bluuebunny/crossref_metadata_2025 huggingface dataset:
Bix4is activated directly by VegT and mediates endoderm formation inXenopusdevelopment
Authors: Casey Elena Silva , Tada Masazumi , Fairclough Lynne , Wylie Christopher C. , Heasman Janet , Smith James C.
Published: April 2021

The maternal T-box gene VegT, whose transcripts are restricted to the vegetal hemisphere of the Xenopus embryo, plays an essential role in early development. Depletion of maternal VegT transcripts causes embryos to develop with no endoderm, while vegetal blastomeres lose the ability to induce mesoderm (Zhang, J., Houston, D. W., King, M. L., Payne, C., Wylie, C. and Heasman, J. (1998) Cell 94, 515-524). The targets of VegT, a transcription activator, must therefore include genes involved both in the specification of endoderm and in the production of mesoderm-inducing signals. We recently reported that the upstream regulatory region of the homeobox-containing gene Bix4 contains T-box binding sites. Here we show that expression of Bix4 requires maternal VegT and that two T-box binding sites are necessary and sufficient for mesodermal and endodermal expression of reporter genes driven by the Bix4 promoter in transgenic Xenopus embryos. Remarkably, a single T-box binding site is able to act as a mesoderm-specific enhancer when placed upstream of a minimal promoter. Finally, we show that Bix4 rescues the formation of endodermal markers in embryos in which VegT transcripts have been ablated but does not restore the ability of vegetal pole blastomeres to induce mesoderm. These results demonstrate that Bix4 acts directly downstream of VegT to specify endodermal differentiation in Xenopus embryos.
Pax6 induces ectopic eyes in a vertebrate
Authors: Chow Robert L. , Altmann Curtis R. , Lang Richard A. , Hemmati-Brivanlou Ali
Published: April 2021

We report here that misexpression of the transcription factor Pax6 in the vertebrate Xenopus laevis leads to the formation of differentiated ectopic eyes. Multiple molecular markers indicated the presence of mature lens fiber cells, ganglion cells, Müller cells, photoreceptors and retinal pigment epithelial cells in a spatial arrangement similar to that of endogenous eyes. Lineage tracing experiments showed that lens, retina and retinal pigment epithelium arose as a consequence of the cell-autonomous function of Pax6. These experiments also reveal that the cell autonomous activity of misexpressed Pax6 causes the ectopic expression of a number of genes including Rx, Otx2, Six3 and endogenous Pax6, each of which has been implicated in eye development. The formation of ectopic and endogenous eyes could be suppressed by coexpression of a dominant-negative form of Pax6. These data show that in vertebrates, as in the invertebrate Drosophila melanogaster, Pax6 is both necessary and sufficient to trigger the cascade of events required for eye formation.
Cell autonomous and non-cell autonomous functions of Otx2 in patterning the rostral brain
Authors: Rhinn Muriel , Dierich Andrée , Meur Marianne Le , Ang Siew-Lan
Published: April 2021

Previous studies have shown that the homeobox gene Otx2 is required first in the visceral endoderm for induction of forebrain and midbrain, and subsequently in the neurectoderm for its regional specification. Here, we demonstrate that Otx2 functions both cell autonomously and non-cell autonomously in neurectoderm cells of the forebrain and midbrain to regulate expression of region-specific homeobox and cell adhesion genes. Using chimeras containing both Otx2 mutant and wild-type cells in the brain, we observe a reduction or loss of expression of Rpx/Hesx1, Wnt1, R-cadherin and ephrin-A2 in mutant cells, whereas expression of En2 and Six3 is rescued by surrounding wild-type cells. Forebrain Otx2 mutant cells subsequently undergo apoptosis. Altogether, this study demonstrates that Otx2 is an important regulator of brain patterning and morphogenesis, through its regulation of candidate target genes such as Rpx/Hesx1, Wnt1, R-cadherin and ephrin-A2.
A new family of growth factors produced by the fat body and active on Drosophila imaginal disc cells
Authors: Kawamura Kazuo , Shibata Tatsuhiro , Saget Olivier , Peel David , Bryant Peter J.
Published: April 2021

By fractionating conditioned medium (CM) from Drosophila imaginal disc cell cultures, we have identified a family of Imaginal Disc Growth Factors (IDGFs), which are the first polypeptide growth factors to be reported from invertebrates. The active fraction from CM, as well as recombinant IDGFs, cooperate with insulin to stimulate the proliferation, polarization and motility of imaginal disc cells. The IDGF family in Drosophila includes at least five members, three of which are encoded by three genes in a tight cluster. The proteins are structurally related to chitinases, but they show an amino acid substitution that is known to abrogate catalytic activity. It therefore seems likely that they have evolved from chitinases but acquired a new growth-promoting function. The IDGF genes are expressed most strongly in the embryonic yolk cells and in the fat body of the embryo and larva. The predicted molecular structure, expression patterns, and mitogenic activity of these proteins suggest that they are secreted and transported to target tissues via the hemolymph. However, the genes are also expressed in embryonic epithelia in association with invagination movements, so the proteins may have local as well as systemic functions. Similar proteins are found in mammals and may constitute a novel class of growth factors.
Conserved regulation of mesenchymal gene expression by Fgf-8 in face and limb development
Authors: Tucker Abigail S. , Khamis Abdul Al , A. Ferguson Christine , Bach Ingolf , Rosenfeld Michael G. , Sharpe Paul T.
Published: April 2021

