bluuebunny/crossref_metadata_2025 huggingface dataset:
The function of the Drosophila fat facets deubiquitinating enzyme in limiting photoreceptor cell number is intimately associated with endocytosis
Authors: Cadavid A.L. , Ginzel A. , Fischer J.A.
Published: April 2021

Fat facets is a deubiquitinating enzyme required in a cell communication pathway that limits to eight the number of photoreceptor cells in each facet of the Drososphila compound eye. Genetic data support a model whereby Faf removes ubiquitin, a polypeptide tag for protein degradation, from a specific ubiquitinated protein thus preventing its degradation. Here, mutations in the liquid facets gene were identified as dominant enhancers of the fat facets mutant eye phenotype. The liquid facets locus encodes epsin, a vertebrate protein associated with the clathrin endocytosis complex. The results of genetic experiments reveal that fat facets and liquid facets facilitate endocytosis and function in common cells to generate an inhibitory signal that prevents ectopic photoreceptor determination. Moreover, it is demonstrated that the fat facets mutant phenotype is extraordinarily sensitive to the level of liquid facets expression. We propose that Liquid facets is a candidate for the critical substrate of Fat facets in the eye.
Integration of FGF and TWIST in calvarial bone and suture development
Authors: Rice David P. C. , Åberg Thomas , Chan Yan-Shun , Tang Zequn , J. Kettunen Päivi , Pakarinen Leila , Maxson Robert E. , Thesleff Irma
Published: April 2021

Mutations in the FGFR1-FGFR3 and TWIST genes are known to cause craniosynostosis, the former by constitutive activation and the latter by haploinsufficiency. Although clinically achieving the same end result, the premature fusion of the calvarial bones, it is not known whether these genes lie in the same or independent pathways during calvarial bone development and later in suture closure. We have previously shown that Fgfr2c is expressed at the osteogenic fronts of the developing calvarial bones and that, when FGF is applied via beads to the osteogenic fronts, suture closure is accelerated (Kim, H.-J., Rice, D. P. C., Kettunen, P. J. and Thesleff, I. (1998)Development 125, 1241-1251). In order to investigate further the role of FGF signalling during mouse calvarial bone and suture development, we have performed detailed expression analysis of the splicing variants of Fgfr1-Fgfr3 and Fgfr4, as well as their potential ligand Fgf2. The IIIc splice variants of Fgfr1-Fgfr3 as well as the IIIb variant of Fgfr2 being expressed by differentiating osteoblasts at the osteogenic fronts (E15). In comparison to Fgf9, Fgf2 showed a more restricted expression pattern being primarily expressed in the sutural mesenchyme between the osteogenic fronts. We also carried out a detailed expression analysis of the helix-loop-helix factors (HLH) Twist and Id1 during calvaria and suture development (E10-P6). Twist and Id1 were expressed by early preosteoblasts, in patterns that overlapped those of the FGF ligands, but as these cells differentiated their expression dramatically decreased. Signalling pathways were further studied in vitro, in E15 mouse calvarial explants. Beads soaked in FGF2 induced Twist and inhibited Bsp, a marker of functioning osteoblasts. Meanwhile, BMP2 upregulated Id1. Id1 is a dominant negative HLH thought to inhibit basic HLH such as Twist. In Drosophila, the FGF receptor FR1 is known to be downstream of Twist. We demonstrated that in Twist+/− mice, FGFR2 protein expression was altered. We propose a model of osteoblast differentiation integrating Twist and FGF in the same pathway, in which FGF acts both at early and late stages. Disruption of this pathway may lead to craniosynostosis.
The transcription factor Lmx1b maintains Wnt1 expression within the isthmic organizer
Authors: Adams Kirk A. , Maida Jennifer M. , Golden Jeffrey A. , Riddle Robert D.
Published: April 2021

