bluuebunny/crossref_metadata_2025_split huggingface dataset:
Analysis of the genetic pathway leading to formation of ectopic apical ectodermal ridges in mouse Engrailed-1 mutant limbs
Authors: Loomis Cynthia A. , Kimmel Robin A. , Tong Chun-Xiang , Michaud Jacques , Joyner Alexandra L. 
Published: April 2021

ABSTRACT
The apical ectodermal ridge (AER), a rim of thickened ectodermal cells at the interface between the dorsal and ventral domains of the limb bud, is required for limb outgrowth and patterning. We have previously shown that the limbs of En1 mutant mice display dorsal-ventral and proximal-distal abnormalities, the latter being reflected in the appearance of a broadened AER and formation of ectopic ventral digits. A detailed genetic analysis of wild-type, En1 and Wnt7a mutant limb buds during AER development has delineated a role for En1 in normal AER formation. Our studies support previous suggestions that AER maturation involves the compression of an early broad ventral domain of limb ectoderm into a narrow rim at the tip and further show that En1 plays a critical role in the compaction phase. Loss of En1 leads to a delay in the distal shift and stratification of cells in the ventral half of the AER. At later stages, this often leads to development of a secondary ventral AER, which can promote formation of an ectopic digit. The second AER forms at the juxtaposition of the ventral border of the broadened mutant AER and the distal border of an ectopic Lmx1b expression domain. Analysis of En1/Wnt7a double mutants demonstrates that the dorsalizing gene Wnt7a is required for the formation of the ectopic AERs in En1 mutants and for ectopic expression of Lmx1b in the ventral mesenchyme. We suggest a model whereby, in En1 mutants, ectopic ventral Wnt7a and/or Lmx1b expression leads to the transformation of ventral cells in the broadened AER to a more dorsal phenotype. This leads to induction of a second zone of compaction ventrally, which in some cases goes on to form an autonomous secondary AER.
Antimorphic goosecoids
Authors: Ferreiro Beatriz , Artinger Michael , Cho Ken W. Y. , Niehrs Christof 
Published: April 2021

ABSTRACT
goosecoid (gsc) is a homeobox gene expressed in the Spemann organizer that has been implicated in vertebrate axis formation. Here antimorphic gscs are described. One antimorphic gsc (MTgsc) was fortuitously created by adding 5 myc epitopes to the N terminus of gsc. The other antimorph (VP16gsc) contains the transcriptional activation domain of VP16. mRNA injection of either antimorph inhibits dorsal gastrulation movements and leads to embryos with severe axial defects. They upregulate ventral gene expression in the dorsal marginal zone and inhibit dorsal mesoderm differentiation. Like the VP16 domain, the N-terminal myc tags act by converting wild-type gsc from a transcriptional repressor into an activator. However, unlike MTgsc, VP16gsc is able at low dose to uncouple head from trunk formation, indicating that different antimorphs may elicit distinct phenotypes. The experiments reveal that gsc and/or gsc-related genes function in axis formation and gastrulation. Moreover, this work warns against using myc tags indiscriminately for labeling DNA-binding proteins.
A Xenopus homologue of aml-1 reveals unexpected patterning mechanisms leading to the formation of embryonic blood
Authors: Tracey W. Daniel , Pepling Melissa E. , Horb Marko E. , Thomsen Gerald H. , Gergen J. Peter 
Published: April 2021

ABSTRACT
The Runt domain gene AML1 is essential for definitive hematopoiesis during murine embryogenesis. We have isolated Xaml, a Xenopus AML1 homologue in order to investigate the patterning mechanisms responsible for the generation of hematopoietic precursors. Xaml is expressed early in the developing ventral blood island in a pattern that anticipates that of later globin. Analysis of globin and Xaml expression in explants, in embryos with perturbed dorsal ventral patterning, and by lineage tracing indicates that the formation of the ventral blood island is more complex than previously thought and involves contributions from both dorsal and ventral tissues. A truncated Xaml protein interferes with primitive hematopoiesis. Based on these results, we propose that Runt domain proteins function in the specification of hematopoietic stem cells in vertebrate embryos.
Cis-regulation downstream of cell type specification: a single compact element controls the complex expression of the CyIIa gene in sea urchin embryos
Authors: Arnone Maria I. , Martin Ellen L. , Davidson Eric H. 
Published: April 2021