Clim-2 (NLI, Lbd1) is one of two related mouse proteins that interact with Lim-domain homeoproteins. In the mouse, embryonic expression of Clim-2 is particularly pronounced in facial ectomesenchyme and limb bud mesenchyme in association with Lim genes, Lhx-6 and Lmx-1 respectively. We show that in common with both these Lim genes, Clim-2 expression is regulated by signals from overlying epithelium. In both the developing face and the limb buds we identify Fgf-8 as the likely candidate signalling molecule that regulates Clim-2 expression. We show that in the mandibular arch, as in the limb, Fgf-8 functions in combination with CD44, a cell surface binding protein, and that blocking CD44 binding results in inhibition of Fgf8-induced expression of Clim-2 and Lhx-6. Regulation of gene expression by Fgf8 in association with CD44 is thus conserved between limb and mandibular arch development.
Fasciclin II and Beaten path modulate intercellular adhesion inDrosophilalarval visual organ development
Authors: Holmes Amy L. , Heilig Joseph S.
Published: April 2021

Previous studies demonstrated that Fasciclin II and Beaten path are necessary for regulating cell adhesion events that are important for motoneuron development in Drosophila. We observe that the cell adhesion molecule Fasciclin II and the secreted anti-adhesion molecule Beaten path have additional critical roles in the development of at least one set of sensory organs, the larval visual organs. Taken together, phenotypic analysis, genetic interactions, expression studies and rescue experiments suggest that, in normal development, secretion of Beaten path by cells of the optic lobes allows the Fasciclin II-expressing larval visual organ cells to detach from the optic lobes as a cohesive cell cluster. Our results also demonstrate that mechanisms guiding neuronal development may be shared between motoneurons and sensory organs, and provide evidence that titration of adhesion and anti-adhesion is critical for early steps in development of the larval visual system.
Control of phyllotaxy in maize by theabphyl1gene
Authors: Jackson David , Hake Sarah
Published: April 2021

Organogenesis in plants occurs at the shoot apical meristem, a group of indeterminate stem cells that are organized during embryogenesis. Regulated initiation of leaves or flowers from the shoot meristem gives rise to the familiar geometric patterns observed throughout the plant kingdom. The mechanism by which these patterns, termed phyllotaxies, are generated, remains unclear. Maize plants initiate leaves singly, alternating from one side to the other in a regular pattern. Here we describe a recessive maize mutant, abphyl1, that initiates leaves in opposite pairs, in a pattern termed decussate phyllotaxy. The decussate shoot meristems are larger than normal throughout development, though the general structure and organization of the meristem is not altered. abph1 mutants are first distinguished during embryogenesis, prior to true leaf initiation, by a larger shoot meristem and coincident larger expression domain of the homeobox gene knotted1. Therefore, the abph1 gene regulates morphogenesis in the embryo, and plays a role in determining the phyllotaxy of the shoot.
Nuclear β-catenin is required to specify vegetal cell fates in the sea urchin embryo
Authors: Logan Catriona Y. , Miller Jeffrey R. , Ferkowicz Michael J. , McClay David R.
Published: April 2021

β-catenin is thought to mediate cell fate specification events by localizing to the nucleus where it modulates gene expression. To ask whether β-catenin is involved in cell fate specification during sea urchin embryogenesis, we analyzed the distribution of nuclear β-catenin in both normal and experimentally manipulated embryos. In unperturbed embryos, β-catenin accumulates in nuclei that include the precursors of the endoderm and mesoderm, suggesting that it plays a role in vegetal specification. Using pharmacological, embryological and molecular approaches, we determined the function of β-catenin in vegetal development by examining the relationship between the pattern of nuclear β-catenin and the formation of endodermal and mesodermal tissues. Treatment of embryos with LiCl, a known vegetalizing agent, caused both an enhancement in the levels of nuclear β-catenin and an expansion in the pattern of nuclear β-catenin that coincided with an increase in endoderm and mesoderm. Conversely, overexpression of a sea urchin cadherin blocked the accumulation of nuclear β-catenin and consequently inhibited the formation of endodermal and mesodermal tissues including micromere-derived skeletogenic mesenchyme. In addition, nuclear β-catenin-deficient micromeres failed to induce a secondary axis when transplanted to the animal pole of uninjected host embryos, indicating that nuclear β-catenin also plays a role in the production of micromere-derived signals. To examine further the relationship between nuclear β-catenin in vegetal nuclei and micromere signaling, we performed both transplantations and deletions of micromeres at the 16-cell stage and demonstrated that the accumulation of β-catenin in vegetal nuclei does not require micromere-derived cues. Moreover, we demonstrate that cell autonomous signals appear to regulate the pattern of nuclear β-catenin since dissociated blastomeres possessed nuclear β-catenin in approximately the same proportion as that seen in intact embryos. Together, these data show that the accumulation of β-catenin in nuclei of vegetal cells is regulated cell autonomously and that this localization is required for the establishment of all vegetal cell fates and the production of micromere-derived signals.
Expression of chicken fibroblast growth factor homologous factor (FHF)-1 and of differentially spliced isoforms of FHF-2 during development and involvement of FHF-2 in chicken limb development
Authors: Munoz-Sanjuan Ignacio , Simandl B. Kay , Fallon John F. , Nathans Jeremy
Published: April 2021

Members of the fibroblast growth factor (FGF) family have been identified as signaling molecules in a variety of developmental processes, including important roles in limb bud initiation, growth and patterning. This paper reports the cloning and characterization of the chicken orthologues of fibroblast growth factor homologous factors-1 and -2 (cFHF-1/cFGF-12 and cFHF-2/cFGF-13, respectively). We also describe the identification of a novel, conserved isoform of FHF-2 in chickens and mammals. This isoform arises by alternative splicing of the first exon of the FHF- 2 gene and is predicted to encode a polypeptide with a distinct amino-terminus.