Cells in the caudal mesencephalon and rostral metencephalon become organized by signals emanating from the isthmus organizer (IsO). The IsO is associated with the isthmus, a morphological constriction of the neural tube which eventually defines the mesencephalic/ metencephalic boundary (MMB). Here we report that the transcription factor Lmx1b is expressed and functions in a distinct region of the IsO. Lmx1b expression is maintained by the glycoprotein Fgf8, a signal capable of mediating IsO signaling. Lmx1b, in turn, maintains the expression of the secreted factor Wnt1. Our conclusions are substantiated by the following: (i) Lmx1b mRNA becomes localized to the isthmus immediately after Fgf8 initiation, (ii) Wnt1 expression is localized to the Lmx1b expression domain, but with slightly later kinetics, (iii) Fgf8-soaked beads generate similar domains of expression for Lmx1b and Wnt1 and (iv) retroviral-mediated expression of Lmx1b (Lmx1b/RCAS) maintains Wnt1 expression in the mesencephalon. Ectopic Lmx1b is insufficient to alter the expression of a number of other genes expressed at the IsO, suggesting that it does not generate a new signaling center. Instead, if we allow Lmx1b/RCAS-infected brains to develop longer, we detect changes in mesencephalic morphology. Since both ectopic and endogenous Lmx1b expression occurs in regions of the isthmus undergoing morphological changes, it could normally play a role in this process. Furthermore, a similar phenotype is not observed in Wnt1/RCAS-infected brains, demonstrating that ectopic Wnt1 is insufficient to mediate the effect of ectopic Lmx1b in our assay. Since Wnt1 function has been linked to the proper segregation of mesencephalic and metencephalic cells, we suggest that Lmx1b and Wnt1 normally function in concert to affect IsO morphogenesis.
The Drosophila homeobox gene optix is capable of inducing ectopic eyes by an eyeless-independent mechanism
Authors: Seimiya Makiko , Gehring Walter J.
Published: April 2021

optix is a new member of the Six/so gene family from Drosophila that contains both a six domain and a homeodomain. Because of its high amino acid sequence similarity with the mouse Six3 gene, optix is considered to be the orthologous gene from Drosophila rather than sine oculis, as previously believed. optix expression was detected in the eye, wing and haltere imaginal discs. Ectopic expression of optix leads to the formation of ectopic eyes suggesting that optix has important functions in eye development. Although optix and sine oculis belong to the same gene family (Six/so) and share a high degree of amino acid sequence identity, there are a number of factors which suggest that their developmental roles are different: (1) the expression patterns of optix and sine oculis are clearly distinct; (2) sine oculis acts downstream of eyeless, whereas optix is expressed independently of eyeless; (3) sine oculis functions synergistically with eyes absent in eye development whereas optix does not; (4) ectopic expression of optix alone, but not of sine oculis can induce ectopic eyes in the antennal disc. These results suggest that optix is involved in eye morphogenesis by an eyeless-independent mechanism.
Functional analysis of the Drosophila Diaphanous FH protein in early embryonic development
Authors: Afshar Katayoun , Stuart Bridget , Wasserman Steven A.
Published: April 2021

The Drosophila Formin Homology (FH) protein Diaphanous has an essential role during cytokinesis. To gain insight into the function of Diaphanous during cytokinesis and explore its role in other processes, we generated embryos deficient for Diaphanous and analyzed three cell-cycle-regulated actin-mediated events during embryogenesis: formation of the metaphase furrow, cellularization and formation of the pole cells. In dia embryos, all three processes are defective. Actin filaments do not organize properly to the metaphase and cellularization furrows and the actin ring is absent from the base of the presumptive pole cells. Furthermore, plasma membrane invaginations that initiate formation of the metaphase furrow and pole cells are missing. Immunolocalization studies of wild-type embryos reveal that Diaphanous localizes to the site where the metaphase furrow is anticipated to form, to the growing tip of cellularization furrows, and to contractile rings. In addition, the dia mutant phenotype reveals a role for Diaphanous in recruitment of myosin II, anillin and Peanut to the cortical region between actin caps. Our findings thus indicate that Diaphanous has a role in actin cytoskeleton organization and is essential for many, if not all, actin-mediated events involving membrane invagination. Based on known biochemical functions of FH proteins, we propose that Diaphanous serves as a mediator between signaling molecules and actin organizers at specific phases of the cell cycle.
Laser-induced gene expression in specific cells of transgenic zebrafish
Authors: Halloran Mary C. , Sato-Maeda Mika , Warren James T. , Su Fengyun , Lele Zsolt , Krone Patrick H. , Kuwada John Y. , Shoji Wataru
Published: April 2021

Over the past few years, a number of studies have described the generation of transgenic lines of zebrafish in which expression of reporters was driven by a variety of promoters. These lines opened up the real possibility that transgenics could be used to complement the genetic analysis of zebrafish development. Transgenic lines in which the expression of genes can be regulated both in space and time would be especially useful. Therefore, we have cloned the zebrafish promoter for the inducible hsp70 gene and made stable transgenic lines of zebrafish that express the reporter green fluorescent protein gene under the control of a hsp70 promoter. At normal temperatures, green fluorescent protein is not detectable in transgenic embryos with the exception of the lens, but is robustly expressed throughout the embryo following an increase in ambient temperature. Furthermore, we have taken advantage of the accessibility and optical clarity of the embryos to express green fluorescent protein in individual cells by focussing a sublethal laser microbeam onto them. The targeted cells appear to develop normally: cells migrate normally, neurons project axons that follow normal pathways, and progenitor cells divide and give rise to normal progeny cells. By generating other transgenic lines in which the hsp70 promoter regulates genes of interest, it should be possible to examine the in vivo activity of the gene products by laser-inducing specific cells to express them in zebrafish embryos. As a first test, we laser-induced single muscle cells to make zebrafish Sema3A1, a semaphorin that is repulsive for specific growth cones, in a hsp70-sema3A1 transgenic line of zebrafish and found that extension by the motor axons was retarded by the induced muscle.
Recruitment of components of Polycomb Group chromatin complexes in Drosophila
Authors: Poux Sylvain , McCabe Donna , Pirrotta Vincenzo
Published: April 2021