ABSTRACT
CyIIa, a cytoskeletal actin gene of Strongylocentrotus purpuratus, is expressed specifically though transiently in the embryonic skeletogenic and secondary mesenchyme and, later in development, is permanently activated in the hindgut and midgut. CyIIa transcription follows, and is therefore downstream of, the initial specification of these embryonic domains. A detailed functional analysis of the cis-regulatory system governing the rate and the location of CyIIa expression during development was carried out using GFP expression constructs. About 4.4 kb of CyIIa sequence including a leader intron were examined for cis-regulatory function. Distal elements scattered over several kb account for 60% of the quantitative output of the expression construct and a strong amplifier of expression is located within the leader intron. However, the complex spatial pattern of CyIIa expression is completely reproduced by a compact upstream regulatory element &lt;450 bp in length. We found no evidence anywhere in the 4.4 kb sequence examined for negative regulators required to repress ectopic expression. The specific site that mediates CyIIa expression in the midgut in late embryos and larvae was identified. This site is the same as that necessary and sufficient for midgut expression of the Endo16 gene late in development, and was shown to bind the same transcription factor. Except for some temporal and quantitative features, the S. purpuratus expression construct is expressed accurately and specifically in the same diverse cell types when introduced into embryos of Lytechinus pictus, which belongs to a different echinoid order. No ectopic expression was observed, in contrast to the result of a similar interspecific gene transfer experiment carried out earlier on a different cytoskeletal actin gene that is expressed much earlier in development. Presentation of the set of transcription factors that activate CyIIa in the differentiated cells in which it is expressed is apparently a conserved feature of these cell types.
The Drosophila Medea gene is required downstream of dpp and encodes a functional homolog of human Smad4
Authors: Hudson John B. , Podos Steven D. , Keith Kallie , Simpson Susan L. , Ferguson Edwin L. 
Published: April 2021

ABSTRACT
The Transforming Growth Factor-β superfamily member decapentaplegic (dpp) acts as an extracellular morphogen to pattern the embryonic ectoderm of the Drosophila embryo. To identify components of the dpp signaling pathway, we screened for mutations that act as dominant maternal enhancers of a weak allele of the dpp target gene zerknüllt. In this screen, we recovered new alleles of the Mothers against dpp (Mad) and Medea genes. Phenotypic analysis of the new Medea mutations indicates that Medea, like Mad, is required for both embryonic and imaginal disc patterning. Genetic analysis suggests that Medea may have two independently mutable functions in patterning the embryonic ectoderm. Complete elimination of maternal and zygotic Medea activity in the early embryo results in a ventralized phenotype identical to that of null dpp mutants, indicating that Medea is required for all dpp-dependent signaling in embryonic dorsal-ventral patterning. Injection of mRNAs encoding DPP or a constitutively activated form of the DPP receptor, Thick veins, into embryos lacking all Medea activity failed to induce formation of any dorsal cell fates, demonstrating that Medea acts downstream of the thick veins receptor. We cloned Medea and found that it encodes a protein with striking sequence similarity to human SMAD4. Moreover, injection of human SMAD4 mRNA into embryos lacking all Medea activity conferred phenotypic rescue of the dorsal-ventral pattern, demonstrating conservation of function between the two gene products.
Isolation and characterization of endothelial progenitor cells from mouse embryos
Authors: Hatzopoulos Antonis K. , Folkman Judah , Vasile Eliza , Eiselen Grant K. , Rosenberg Robert D. 
Published: April 2021