Whole-mount in situ hybridization reveals restricted domains of expression of cFHF-1 and cFHF-2 in the developing neural tube, peripheral sensory ganglia and limb buds, and shows that the two cFHF-2 transcript isoforms are present in non-overlapping spatial distributions in the neural tube and adjacent structures. In the developing limbs, cFHF-1 is confined to the posterior mesoderm in an area that encompasses the zone of polarizing activity and cFHF-2 is confined to the distal anterior mesoderm in a region that largely overlaps the progress zone. Ectopic cFHF-2 expression is induced adjacent to grafts of cells expressing Sonic Hedgehog and the zone of cFHF-2 expression is expanded in talpid2 embryos. In the absence of the apical ectodermal ridge or in wingless or limbless mutant embryos, expression of cFHF-1 and cFHF-2 is lost from the limb bud. A role for cFHF-2 in the patterning and growth of skeletal elements is implied by the observation that engraftment of developing limb buds with QT6 cells expressing a cFHF-2 isoform that is normally expressed in the limb leads to a variety of morphological defects. Finally, we show that a secreted version of cFHF-2 activates the expression of HoxD13, HoxD11, Fgf-4 and BMP-2 ectopically, consistent with cFHF-2 playing a role in anterior-posterior patterning of the limb.
Dfrizzled-3, a new Drosophila Wnt receptor, acting as an attenuator of Wingless signaling in wingless hypomorphic mutants
Authors: Sato Atsushi , Kojima Tetsuya , Ui-Tei Kumiko , Miyata Yuhei , Saigo Kaoru
Published: April 2021

In Drosophila, two Frizzled proteins, Frizzled and Dfrizzled-2, have been reported to serve as receptors of Wingless. Here, we identified the third member of the Drosophila Frizzled family (Dfrizzled-3). In contrast to Dfrizzled-2, Dfrizzled-3 was transcriptionally upregulated by Wingless signaling. Although Dfrizzled-3 was capable of binding to Wingless in vitro, Wingless-dependent Armadillo/β-catenin stabilization occurred much less effectively in Drosophila cells transfected with Dfrizzled-3 than in those with Dfrizzled-2. Flies lacking Dfrizzled-3 activity were viable and fertile, with few morphological defects. Genetic and immunochemical analysis indicated that the absence of Dfrizzled-3 activity suppresses the effects of hypomorphic wingless mutations such as failure of wing and antenna formation and restores target gene expression to the normal levels without change in wingless expression. Wingless signaling may thus be attenuated by Dfrizzled-3 at least in wingless hypomorphic mutants.
Interplay of Notch and FGF signaling restricts cell fate and MAPK activation in the Drosophila trachea
Authors: Ikeya Tomoatsu , Hayashi Shigeo
Published: April 2021

The patterned branching in the Drosophila tracheal system is triggered by the FGF-like ligand Branchless that activates a receptor tyrosine kinase Breathless and the MAP kinase pathway. A single fusion cell at the tip of each fusion branch expresses the zinc-finger gene escargot, leads branch migration in a stereotypical pattern and contacts with another fusion cell to mediate fusion of the branches. A high level of MAP kinase activation is also limited to the tip of the branches. Restriction of such cell specialization events to the tip is essential for tracheal tubulogenesis. Here we show that Notch signaling plays crucial roles in the singling out process of the fusion cell. We found that Notch is activated in tracheal cells by Branchless signaling through stimulation of Delta expression at the tip of tracheal branches and that activated Notch represses the fate of the fusion cell. In addition, Notch is required to restrict activation of MAP kinase to the tip of the branches, in part through the negative regulation of Branchless expression. Notch-mediated lateral inhibition in sending and receiving cells is thus essential to restrict the inductive influence of Branchless on the tracheal tubulogenesis.
Vertebrate Sprouty genes are induced by FGF signaling and can cause chondrodysplasia when overexpressed
Authors: Minowada George , Jarvis Lesley A. , Chi Candace L. , Neubüser Annette , Sun Xin , Hacohen Nir , Krasnow Mark A. , Martin Gail R.
Published: April 2021

The Drosophila sprouty gene encodes an antagonist of FGF and EGF signaling whose expression is induced by the signaling pathways that it inhibits. Here we describe a family of vertebrate Sprouty homologs and demonstrate that the regulatory relationship with FGF pathways has been conserved. In both mouse and chick embryos, Sprouty genes are expressed in intimate association with FGF signaling centers. Gain- and loss-of-function experiments demonstrate that FGF signaling induces Sprouty gene expression in various tissues. Sprouty overexpression obtained by infecting the prospective wing territory of the chick embryo with a retrovirus containing a mouse Sprouty gene causes a reduction in limb bud outgrowth and other effects consistent with reduced FGF signaling from the apical ectodermal ridge. At later stages of development in the infected limbs there was a dramatic reduction in skeletal element length due to an inhibition of chondrocyte differentiation. The results provide evidence that vertebrate Sprouty proteins function as FGF-induced feedback inhibitors, and suggest a possible role for Sprouty genes in the pathogenesis of specific human chondrodysplasias caused by activating mutations in Fgfr3.
The role of the NK-homeobox gene slouch (S59) in somatic muscle patterning
Authors: Knirr Stefan , Azpiazu Natalia , Frasch Manfred
Published: April 2021

