus. AmphiPax2/5/8 is initially expressed well back in the rhombospinal domain and not where a MHB would be expected. In contrast, most of the other expression domains of AmphiPax2/5/8 correspond to expression domains of vertebrate Pax2, Pax5 and Pax8 in structures that are probably homologous – support cells of the eye, nephridium, thyroid-like structures and pharyngeal gill slits; although AmphiPax2/5/8 is not transcribed in any structures that could be interpreted as homologues of vertebrate otic placodes or otic vesicles. In sum, the developmental expression of AmphiPax2/5/8 indicates that the amphioxus central nervous system lacks a MHB resembling the vertebrate isthmic region. Additional gene expression data for the developing ascidian and amphioxus nervous systems would help determine whether a MHB is a basal chordate character secondarily lost in amphioxus. The alternative is that the MHB is a vertebrate innovation.
Differential transcriptional control as the major molecular event in generating Otx1−/− and Otx2−/− divergent phenotypes
Authors: Acampora Dario , Avantaggiato Virginia , Tuorto Francesca , Barone Paolo , Perera Marzia , Choo Daniel , Wu Doris , Corte Giorgio , Simeone Antonio
Published: April 2021

Otx1 and Otx2, two murine homologs of the Drosophila orthodenticle (otd) gene, show a limited amino acid sequence divergence. Their embryonic expression patterns overlap in spatial and temporal profiles with two major exceptions: until 8 days post coitum (d.p.c.) only Otx2 is expressed in gastrulating embryos, and from 11 d.p.c. onwards only Otx1 is transcribed within the dorsal telencephalon. Otx1 null mice exhibit spontaneous epileptic seizures and multiple abnormalities affecting primarily the dorsal telencephalic cortex and components of the acoustic and visual sense organs. Otx2 null mice show heavy gastrulation abnormalities and lack the rostral neuroectoderm corresponding to the forebrain, midbrain and rostral hindbrain. In order to define whether these contrasting phenotypes reflect differences in expression pattern or coding sequence of Otx1 and Otx2 genes, we replaced Otx1 with a human Otx2 (hOtx2) full- coding cDNA. Interestingly, homozygous mutant mice (hOtx21/hOtx21) fully rescued epilepsy and corticogenesis abnormalities and showed a significant improvement of mesencephalon, cerebellum, eye and lachrymal gland defects. In contrast, the lateral semicircular canal of the inner ear was never recovered, strongly supporting an Otx1-specific requirement for the specification of this structure. These data indicate an extended functional homology between OTX1 and OTX2 proteins and provide evidence that, with the exception of the inner ear, in Otx1 and Otx2 null mice contrasting phenotypes stem from differences in expression patterns rather than in amino acid sequences.
The zebrafish bozozok locus encodes Dharma, a homeodomain protein essential for induction of gastrula organizer and dorsoanterior embryonic structures
Authors: Fekany Kimberly , Yamanaka Yojiro , Leung TinChung , Sirotkin Howard I. , Topczewski Jacek , Gates Michael A. , Hibi Masahiko , Renucci Armand , Stemple Derek , Radbill Andy , Schier Alexander F. , Driever Wolfgang , Hirano Toshio , Talbot William S. , Solnica-Krezel Lilianna
Published: April 2021

The dorsal gastrula organizer plays a fundamental role in establishment of the vertebrate axis. We demonstrate that the zebrafish bozozok (boz) locus is required at the blastula stages for formation of the embryonic shield, the equivalent of the gastrula organizer and expression of multiple organizer-specific genes. Furthermore, boz is essential for specification of dorsoanterior embryonic structures, including notochord, prechordal mesendoderm, floor plate and forebrain. We report that boz mutations disrupt the homeobox gene dharma. Overexpression of boz in the extraembryonic yolk syncytial layer of boz mutant embryos is sufficient for normal development of the overlying blastoderm, revealing an involvement of extraembryonic structures in anterior patterning in fish similarly to murine embryos. Epistatic analyses indicate that boz acts downstream of β-catenin and upstream to TGF-β signaling or in a parallel pathway. These studies provide genetic evidence for an essential function of a homeodomain protein in β-catenin-mediated induction of the dorsal gastrula organizer and place boz at the top of a hierarchy of zygotic genes specifying the dorsal midline of a vertebrate embryo.
The Netrin receptor Frazzled is required in the target for establishment of retinal projections in the Drosophila visual system
Authors: Gong Qizhi , Rangarajan Radha , Seeger Mark , Gaul Ulrike
Published: April 2021

Retinal axons in Drosophila make precise topographic connections with their target cells in the optic lobe. Here we investigate the role of the Netrins and their receptor Frazzled in the establishment of retinal projections. We find that the Netrins, although expressed in the target, are not required for retinal projections. Surprisingly, Frazzled, found on both retinal fibers and target cells, is required in the target for attracting retinal fibers, while playing at best a redundant role in the retinal fibers themselves; this finding demonstrates that target attraction is necessary for topographic map formation. Finally, we show that Frazzled is not required for the differentiation of cells in the target. Our data suggest that Frazzled does not function as a Netrin receptor in attracting retinal fibers to the target; nor does it seem to act as a homotypic cell adhesion molecule. We favor the possibility that Frazzled in the target interacts with a component on the surface of retinal fibers, possibly another Netrin receptor.