Polycomb Group complexes assemble at polycomb response elements (PREs) in vivo and silence genes in the surrounding chromatin. To study the recruitment of silencing complexes, we have targeted various Polycomb Group (PcG) proteins by fusing them to the LexA DNA binding domain. When LexA-PC, -PSC, -PH or -SU(Z)2 are targeted to a reporter gene, they recruit functional PcG-silencing complexes that recapitulate the silencing behavior of a PRE: silencing is sensitive to the state of activity of the target chromatin. When the target is transcriptionally active, silencing is not established but when the target is not active at syncytial blastoderm, it becomes silenced. The repressed state persists through embryonic development but cannot be maintained in larval imaginal discs even when the LexA-PcG fusion is constitutively expressed, suggesting a discontinuity in the mechanism of repression. These proteins also interact with other PC-containing complexes in embryonic nuclear extracts. In contrast LexA-PHO is neither able to silence nor to interact with PC-containing complexes. Analysis of pho mutant embryos and of PRE constructs whose PHO-binding sites are mutated suggests that, while PHO is important for silencing in imaginal discs, it is not necessary for embryonic PcG silencing.
Heparan sulfate proteoglycans are critical for the organization of the extracellular distribution of Wingless
Authors: Baeg Gyeong-Hun , Lin Xinhua , Khare Narmada , Baumgartner Stefan , Perrimon Norbert
Published: April 2021

Recent studies in Drosophila have shown that heparan sulfate proteoglycans (HSPGs) are required for Wingless (Wg/Wnt) signaling. In addition, genetic and phenotypic analyses have implicated the glypican gene dally in this process. Here, we report the identification of another Drosophila glypican gene, dally-like (dly) and show that it is also involved in Wg signaling. Inhibition of dly gene activity implicates a function for DLY in Wg reception and we show that overexpression of DLY leads to an accumulation of extracellular Wg. We propose that DLY plays a role in the extracellular distribution of Wg. Consistent with this model, a dramatic decrease of extracellular Wg was detected in clones of cells that are deficient in proper glycosaminoglycan biosynthesis. We conclude that HSPGs play an important role in organizing the extracellular distribution of Wg.
The dermomyotome dorsomedial lip drives growth and morphogenesis of both the primary myotome and dermomyotome epithelium
Authors: Ordahl Charles P. , Berdougo Eli , Venters Sara J. , Denetclaw Wilfred F.
Published: April 2021

The cellular and molecular mechanisms that govern early muscle patterning in vertebrate development are unknown. The earliest skeletal muscle to organize, the primary myotome of the epaxial domain, is a thin sheet of muscle tissue that expands in each somite segment in a lateral-to-medial direction in concert with the overlying dermomyotome epithelium. Several mutually contradictory models have been proposed to explain how myotome precursor cells, which are known to reside within the dermomyotome, translocate to the subjacent myotome layer to form this first segmented muscle tissue of the body. Using experimental embryology to discriminate among these models, we show here that ablation of the dorsomedial lip (DML) of the dermomyotome epithelium blocks further primary myotome growth while ablation of other dermomyotome regions does not. Myotome growth and morphogenesis can be restored in a DML-ablated somite of a host embryo by transplantation of a second DML from a donor embryo. Chick-quail marking experiments show that new myotome cells in such recombinant somites are derived from the donor DML and that cells from other regions of the somite are neither present nor required. In addition to the myotome, the transplanted DML also gives rise to the dermomyotome epithelium overlying the new myotome growth region and from which the mesenchymal dermatome will later emerge. These results demonstrate that the DML is a cellular growth engine that is both necessary and sufficient to drive the growth and morphogenesis of the primary myotome and simultaneously drive that of the dermomyotome, an epithelium containing muscle, dermis and possibly other potentialities.
REF-1, a protein with two bHLH domains, alters the pattern of cell fusion inC. elegansby regulating Hox protein activity
Authors: Alper Scott , Kenyon Cynthia
Published: April 2021