In the Drosophila embryo, a distinct class of myoblasts, designated as muscle founders, prefigures the mature pattern of somatic body wall muscles. Each founder cell appears to be instrumental in generating a single larval muscle with a defined identity. The NK homeobox gene S59 was the first of a growing number of proposed ‘identity genes’ that have been found to be expressed in stereotyped patterns in specific subsets of muscle founders and their progenitor cells and are thought to control their developmental fates. In the present study, we describe the effects of gain- and loss-of-function experiments with S59. We find that a null mutation in the gene encoding S59, which we have named slouch (slou), disrupts the development of all muscles that are derived from S59-expressing founder cells. The observed phenotypes upon mutation and ectopic expression of slouch include transformations of founder cell fates, thus confirming that slouch (S59) functions as an identity gene in muscle development. These fate transformations occur between sibling founder cells as well as between neighboring founders that are not lineage-related. In the latter case, we show that slouch (S59) activity is required cell-autonomously to repress the expression of ladybird (lb) homeobox genes, thereby preventing specification along the lb pathway. Together, these findings provide new insights into the regulatory interactions that establish the somatic muscle pattern.
Clonal analysis ofDrosophilaembryonic neuroblasts: neural cell types, axon projections and muscle targets
Authors: Schmid Aloisia , Chiba Akira , Doe Chris Q.
Published: April 2021

An experimental analysis of neurogenesis requires a detailed understanding of wild-type neural development. Recent DiI cell lineage studies have begun to elucidate the family of neurons and glia produced by each Drosophila embryonic neural precursor (neuroblast). Here we use DiI labeling to extend and clarify previous studies, but our analysis differs from previous studies in four major features: we analyze and compare lineages of every known embryonic neuroblast; we use an in vivo landmark (engrailed-GFP) to increase the accuracy of neuroblast identification; we use confocal fluorescence and Nomarski microscopy to collect three-dimensional data in living embryos simultaneously for each DiI-labeled clone, the engrailed-GFP landmark, and the entire CNS and muscle target field (Nomarski images); and finally, we analyze clones very late in embryonic development, which reveals novel cell types and axon/dendrite complexity.

We identify the parental neuroblasts for all the cell types of the embryonic CNS: motoneurons, intersegmental interneurons, local interneurons, glia and neurosecretory cells (whose origins had never been determined). We identify muscle contacts for every thoracic and abdominal motoneuron at stage 17. We define the parental neuroblasts for neurons or glia expressing well-known molecular markers or neurotransmitters. We correlate Drosophila cell lineage data with information derived from other insects. In addition, we make the following novel conclusions: (1) neuroblasts at similar dorsoventral positions, but not anteroposterior positions, often generate similar cell lineages, and (2) neuroblasts at similar dorsoventral positions often produce the same motoneuron subtype: ventral neuroblasts typically generate motoneurons with dorsal muscle targets, while dorsal neuroblasts produce motoneurons with ventral muscle targets.

Lineage data and movies can be found at http://www.biologists.com/Development/movies/dev8623.html/ and http://www.uoneuro.uoregon.edu/doelab/lineages.
Patterning the ascidian nervous system: structure, expression and transgenic analysis of theCiHox3gene
Authors: Locascio Annamaria , Aniello Francesco , Amoroso Alessandro , Manzanares Miguel , Krumlauf Robb , Branno Margherita
Published: April 2021

Hox genes play a fundamental role in the establishment of chordate body plan, especially in the anteroposterior patterning of the nervous system. Particularly interesting are the anterior groups of Hox genes (Hox1-Hox4) since their expression is coupled to the control of regional identity in the anterior regions of the nervous system, where the highest structural diversity is observed. Ascidians, among chordates, are considered a good model to investigate evolution of Hox gene, organisation, regulation and function. We report here the cloning and the expression pattern of CiHox3, a Ciona intestinalis anterior Hox gene homologous to the paralogy group 3 genes. In situ hybridization at the larva stage revealed that CiHox3 expression was restricted to the visceral ganglion of the central nervous system. The presence of a sharp posterior boundary and the absence of transcript in mesodermal tissues are distinctive features of CiHox3 expression when compared to the paralogy group 3 in other chordates. We have investigated the regulatory elements underlying CiHox3 neural-specific expression and, using transgenic analysis, we were able to isolate an 80 bp enhancer responsible of CiHox3 activation in the central nervous system (CNS). A comparative study between mouse and Ciona Hox3 promoters demonstrated that divergent mechanisms are involved in the regulation of these genes in vertebrates and ascidians.
Mode of action of VegT in mesoderm and endoderm formation
Authors: Clements Debbie , Friday Rosalind V. , Woodland Hugh R.
Published: April 2021