ABSTRACT
The cardiovascular system develops early in embryogenesis from cells of mesodermal origin. To study the molecular and cellular processes underlying this transition, we have isolated mesodermal cells from murine embryos at E7.5 with characteristic properties of endothelial progenitors by using a combination of stromal cell layers and growth conditions. The isolated embryonic cells displayed unlimited stem-cell-like growth potential and a stable phenotype in culture. RNA analysis revealed that the embryonic cells express the endothelial-specific genes tie-2 and thrombomodulin (TM) as well as the early mesodermal marker fgf-3. The GSL I-B4 isolectin, a marker of early endothelial cells, specifically binds to the isolated cells. The in vitro differentiation with retinoic acid and cAMP led to a 5– to 10-fold induction of flk-1, von Willebrand Factor (vWF), TM, GATA-4 and GATA-6. Electron microscopy revealed that in vitro differentiation is associated with increased amounts of rER and Golgi, and a dramatic increase in secretory vesicles packed with vWF. When cultured in Matrigel, the embryonic cells assume the characteristic endothelial cobblestone morphology and form tubes. Injection into chicken embryos showed incorporation of the embryonic cells in the endocardium and the brain vasculature. The expression of TM, tie-2, GATA-4 and GATA-6 suggests that the isolated embryonic endothelial cell progenitors are derived from the proximal lateral mesoderm where the pre-endocardial tubes form. The properties of the endothelial cell progenitors described here provide a novel approach to analyze mediators, signaling pathways and transcriptional control in early vascular development.
virilizer regulates Sex-lethal in the germline of Drosophila melanogaster
Authors: Schütt Corina , Hilfiker Andres , Nöthiger Rolf 
Published: April 2021

ABSTRACT
In Drosophila, the gene Sex-lethal (Sxl) is required for female development. It controls sexual differentiation in the soma, dosage compensation and oogenesis. The continuous production of SXL proteins in XX animals is maintained by autoregulation and depends on virilizer (vir). This gene is required in somatic cells for the female-specific splicing of Sxl primary transcripts and for an unknown vital process in both sexes. In the soma, clones of XX cells lacking Sxl or vir are sexually transformed and form male structures; in the germline, XX cells mutant for Sxl extensively proliferate, but are unable to differentiate.
We now studied the role of vir in the germline by generating germline chimeras. We found that XX germ cells mutant for vir, in contrast to cells mutant for Sxl, perform oogenesis. We show that the early production of SXL in undifferentiated germ cells is independent of vir while, later in oogenesis, expression of Sxl becomes dependent on vir. We conclude that the early SXL proteins are sufficient for the production of eggs whereas the later SXL proteins are dispensable for this process.
However, vir must be active in the female germline to allow normal embryonic development because maternal products of vir are required for the early post-transcriptional regulation of Sxl in XX embryos and for a vital process in embryos of both sexes.
The Drosophila gene Medea demonstrates the requirement for different classes of Smads in dpp signaling
Authors: Das Pradeep , L. Maduzia Lisa , Wang Huang , L. Finelli Alyce , Cho Seo-Hee , Smith Marsha M. , Padgett Richard W. 
Published: April 2021

ABSTRACT
Signals from transforming growth factor-β (TGF-β) ligands are transmitted within the cell by members of the Smad family, which can be grouped into three classes based on sequence similarities. Our previous identification of both class I and II Smads functioning in a single pathway in C. elegans, raised the issue of whether the requirement for Smads derived from different classes is a general feature of TGF-β signaling. We report here the identification of a new Drosophila class II Smad, Medea, a close homolog of the human tumor-suppressor gene DPC4. Embryos from germline clones of both Medea and Mad (a class I Smad) are ventralized, as are embryos null for the TGF-β-like ligand decapentaplegic (dpp). Loss of Medea also blocks dpp signaling during later development, suggesting that Medea, like Mad, is universally required for dpp signaling. Furthermore, we show that the necessity for these two closely related, non-redundant Smads, is due to their different signaling properties – upon activation of the Dpp pathway, Mad is required to actively translocate Medea into the nucleus. These results provide a paradigm for, and distinguish between, the requirement for class I and II Smads in Dpp/BMP signaling.
Regulation of bHLH-PAS protein subcellular localization during Drosophila embryogenesis
Authors: Ward Mary P. , Mosher Jack T. , Crews Stephen T. 
Published: April 2021