Hox genes control the choice of cell fates along the anteroposterior (AP) body axis of many organisms. In C. elegans, two Hox genes, lin-39 and mab-5, control the cell fusion decision of the 12 ventrally located Pn.p cells. Specific Pn.p cells fuse with an epidermal syncytium, hyp7, in a sexually dimorphic pattern. In hermaphrodites, Pn.p cells in the mid-body region remain unfused whereas in males, Pn.p cells adopt an alternating pattern of syncytial and unfused fates. The complexity of these fusion patterns arises because the activities of these two Hox proteins are regulated in a sex-specific manner. MAB-5 activity is inhibited in hermaphrodite Pn.p cells and thus MAB-5 normally only affects the male Pn.p fusion pattern. Here we identify a gene, ref-1, that regulates the hermaphrodite Pn.p cell fusion pattern largely by regulating MAB-5 activity in these cells. Mutation of ref-1 also affects the fate of other epidermal cells in distinct AP body regions. ref-1 encodes a protein with two basic helix-loop-helix domains distantly related to those of the hairy/Enhancer of split family. ref-1, and another hairy homolog, lin-22, regulate similar cell fate decisions in different body regions along the C. elegans AP body axis.
A BAC transgenic analysis of the Mrf4/Myf5 locus reveals interdigitated elements that control activation and maintenance of gene expression during muscle development
Authors: Carvajal Jaime J. , Cox David , Summerbell Dennis , Rigby Peter W. J.
Published: April 2021

The muscle-specific transcription factors Myf5 and Mrf4 are two of the four myogenic regulatory factors involved in the transcriptional cascade responsible for skeletal myogenesis in the vertebrate embryo. Myf5 is the first of these four genes to be expressed in the mouse. We have previously described discrete enhancers that drive Myf5 expression in epaxial and hypaxial somites, branchial arches and central nervous system, and argued that additional elements are required for proper expression (Summerbell, D., Ashby, P. R., Coutelle, O., Cox, D., Yee, S. P. and Rigby, P. W. J. (2000) Development 127, 3745–3757). We have now investigated the transcriptional regulation of both Myf5 and Mrf4 using bacterial artificial chromosome transgenesis. We show that a clone containing Myf5 and 140 kb of upstream sequences is sufficient to recapitulate the known expression patterns of both genes. Our results confirm and reinforce the conclusion of our earlier studies, that Myf5 expression is regulated differently in each of a considerable number of populations of muscle progenitors, and they begin to illuminate the evolutionary origins of this complex regulation. We further show that separate elements are involved in the activation and maintenance of expression in the various precursor populations, reflecting the diversity of the signals that control myogenesis. Mrf4 expression requires at least four elements, one of which may be shared with Myf5, providing a possible explanation for the linkage of these genes throughout vertebrate phylogeny. Further complexity is revealed by the demonstration that elements which control Mrf4 and Myf5 are embedded in an unrelated neighbouring gene.
Non-imprinted Igf2r expression decreases growth and rescues the Tme mutation in mice
Authors: Wutz A. , Theussl H. C. , Dausman J. , Jaenisch R. , Barlow D. P. , Wagner E. F.
Published: April 2021

In the mouse the insulin-like growth factor receptor type 2 gene (Igf2r) is imprinted and maternally expressed. Igf2r encodes a trans-membrane receptor that transports mannose-6-phosphate tagged proteins and insulin-like growth factor 2 to lysosomes. During development the receptor reduces the amount of insulin-like growth factors and thereby decreases embryonic growth. The dosage of the gene is tightly regulated by genomic imprinting, leaving only the maternal copy of the gene active. Although the function of Igf2r in development is well established, the function of imprinting the gene remains elusive. Gene targeting experiments in mouse have demonstrated that the majority of genes are not sensitive to gene dosage, and mice heterozygous for mutations generally lack phenotypic alterations. To investigate whether reduction of Igf2r gene dosage by genomic imprinting has functional consequences for development we generated a non-imprinted allele (R2Δ). We restored biallelic expression to Igf2r by deleting a critical element for repression of the paternal allele (region 2) in mouse embryonic stem cells. Maternal inheritance of the R2Δ allele has no phenotype; however, paternal inheritance results in bialleleic expression of Igf2r, which causes a 20% reduction in weight late in embryonic development that persists into adulthood. Paternal inheritance of the R2Δ allele rescues the lethality of a maternally inherited Igf2r null allele and a maternally inherited Tme (T-associated maternal effect) mutation. These data show that the biological function of imprinting Igf2r is to increase birth weight and they also establish Igf2r as the Tme gene.
Interactions between Wnt and Vg1 signalling pathways initiate primitive streak formation in the chick embryo
Authors: Skromne Isaac , Stern Claudio D.
Published: April 2021