mRNA encoding the T-box transcription factor VegT is located throughout the vegetal pole of the Xenopus egg and is believed to play an important part in endoderm and mesoderm formation. We find that VegT generates endoderm both by cell-autonomous action and by generating TGF-β family signals, the latter being entirely responsible for its mesoderm-inducing activity. Signalling molecules induced cell-autonomously by VegT include derrière, Xnr4 and activin B. Xnr1 and Xnr2 are also induced, but primarily in a non-autonomous manner. All of these signalling molecules are found in the blastula and gastrula vegetal pole and induce both endoderm and mesoderm in the animal cap assay, and hence are good candidates both for the endogenous zygotic mesoderm-inducing signal and for reinforcing the vegetal expression of endoderm markers.
Translational regulation of oskar mRNA occurs independent of the cap and poly(A) tail in Drosophila ovarian extracts
Authors: Lie Yung S. , Macdonald Paul M.
Published: April 2021

Translational regulation plays a prominent role in Drosophila body patterning. Progress in elucidating the underlying mechanisms has been limited by the lack of a homologous in vitro system that supports regulation. Here we show that extracts prepared from Drosophila tissues are competent for translation. Ovarian extracts, but not embryonic extracts, support the Bruno response element- and Bruno-dependent repression of oskar mRNA translation, which acts in vivo to prevent protein synthesis from transcripts not localized to the posterior pole of the oocyte. Consistent with suggestive evidence from in vivo experiments, regulation in vitro does not involve changes in poly(A) tail length. Moreover, inhibition studies strongly suggest that repression does not interfere with the process of 5′ cap recognition. Translational regulation mediated through the Bruno response elements is thus likely to occur via a novel mechanism.
Key roles of retinoic acid receptors alpha and beta in the patterning of the caudal hindbrain, pharyngeal arches and otocyst in the mouse
Authors: Dupé Valérie , Ghyselinck Norbert B. , Wendling Olivia , Chambon Pierre , Mark Manuel
Published: April 2021

Mouse fetuses carrying targeted inactivations of both the RARα and the RARβ genes display a variety of malformations in structures known to be partially derived from the mesenchymal neural crest originating from postotic rhombomeres (e.g. thymus and great cephalic arteries) (Ghyselinck, N., Dupé, V., Dierich, A., Messaddeq, N., Garnier, J.M., Rochette-Egly, C., Chambon, P. and Mark M. (1997). Int. J. Dev. Biol. 41, 425-447). In a search for neural crest defects, we have analysed the rhombomeres, cranial nerves and pharyngeal arches of these double null mutants at early embryonic stages. The mutant posttic cranial nerves are disorganized, indicating that RARs are involved in the patterning of structures derived from neurogenic neural crest, even though the lack of RARα and RARβ has no detectable effect on the number and migration path of neural crest cells. Interestingly, the double null mutation impairs early developmental processes known to be independent of the neural crest e.g., the initial formation of the 3rd and 4th branchial pouches and of the 3rd, 4th and 6th arch arteries. The double mutation also results in an enlargement of rhombomere 5, which is likely to be responsible for the induction of supernumerary otic vesicles, in a disappearance of the rhombomere 5/6 boundary, and in profound alterations of rhombomere identities. In the mutant hindbrain, the expression domain of kreisler is twice its normal size and the caudal stripe of Krox-20 extends into the presumptive rhombomeres 6 and 7 region. In this region, Hoxb-1 is ectopically expressed, Hoxb-3 is ectopically up-regulated and Hoxd-4 expression is abolished. These data, which indicate that retinoic acid signaling through RARα and/or RARβ is essential for the specification of rhombomere identities and for the control of caudal hindbrain segmentation by restricting the expression domains of kreisler and of Krox-20, also strongly suggest that this signaling plays a crucial role in the posteriorization of the hindbrain neurectoderm.
A novel fork head gene mediates early steps during Xenopus lens formation
Authors: Kenyon Kristy L. , Moody Sally A. , Jamrich Milan
Published: April 2021

Xlens1 is a novel Xenopus member of the fork head gene family, named for its nearly restricted expression in the anterior ectodermal placode, presumptive lens ectoderm (PLE), and anterior epithelium of the differentiated lens. The temporal and spatial restriction of its expression suggests that: (1) Xlens1 is transcribed initially at neural plate stages in response to putative signals from the anterior neural plate that transform lens-competent ectoderm to lens-biased ectoderm; (2) further steps in the process of lens-forming bias restrict Xlens1 expression to the presumptive lens ectoderm (PLE) during later neural plate stages; (3) interactions with the optic vesicle maintain Xlens1 expression in the lens placode; and (4) Xlens1 expression is downregulated as committed lens cells undergo terminal differentiation. Induction assays demonstrate that pax6 induces Xlens1 expression, but unlike pax6, Xlens1 cannot induce the expression of the lens differentiation marker β-crystallin. In the whole embryo, overexpression of Xlens1 in the lens ectoderm causes it to thicken and maintain gene expression characteristics of the PLE. Also, this overexpression suppresses differentiation in the lens ectoderm, suggesting that Xlens1 functions to maintain specified lens ectoderm in an undifferentiated state. Misexpression of Xlens1 in other regions causes hypertrophy of restricted tissues but only occasionally leads ectopic sites of γ-crystallin protein expression in select anterior head regions. These results indicate that Xlens1 expression alone does not specify lens ectoderm. Lens specification and differentiation likely depends on a combination of other gene products and an appropriate level of Xlens1 activity.
Regulation of Hox target genes by a DNA bound Homothorax/Hox/Extradenticle complex
Authors: Ryoo Hyung Don , Marty Thomas , Casares Fernando , Affolter Markus , Mann Richard S.
Published: April 2021