ABSTRACT
The Drosophila Single-minded and Tango basic-helix-loop-helix-PAS protein heterodimer controls transcription and embryonic development of the CNS midline cells, while the Trachealess and Tango heterodimer controls tracheal cell and salivary duct transcription and development. Expression of both single-minded and trachealess is highly restricted to their respective cell lineages, however tango is broadly expressed. The developmental control of subcellular localization of these proteins is investigated because of their similarity to the mammalian basic-helix-loop-helix-PAS Aromatic hydrocarbon receptor whose nuclear localization is dependent on ligand binding. Confocal imaging of Single-minded and Trachealess protein localization indicate that they accumulate in cell nuclei when initially synthesized in their respective cell lineages and remain nuclear throughout embryogenesis. Ectopic expression experiments show that Single-minded and Trachealess are localized to nuclei in cells throughout the ectoderm and mesoderm, indicating that nuclear accumulation is not regulated in a cell-specific fashion and unlikely to be ligand dependent. In contrast, nuclear localization of Tango is developmentally regulated; it is localized to the cytoplasm in most cells except the CNS midline, salivary duct, and tracheal cells where it accumulates in nuclei. Genetic and ectopic expression experiments indicate that Tango nuclear localization is dependent on the presence of a basic-helix-loop-helix-PAS protein such as Single-minded or Trachealess. Conversely, Drosophila cell culture experiments show that Single-minded and Trachealess nuclear localization is dependent on Tango since they are cytoplasmic in the absence of Tango. These results suggest a model in which Single-minded and Trachealess dimerize with Tango in the cytoplasm of the CNS midline cells and trachea, respectively, and the dimeric complex accumulates in nuclei in a ligand-independent mode and regulates lineage-specific transcription. The lineage-specific action of Single-minded and Trachealess derives from transcriptional activation of their genes in their respective lineages, not from extracellular signaling.
The CArG boxes in the promoter of the Arabidopsis floral organ identity gene APETALA3 mediate diverse regulatory effects
Authors: Tilly Joline J. , Allen David W. , Jack Thomas 
Published: April 2021

ABSTRACT
APETALA3 is a MADS box gene required for normal development of the petals and stamens in the Arabidopsis flower. Studies in yeast, mammals and plants demonstrate that MADS domain transcription factors bind with high affinity to a consensus sequence called the CArG box. The APETALA3 promoter contains three close matches to the consensus CArG box sequence. To gain insights into the APETALA3 regulatory circuitry, we have analyzed the APETALA3 promoter using AP3::uidA(GUS) fusions. 496 base pairs of APETALA3 promoter sequence 5′ to the transcriptional start directs GUS activity in the same temporal and spatial expression pattern as the APETALA3 RNA and protein in wild-type flowers. A synthetic promoter consisting of three tandem repeats of a 143 base pair sequence directs reporter gene activity exclusively to petals and stamens in the flower. We have analyzed the role of the CArG boxes by site-specific mutagenesis and find that the three CArG boxes mediate discrete regulatory effects. Mutations in CArG1 result in a decrease in reporter expression suggesting that CArG1 is the binding site for a positively acting factor or factors. Mutations in CArG2 result in a decrease in reporter expression in petals, but the expression pattern in stamens is unchanged. By contrast, mutations in CArG3 result in an increase in the level of reporter gene activity during early floral stages suggesting that CArG3 is the binding site for a negatively acting factor.
crooked legs encodes a family of zinc ﬁnger proteins required for leg morphogenesis and ecdysone-regulated gene expression during Drosophila metamorphosis
Authors: D′Avino Pier Paolo , Thummel Carl S. 
Published: April 2021

ABSTRACT
Drosophila imaginal discs undergo extensive pattern formation during larval development, resulting in each cell acquiring a speciﬁc adult fate. The ﬁnal manifestation of this pattern into adult structures is dependent on pulses of the steroid hormone ecdysone during metamorphosis, which trigger disc eversion, elongation and differentiation. We have deﬁned genetic criteria that allow us to screen for ecdysone-inducible regulatory genes that are required for this transformation from patterned disc to adult structure. We describe here the ﬁrst genetic locus isolated using these criteria: crooked legs (crol). crol mutants die during pupal development with defects in adult head eversion and leg morphogenesis. The crol gene is induced by ecdysone during the onset of metamorphosis and encodes at least three protein isoforms that contain 12-18 C2H2 zinc ﬁngers. Consistent with this sequence motif, crol mutations have stage-speciﬁc effects on ecdysone-regulated gene expression. The EcR ecdysone receptor, and the BR-C, E74 and E75 early regulatory genes, are submaximally induced in crol mutants in response to the prepupal ecdysone pulse. These changes in gene activity are consistent with the crol lethal phenotypes and provide a basis for understanding the molecular mechanisms of crol action. The genetic criteria described here provide a new direction for identifying regulators of adult tissue development during insect metamorphosis.
Proximodistal axis formation in the Drosophila leg: subdivision into proximal and distal domains by Homothorax and Distal-less
Authors: Wu Jun , Cohen Stephen M. 
Published: April 2021