The posterior marginal zone (PMZ) of the chick embryo has Nieuwkoop centre-like properties: when transplanted to another part of the marginal zone, it induces a complete embryonic axis, without making a cellular contribution to the induced structures. However, when the PMZ is removed, the embryo can initiate axis formation from another part of the remaining marginal zone. Chick Vg1 can mimic the axis-inducing ability of the PMZ, but only when misexpressed somewhere within the marginal zone. We have investigated the properties that define the marginal zone as a distinct region. We show that the competence of the marginal zone to initiate ectopic primitive streak formation in response to cVg1 is dependent on Wnt activity. First, within the Wnt family, only Wnt8C is expressed in the marginal zone, in a gradient decreasing from posterior to anterior. Second, misexpression of Wnt1 in the area pellucida enables this region to form a primitive streak in response to cVg1. Third, the Wnt antagonists Crescent and Dkk-1 block the primitive streak-inducing ability of cVg1 in the marginal zone. These findings suggest that Wnt activity defines the marginal zone and allows cVg1 to induce an axis. We also present data suggesting some additional complexity: first, the Vg1 and Wnt pathways appear to regulate the expression of downstream components of each other’s pathway; and second, misexpression of different Wnt antagonists suggests that different classes of Wnts may cooperate with each other to regulate axis formation in the normal embryo.
Involvement of a proline-rich motif and RING-H2 finger of Deltex in the regulation of Notch signaling
Authors: Matsuno Kenji , Ito Mikiko , Hori Kazuya , Miyashita Fumiyasu , Suzuki Satoshi , Kishi Noriyuki , Artavanis-Tsakonas Spyros , Okano Hideyuki
Published: April 2021

The Notch pathway is an evolutionarily conserved signaling mechanism that is essential for cell-cell interactions. The Drosophila deltex gene regulates Notch signaling in a positive manner, and its gene product physically interacts with the intracellular domain of Notch through its N-terminal domain. Deltex has two other domains that are presumably involved in protein-protein interactions: a proline-rich motif that binds to SH3-domains, and a RING-H2 finger motif. Using an overexpression assay, we have analyzed the functional involvement of these Deltex domains in Notch signaling. The N-terminal domain of Deltex that binds to the CDC10/Ankyrin repeats of the Notch intracellular domain was indispensable for the function of Deltex. A mutant form of Deltex that lacked the proline-rich motif behaved as a dominant-negative form. This dominant-negative Deltex inhibited Notch signaling upstream of an activated, nuclear form of Notch and downstream of full-length Notch, suggesting the dominant-negative Deltex might prevent the activation of the Notch receptor. We found that Deltex formed a homo-multimer, and mutations in the RING-H2 finger domain abolished this oligomerization. The same mutations in the RING-H2 finger motif of Deltex disrupted the function of Deltex in vivo. However, when the same mutant was fused to a heterologous dimerization domain (Glutathione-S-Transferase), the chimeric protein had normal Deltex activity. Therefore, oligomerization mediated by the RING-H2 finger motif is an integral step in the signaling function of Deltex.
Manipulation of leaf shape by modulation of cell division
Authors: Wyrzykowska Joanna , Pien Stéphane , Shen Wen Hui , Fleming Andrew J.
Published: April 2021

The role of cell division as a causal element in plant morphogenesis is debatable, with accumulating evidence supporting the action of cell division-independent mechanisms. To directly test the morphogenic function of cell division, we have utilised a microinduction technique to locally and transiently manipulate the expression in transgenic plants of two genes encoding putative effectors of the cell cycle, a tobacco A-type cyclin and a yeast cdc25. The results show that local expression of these genes leads to modulation of cell division patterns. Moreover, whereas altered cell division in the apical meristem had no influence on organogenesis, local induction of cell proliferation on the flanks of young leaf primordia led to a dramatic change in lamina development and, thus, leaf shape. These data indicate that the role of cell division in plant morphogenesis is context dependent and identify cell division in the leaf primordium as a potential target for factors regulating leaf shape.
EGFR signalling inhibits Capicua-dependent repression during specification ofDrosophilawing veins
Authors: Roch Fernando , Jiménez Gerardo , Casanova Jordi
Published: April 2021