To regulate their target genes, the Hox proteins of Drosophila often bind to DNA as heterodimers with the homeodomain protein Extradenticle (EXD). For EXD to bind DNA, it must be in the nucleus, and its nuclear localization requires a third homeodomain protein, Homothorax (HTH). Here we show that a conserved N-terminal domain of HTH directly binds to EXD in vitro, and is sufficient to induce the nuclear localization of EXD in vivo. However, mutating a key DNA binding residue in the HTH homeodomain abolishes many of its in vivo functions. HTH binds to DNA as part of a HTH/Hox/EXD trimeric complex, and we show that this complex is essential for the activation of a natural Hox target enhancer. Using a dominant negative form of HTH we provide evidence that similar complexes are important for several Hox- and exd-mediated functions in vivo. These data suggest that Hox proteins often function as part of a multiprotein complex, composed of HTH, Hox, and EXD proteins, bound to DNA.
Defects in mouse mammary gland development caused by conditional haploinsufficiency of Patched-1
Authors: Lewis Michael T. , Ross Sarajane , Strickland Phyllis A. , Sugnet Charles W. , Jimenez Elsa , Scott Matthew P. , Daniel Charles W.
Published: April 2021

In vertebrates, the hedgehog family of cell signaling proteins and associated downstream network components play an essential role in mediating tissue interactions during development and organogenesis. Loss-of-function or misexpression mutation of hedgehog network components can cause birth defects, skin cancer and other tumors. The mammary gland is a specialized skin derivative requiring epithelial-epithelial and epithelial-stromal tissue interactions similar to those required for development of other organs, where these interactions are often controled by hedgehog signaling. We have investigated the role of the Patched-1 (Ptc1) hedgehog receptor gene in mammary development and neoplasia. Haploinsufficiency at the Ptc1 locus results in severe histological defects in ductal structure, and minor morphological changes in terminal end buds in heterozygous postpubescent virgin animals. Defects are mainly ductal hyperplasias and dysplasias characterized by multilayered ductal walls and dissociated cells impacting ductal lumens. This phenotype is 100% penetrant.

Remarkably, defects are reverted during late pregnancy and lactation but return upon involution and gland remodeling. Whole mammary gland transplants into athymic mice demonstrates that the observed dysplasias reflect an intrisic developmental defect within the gland. However, Ptc1-induced epithelial dysplasias are not stable upon transplantation into a wild-type epithelium-free fat pad, suggesting stromal (or epithelial and stromal) function of Ptc1. Mammary expression of Ptc1 mRNA is both epithelial and stromal and is developmentally regulated. Phenotypic reversion correlates with developmentally regulated and enhanced expression of Indian hedgehog (Ihh) during pregnancy and lactation. Data demonstrate a critical mammary role for at least one component of the hedgehog signaling network and suggest that Ihh is the primary hedgehog gene active in the gland.
Involvement of the small GTPases XRhoA and XRnd1 in cell adhesion and head formation in early Xenopus development
Authors: Wünnenberg-Stapleton Katrin , Blitz Ira L. , Hashimoto Chikara , Cho Ken W. Y.
Published: April 2021

The Rho family of small GTPases regulates a variety of cellular functions, including the dynamics of the actin cytoskeleton, cell adhesion, transcription, cell growth and membrane trafficking. We have isolated the first Xenopus homologs of the Rho-like GTPases RhoA and Rnd1 and examined their potential roles in early Xenopus development. We found that Xenopus Rnd1 (XRnd1) is expressed in tissues undergoing extensive morphogenetic changes, such as marginal zone cells involuting through the blastopore, somitogenic mesoderm during somite formation and neural crest cells. XRnd1 also causes a severe loss of cell adhesion in overexpression experiments. These data and the expression pattern suggest that XRnd1 regulates morphogenetic movements by modulating cell adhesion in early embryos. Xenopus RhoA (XRhoA) is a potential XRnd1 antagonist, since overexpression of XRhoA increases cell adhesion in the embryo and reverses the disruption of cell adhesion caused by XRnd1. In addition to the potential roles of XRnd1 and XRhoA in the regulation of cell adhesion, we find a role for XRhoA in axis formation. When coinjected with dominant-negative BMP receptor (tBR) in the ventral side of the embryo, XRhoA causes the formation of head structures resembling the phenotype seen after coinjection of wnt inhibitors with dominant-negative BMP receptor. Since dominant-negative XRhoA is able to reduce the formation of head structures, we propose that XRhoA activity is essential for head formation. Thus, XRhoA may have a dual role in the embryo by regulating cell adhesion properties and pattern formation.
Pax1 and Pax9 synergistically regulate vertebral column development
Authors: Peters Heiko , Wilm Bettina , Sakai Norio , Imai Kenji , Maas Richard , Balling Rudi
Published: April 2021

The paralogous genes Pax1 and Pax9 constitute one group within the vertebrate Pax gene family. They encode closely related transcription factors and are expressed in similar patterns during mouse embryogenesis, suggesting that Pax1 and Pax9 act in similar developmental pathways. We have recently shown that mice homozygous for a defined Pax1 null allele exhibit morphological abnormalities of the axial skeleton, which is not affected in homozygous Pax9 mutants. To investigate a potential interaction of the two genes, we analysed Pax1/Pax9 double mutant mice. These mutants completely lack the medial derivatives of the sclerotomes, the vertebral bodies, intervertebral discs and the proximal parts of the ribs. This phenotype is much more severe than that of Pax1 single homozygous mutants. In contrast, the neural arches, which are derived from the lateral regions of the sclerotomes, are formed. The analysis of Pax9 expression in compound mutants indicates that both spatial expansion and upregulation of Pax9 expression account for its compensatory function during sclerotome development in the absence of Pax1.