Abstract
The developing legs of Drosophila are subdivided into proximal and distal domains by the activity of the homeodomain proteins Homothorax (Hth) and Distal-less (Dll). The expression domains of Dll and Hth are initially reciprocal. Wingless and Dpp define both domains by activating Dll and by repressing Hth in the distal region of the disc. Wg and Dpp do not act through Dll to repress Hth. Hth functions to reduce the sensitivity of proximal cells to Wg and Dpp. This serves to limit the effective range of these signals in regulating later-acting genes such as Dac. We present evidence that proximal and distal cells tend to sort- out from one another. Cells forced to express Hth are unable to mix with distal cells. Likewise, cells forced to express Dll are unable to mix with proximal cells. Clones of cells unable to express Dll in the distal region sort-out from the disc. Clones of cells unable to express Hth lose the specialized population of cells at the interface between proximal and distal territories and cause fusion between body wall and leg segments. These observations suggest that sorting-out behavior of Hth- and Dll-expressing cells contributes to subdivision of the leg into proximal and distal domains.
Competence, specification and induction of Pax-3 in the trigeminal placode
Authors: Baker Clare V. H. , Stark Michael R. , Marcelle Christophe , Bronner-Fraser Marianne 
Published: April 2021

ABSTRACT
Placodes are discrete regions of thickened ectoderm that contribute extensively to the peripheral nervous system in the vertebrate head. The paired-domain transcription factor Pax-3 is an early molecular marker for the avian ophthalmic trigeminal (opV) placode, which forms sensory neurons in the ophthalmic lobe of the trigeminal ganglion. Here, we use collagen gel cultures and heterotopic quail- chick grafts to examine the competence, specification and induction of Pax-3 in the opV placode. At the 3-somite stage, the whole head ectoderm rostral to the first somite is competent to express Pax-3 when grafted to the opV placode region, though competence is rapidly lost thereafter in otic-level ectoderm. Pax-3 specification in presumptive opV placode ectoderm occurs by the 8-somite stage, concomitant with robust Pax-3 expression. From the 8-somite stage onwards, significant numbers of cells are committed to express Pax-3. The entire length of the neural tube has the ability to induce Pax-3 expression in competent head ectoderm and the inductive interaction is direct. We propose a detailed model for Pax-3 induction in the opV placode.
A comparative molecular approach to mesodermal patterning in basal deuterostomes: the expression pattern of Brachyury in the enteropneust hemichordate Ptychodera flava
Authors: Peterson Kevin J. , Cameron R. Andrew , Tagawa Kunifumi , Satoh Noriyuki , Davidson Eric H. 
Published: April 2021

ABSTRACT
This work concerns the formation of mesoderm in the development of an enteropneust hemichordate, Ptychodera flava, and the expression of the Brachyury gene during this process. Brachyury expression occurs in two distinct phases. In the embryo, Brachyury is transcribed during gastrulation in the future oral and anal regions of the gut, but transcripts are no longer detected by 2 weeks of development. Brachyury expression is not detected during the 5 months of larval planktonic existence. During this time, the adult coeloms begin to develop, originating as coalescences of cells that appear to delaminate from the wall of the gut. Brachyury expression cannot be detected again until metamorphosis, when transcripts appear in the mesoderm of the adult proboscis, collar and the very posterior region of the trunk. It is also expressed in the posterior end of the gut. At no time is Brachyury expressed in the stomochord, the putative homologue of the chordate notochord. These observations illuminate the process of maximal indirect development in Ptychodera and, by comparison with patterns of Brachyury expression in the indirect development of echinoderms, their sister group, they reveal the evolutionary history of Brachyury utilization in deuterostomes.