Localised activation of the Ras/Raf pathway by Epidermal Growth Factor Receptor (EGFR) signalling specifies the formation of veins in the Drosophila wing. However, little is known about how the EGFR signal regulates transcriptional responses during the vein/intervein cell fate decision. We provide evidence that EGFR signalling induces expression of vein-specific genes by inhibiting the Capicua (Cic) HMG-box repressor, a known regulator of embryonic body patterning. Lack of Cic function causes ectopic expression of EGFR targets such as argos, ventral veinless and decapentaplegic and leads to formation of extra vein tissue. In vein cells, EGFR signalling downregulates Cic protein levels in the nucleus and relieves repression of vein-specific genes, whereas intervein cells maintain high levels of Cic throughout larval and pupal development, repressing the expression of vein-specific genes and allowing intervein differentiation. However, regulation of some EGFR targets such as rhomboid appears not to be under direct control of Cic, suggesting that EGFR signalling branches out in the nucleus and controls different targets via distinct mediator factors. Our results support the idea that localised inactivation of transcriptional repressors such as Cic is a rather general mechanism for regulation of target gene expression by the Ras/Raf pathway.
reaperis required for neuroblast apoptosis duringDrosophiladevelopment
Authors: Peterson Christian , Carney Ginger E. , Taylor Barbara J. , White Kristin
Published: April 2021

Developmentally regulated apoptosis in Drosophila requires the activity of the reaper (rpr), grim and head involution defective (hid) genes. The expression of these genes is differentially regulated, suggesting that there are distinct requirements for their proapoptotic activity in response to diverse developmental and environmental inputs. To examine this hypothesis, a mutation that removes the rpr gene was generated. In flies that lack rpr function, most developmental apoptosis was unaffected. However, the central nervous systems of rpr null flies were very enlarged. This was due to the inappropriate survival of both larval neurons and neuroblasts. Importantly, neuroblasts rescued from apoptosis remained functional, continuing to proliferate and generating many extra neurons. Males mutant for rpr exhibited behavioral defects resulting in sterility. Although both the ecdysone hormone receptor complex and p53 directly regulate rpr transcription, rpr was found to play a limited role in inducing apoptosis in response to either of these signals.
Requirement for downregulation of kreisler during late patterning of the hindbrain
Authors: Theil Thomas , Ariza-McNaughton Linda , Manzanares Miguel , Brodie Jim , Krumlauf Robb , Wilkinson David G.
Published: April 2021

Pattern formation in the hindbrain is governed by a segmentation process that provides the basis for the organisation of cranial motor nerves. A cascade of transcriptional activators, including the bZIP transcription factor encoded by the kreisler gene controls this segmentation process. In kreisler mutants, r5 fails to form and this correlates with abnormalities in the neuroanatomical organisation of the hindbrain. Studies of Hox gene regulation suggest that kreisler may regulate the identity as well as the formation of r5, but such a role cannot be detected in kreisler mutants since r5 is absent. To gain further insights into the function of kreisler we have generated transgenic mice in which kreisler is ectopically expressed in r3 and for an extended period in r5. In these transgenic mice, the Fgf3, Krox20, Hoxa3 and Hoxb3 genes have ectopic or prolonged expression domains in r3, indicating that it acquires molecular characteristics of r5. Prolonged kreisler expression subsequently causes morphological alterations of r3/r5 that are due to an inhibition of neuronal differentiation and migration from the ventricular zone to form the mantle layer. We find that these alterations in r5 correlate with an arrest of facial branchiomotor neurone migration from r4 into the caudal hindbrain, which is possibly due to the deficiency in the mantle layer through which they normally migrate. We propose that the requirement for the downregulation of segmental kreisler expression prior to neuronal differentiation reflects the stage-specific roles of this gene and its targets.
TLP-1 is an asymmetric cell fate determinant that responds to Wnt signals and controls male tail tip morphogenesis inC. elegans
Authors: Zhao Xiaojun , Yang Ying , Fitch David H. A. , Herman Michael A.
Published: April 2021

We have isolated mutations defining a new gene, tlp-1, that affect asymmetric cell fates and morphogenesis during the development of the C. elegans tail. tlp-1 mutations cause defects in the specification of asymmetric cell fates in the descendants of the T blast cell, whose polarity is controlled by Wnt signaling and cause abnormal male tail development leading to the formation of a posterior protrusion reminiscent of ‘leptoderan’, or pointy tailed, nematode species. In wild-type C. elegans males, which have a ‘peloderan’ or rounded tail, retraction of the tail tip hypodermis involves a temporally ordered set of cell fusions and changes in cell shape that appear to be heterochronically delayed in tlp-1 males, suggesting that subtle changes in these events can bring about evolutionary changes in morphology. tlp-1 encodes a C2H2 zinc-finger protein that is a member of the Sp family of transcription factors. A TLP-1::GFP fusion protein is expressed in the nuclei of many cells during early embryogenesis and then becomes restricted primarily to posterior cells. At hatching, it is expressed in several head neurons, the posterior intestine cells, tail hypodermal cells, the T cells and specific T-cell descendents in a pattern that suggests TLP-1 may be asymmetrically expressed during the divisions of the T cell lineage. Furthermore, the asymmetry of TLP-1 expression and function appears to be controlled by Wnt signals that control T cell polarity. These results suggest that tlp-1 encodes a transcription factor required for cellular asymmetry that functions downstream of Wnt signals that control cell polarity, as well as in cell fusion and patterning in the C. elegans tail.
Comparison of the generic neuronal differentiation and neuron subtype specification functions of mammalianachaete-scuteandatonalhomologs in cultured neural progenitor cells
Authors: Lo Liching , Dormand Emma , Greenwood Amy , Anderson David J.
Published: April 2021