In Pax1/Pax9 double homozygous mutants, formation and anteroposterior polarity of sclerotomes, as well as induction of a chondrocyte-specific cell lineage, appear normal. However, instead of a segmental arrangement of vertebrae and intervertebral disc anlagen, a loose mesenchyme surrounding the notochord is formed. The gradual loss of Sox9 and Collagen II expression in this mesenchyme indicates that the sclerotomes are prevented from undergoing chondrogenesis. The first detectable defect is a low rate of cell proliferation in the ventromedial regions of the sclerotomes after sclerotome formation but before mesenchymal condensation normally occurs. At later stages, an increased number of cells undergoing apoptosis further reduces the area normally forming vertebrae and intervertebral discs. Our results reveal functional redundancy between Pax1 and Pax9 during vertebral column development and identify an early role of Pax1 and Pax9 in the control of cell proliferation during early sclerotome development. In addition, our data indicate that the development of medial and lateral elements of vertebrae is regulated by distinct genetic pathways.
double-time is identical to discs overgrown, which is required for cell survival, proliferation and growth arrest in Drosophila imaginal discs
Authors: Zilian Olav , Frei Erich , Burke Richard , Brentrup Doris , Gutjahr Thomas , Bryant Peter J. , Noll Markus
Published: April 2021

We have isolated the discs overgrown gene of Drosophila and shown that it encodes a homolog of the Casein kinase Iδ/ε subfamily and is identical to the double-time gene. However, in contrast to the weak double-time alleles, which appear to affect only the circadian rhythm, discs overgrown alleles, including bona fide null alleles, show strong effects on cell survival and growth control in imaginal discs. Analysis of their phenotypes and molecular lesions suggests that the Discs overgrown protein is a crucial component in the mechanism that links cell survival during proliferation to growth arrest in imaginal discs. This work provides the first analysis in a multicellular organism of Casein kinase Iδ/ε functions necessary for survival. Since the amino acid sequences and three-dimensional structures of Casein kinase Iδ/ε enzymes are highly conserved, the results suggest that these proteins may also function in controlling cell growth and survival in other organisms.
Wingless transduction by the Frizzled and Frizzled2 proteins of Drosophila
Authors: Chen Chiann-mun , Struhl Gary
Published: April 2021

Wingless (Wg) protein is a founding member of the Wnt family of secreted proteins which have profound organizing roles in animal development. Two members of the Frizzled (Fz) family of seven-pass transmembrane proteins, Drosophila Fz and Fz2, can bind Wg and are candidate Wg receptors. However, null mutations of the fz gene have little effect on Wg signal transduction and the lack of mutations in the fz2 gene has thus far prevented a rigorous examination of its role in vivo. Here we describe the isolation of an amber mutation of fz2 which truncates the coding sequence just after the amino-terminal extracellular domain and behaves genetically as a loss-of-function allele. Using this mutation, we show that Wg signal transduction is abolished in virtually all cells lacking both Fz and Fz2 activity in embryos as well as in the wing imaginal disc. We also show that Fz and Fz2 are functionally redundant: the presence of either protein is sufficient to confer Wg transducing activity on most or all cells throughout development. These results extend prior evidence of a ligand-receptor relationship between Wnt and Frizzled proteins and suggest that Fz and Fz2 are the primary receptors for Wg in Drosophila.
An adenylyl cyclase that functions during late development of Dictyostelium
Authors: Söderbom Fredrik , Anjard Christophe , Iranfar Negin , Fuller Danny , Loomis William F.
Published: April 2021

A variety of extracellular signals lead to the accumulation of cAMP which can act as a second message within cells by activating protein kinase A (PKA). Expression of many of the essential developmental genes in Dictyostelium discoideum are known to depend on PKA activity. Cells in which the receptor-coupled adenylyl cyclase gene, acaA, is genetically inactivated grow well but are unable to develop. Surprisingly, acaA− mutant cells can be rescued by developing them in mixtures with wild-type cells, suggesting that another adenylyl cyclase is present in developing cells that can provide the internal cAMP necessary to activate PKA. However, the only other known adenylyl cyclase gene in Dictyostelium, acgA, is only expressed during germination of spores and plays no role in the formation of fruiting bodies. By screening morphological mutants generated by Restriction Enzyme Mediated Integration (REMI) we discovered a novel adenylyl cyclase gene, acrA, that is expressed at low levels in growing cells and at more than 25-fold higher levels during development. Growth and development up to the slug stage are unaffected in acrA− mutant strains but the cells make almost no viable spores and produce unnaturally long stalks. Adenylyl cyclase activity increases during aggregation, plateaus during the slug stage and then increases considerably during terminal differentiation. The increase in activity following aggregation fails to occur in acrA− cells. As long as ACA is fully active, ACR is not required until culmination but then plays a critical role in sporulation and construction of the stalk.
The transcription factor GATA3 is a downstream effector of Hoxb1 specification in rhombomere 4
Authors: Pata Illar , Studer Michèle , Doorninck J. Hikke van , Briscoe James , Kuuse Sulev , Engel J. Douglas , Grosveld Frank , Karis Alar
Published: April 2021

In this paper, we show that the transcription factor GATA3 is dynamically expressed during hindbrain development. Function of GATA3 in ventral rhombomere (r) 4 is dependent on functional GATA2, which in turn is under the control of Hoxb1. In particular, the absence of Hoxb1 results in the loss of GATA2 expression in r4 and the absence of GATA2 results in the loss of GATA3 expression. The lack of GATA3 expression in r4 inhibits the projection of contralateral vestibuloacoustic efferent neurons and the migration of facial branchiomotor neurons similar to Hoxb1-deficient mice. Ubiquitous expression of Hoxb1 in the hindbrain induces ectopic expression of GATA2 and GATA3 in ventral r2 and r3. These findings demonstrate that GATA2 and GATA3 lie downstream of Hoxb1 and provide the first example of Hox pathway transcription factors within a defined population of vertebrate motor neurons.
Anterior movement of ventral diencephalic precursors separates the primordial eye field in the neural plate and requires cyclops
Authors: Varga Zoltán M. , Wegner Jeremy , Westerfield Monte
Published: April 2021

A currently favored hypothesis postulates that a single field of cells in the neural plate forms bilateral retinas. To learn how retinal precursors segregate, we followed individual labeled neural plate cells in zebrafish. In the late gastrula, a single field of odd-paired-like-expressing cells contributed to both retinas, bordered posteriorly by diencephalic precursors expressing mariposa. Median mariposa-expressing cells moved anteriorly, separating the eyes, and formed ventral anterior diencephalon, the presumptive hypothalamus. In cyclops mutants, corresponding cells failed to move anteriorly, a ventral diencephalon never formed, and the eyes remained fused. Ablation of the region containing these cells induced cyclopia in wild types. Our results indicate that movement of a median subpopulation of diencephalic precursors separates retinal precursors into left and right eyes. Wild-type cyclops gene function is required for these morphogenetic movements.
The actin cytoskeleton is required to elaborate and maintain spatial patterning during trichome cell morphogenesis in Arabidopsis thaliana
Authors: Mathur Jaideep , Spielhofer Pius , Kost Benedikt , Chua Nam-Hai
Published: April 2021

Arabidopsis thaliana trichomes provide an attractive model system to dissect molecular processes involved in the generation of shape and form in single cell morphogenesis in plants. We have used transgenic Arabidopsis plants carrying a GFP-talin chimeric gene to analyze the role of the actin cytoskeleton in trichome cell morphogenesis. We found that during trichome cell development the actin microfilaments assumed an increasing degree of complexity from fine filaments to thick, longitudinally stretched cables. Disruption of the F-actin cytoskeleton by actin antagonists produced distorted but branched trichomes which phenocopied trichomes of mutants belonging to the ‘distorted’ class. Subsequent analysis of the actin cytoskeleton in trichomes of the distorted mutants, alien, crooked, distorted1, gnarled, klunker and wurm uncovered actin organization defects in each case. Treatments of wild-type seedlings with microtubule-interacting drugs elicited a radically different trichome phenotype characterized by isotropic growth and a severe inhibition of branch formation; these trichomes did not show defects in actin cytoskeleton organization. A normal actin cytoskeleton was also observed in trichomes of the zwichel mutant which have reduced branching. ZWICHEL, which was previously shown to encode a kinesin-like protein is thought to be involved in microtubule-linked processes. Based on our results we propose that microtubules establish the spatial patterning of trichome branches whilst actin microfilaments elaborate and maintain the overall trichome pattern during development.
Decoding vectorial information from a gradient: sequential roles of the receptors Frizzled and Notch in establishing planar polarity in the Drosophila eye
Authors: Tomlinson Andrew , Struhl Gary
Published: April 2021

The Drosophila eye is composed of several hundred ommatidia that can exist in either of two chiral forms, depending on position: ommatidia in the dorsal half of the eye adopt one chiral form, whereas ommatidia in the ventral half adopt the other. Chirality appears to be specified by a polarizing signal with a high activity at the interface between the two halves (the ‘equator’), which declines in opposite directions towards the dorsal and ventral poles. Here, using genetic mosaics, we show that this polarizing signal is decoded by the sequential use of two receptor systems. The first depends on the seven-transmembrane receptor Frizzled (Fz) and distinguishes between the two members of the R3/R4 pair of presumptive photoreceptor cells, predisposing the cell that is located closer to the equator and having higher Fz activity towards the R3 photoreceptor fate and the cell further away towards the R4 fate. This bias is then amplified by subsequent interactions between the two cells mediated by the receptor Notch (N) and its ligand Delta (Dl), ensuring that the equatorial cell becomes the R3 photoreceptor while the polar cell becomes the R4 photoreceptor. As a consequence of this reciprocal cell fate decision, the R4 cell moves asymmetrically relative to the R3 cell, initiating the appropriate chiral pattern of the remaining cells of the ommatidium.