In the vertebrate peripheral nervous system, the proneural genes neurogenin 1 and neurogenin 2 (Ngn1 and Ngn2), and Mash1 are required for sensory and autonomic neurogenesis, respectively. In cultures of neural tube-derived, primitive PNS progenitors NGNs promote expression of sensory markers and MASH1 that of autonomic markers. These effects do not simply reflect enhanced neuronal differentiation, suggesting that both bHLH factors also specify neuronal identity like their Drosophila counterparts. At high concentrations of BMP2 or in neural crest stem cells (NCSCs), however, NGNs like MASH1 promote only autonomic marker expression. These data suggest that that the identity specification function of NGNs is more sensitive to context than is that of MASH1. In NCSCs, MASH1 is more sensitive to Notch-mediated inhibition of neurogenesis and cell cycle arrest, than are the NGNs. Thus, the two proneural genes differ in other functional properties besides the neuron subtype identities they can promote. These properties may explain cellular differences between MASH1- and NGN-dependent lineages in the timing of neuronal differentiation and cell cycle exit.
The MAPK pathway triggers activation of Nek2 during chromosome condensation in mouse spermatocytes
Authors: Di Agostino Silvia , Rossi Pellegrino , Geremia Raffaele , Sette Claudio
Published: April 2021

Chromosome condensation during the G2/M progression of mouse pachytene spermatocytes induced by the phosphatase inhibitor okadaic acid (OA) requires the activation of the MAPK Erk1. In many cell systems, p90Rsks are the main effectors of Erk1/2 function. We have identified p90Rsk2 as the isoform that is specifically expressed in mouse spermatocytes and have shown that it is activated during the OA-triggered meiotic G2/M progression. By using the MEK inhibitor U0126, we have demonstrated that activation of p90Rsk2 during meiotic progression requires activation of the MAPK pathway. Immunofluorescence analysis indicates that activated Erks and p90Rsk2 are tightly associated with condensed chromosomes during the G2/M transition in meiotic cells. We also found that active p90Rsk2 was able to phosphorylate histone H3 at Ser10 in vitro, but that the activation of the Erk1/p90Rsk2 pathway was not necessary for phosphorylation of H3 in vivo. Furthermore, phosphorylation of H3 was not sufficient to cause condensation of meiotic chromosomes in mouse spermatocytes. Other proteins known to associate with chromatin may represent effectors of Erk1 and p90Rsk2 during chromosome condensation. Nek2 (NIMA-related kinase 2), which associates with chromosomes, plays an active role in chromatin condensation and is stimulated by treatment of pachytene spermatocytes with okadaic acid. We show that inhibition of the MAPK pathway by preincubation of spermatocytes with U0126 suppresses Nek2 activation, and that incubation of spermatocyte cell extracts with activated p90Rsk2 causes stimulation of Nek2 kinase activity. Furthermore, we show that the Nek2 kinase domain is a substrate for p90Rsk2 phosphorylation in vitro. These data establish a connection between the Erk1/p90Rsk2 pathway, Nek2 activation and chromosome condensation during the G2/M transition of the first meiotic prophase.
Early embryonic expression ofFGF4/6/9gene and its role in the induction of mesenchyme and notochord inCiona savignyiembryos
Authors: Imai Kaoru S. , Satoh Nori , Satou Yutaka
Published: April 2021

In early Ciona savignyi embryos, nuclear localization of β-catenin is the first step of endodermal cell specification, and triggers the activation of various target genes. A cDNA for Cs-FGF4/6/9, a gene activated downstream of β-catenin signaling, was isolated and shown to encode an FGF protein with features of both FGF4/6 and FGF9/20. The early embryonic expression of Cs-FGF4/6/9 was transient and the transcript was seen in endodermal cells at the 16- and 32-cell stages, in notochord and muscle cells at the 64-cell stage, and in nerve cord and muscle cells at the 110-cell stage; the gene was then expressed again in cells of the nervous system after neurulation. When the gene function was suppressed with a specific antisense morpholino oligo, the differentiation of mesenchyme cells was completely blocked, and the fate of presumptive mesenchyme cells appeared to change into that of muscle cells. The inhibition of mesenchyme differentiation was abrogated by coinjection of the morpholino oligo and synthetic Cs-FGF4/6/9 mRNA. Downregulation of β-catenin nuclear localization resulted in the absence of mesenchyme cell differentiation due to failure of the formation of signal-producing endodermal cells. Injection of synthetic Cs-FGF4/6/9 mRNA in β-catenin-downregulated embryos evoked mesenchyme cell differentiation. These results strongly suggest that Cs-FGF4/6/9 produced by endodermal cells acts an inductive signal for the differentiation of mesenchyme cells. On the other hand, the role of Cs-FGF4/6/9 in the induction of notochord cells is partial; the initial process of the induction was inhibited by Cs-FGF4/6/9 morpholino oligo, but notochord-specific genes were expressed later to form a partial notochord.
A positive role for Patched-Smoothened signaling in promoting cell proliferation during normal head development inDrosophila
Authors: Shyamala Baragur V. , Bhat Krishna Moorthi
Published: April 2021

The transmembrane receptor Patched regulates several developmental processes in both invertebrates and vertebrates. In vertebrates, Patched also acts as a tumor suppressor. The Patched pathway normally operates by negatively regulating Smoothened, a G-protein-coupled receptor; binding of Hedgehog ligand to Patched relieves this negative interaction and allows signaling by Smoothened. We show that Ptc regulates Drosophila head development by promoting cell proliferation in the eye-antennal disc. During head morphogenesis, Patched positively interacts with Smoothened, which leads to the activation of Activin type I receptor Baboon and stimulation of cell proliferation in the eye-antennal disc. Thus, loss of Ptc or Smoothened activity affects cell proliferation in the eye-antennal disc and results in adult head capsule defects. Similarly, reducing the dose of smoothened in a patched background enhances the head defects. Consistent with these results, gain-of-function Hedgehog interferes with the activation of Baboon by Patched and Smoothened, leading to a similar head capsule defect. Expression of an activated form of Baboon in the patched domain in a patched mutant background completely rescues the head defects. These results provide insight into head morphogenesis, a process we know very little about, and reveal an unexpected non-canonical positive signaling pathway in which Patched and Smoothened function to promote cell proliferation as opposed to repressing it.
Reciprocal functions of theDrosophilaYellow and Ebony proteins in the development and evolution of pigment patterns
Authors: Wittkopp Patricia J. , True John R. , Carroll Sean B.
Published: April 2021

Body coloration affects how animals interact with the environment. In insects, the rapid evolution of black and brown melanin patterns suggests that these are adaptive traits. The developmental and molecular mechanisms that generate these pigment patterns are largely unknown. We demonstrate that the regulation and function of the yellow and ebony genes in Drosophila melanogaster play crucial roles in this process. The Yellow protein is required to produce black melanin, and is expressed in a pattern that correlates with the distribution of this pigment. Conversely, Ebony is required to suppress some melanin formation, and is expressed in cells that will produce both melanized and non-melanized cuticle. Ectopic expression of Ebony inhibits melanin formation, but increasing Yellow expression can overcome this effect. In addition, ectopic expression of Yellow is sufficient to induce melanin formation, but only in the absence of Ebony. These results suggest that the patterns and levels of Yellow and Ebony expression together determine the pattern and intensity of melanization. Based on their functions in Drosophila melanogaster, we propose that changes in the expression of Yellow and/or Ebony may have evolved with melanin patterns. Consistent with our hypothesis, we find that Yellow and Ebony are expressed in complementary spatial patterns that correlate with the formation of an evolutionary novel, male-specific pigment pattern in Drosophila biarmipes wings. These findings provide a developmental and genetic framework for understanding the evolution of melanin patterns.
Pelle kinase is activated by autophosphorylation during Toll signaling inDrosophila
Authors: Shen Baohe , Manley James L.
Published: April 2021

The Drosophila Pelle kinase plays a key role in the evolutionarily conserved Toll signaling pathway, but the mechanism responsible for its activation has been unknown. We present in vivo and in vitro evidence establishing an important role for concentration-dependent autophosphorylation in the signaling process. We first show that Pelle phosphorylation can be detected transiently in early embryos, concomitant with activation of signaling. Importantly, Pelle phosphorylation is enhanced in a gain-of-function Toll mutant (Toll10b), but decreased by loss-of-function Toll alleles. Next we found that Pelle is phosphorylated in transfected Schneider L2 cells in a concentration-dependent manner such that significant modification is observed only at high Pelle concentrations, which coincide with levels required for phosphorylation and activation of the downstream target, Dorsal. Pelle phosphorylation is also enhanced in L2 cells co-expressing Toll10b, and is dependent on Pelle kinase activity. In vitro kinase assays revealed that recombinant, autophosphorylated Pelle is far more active than unphosphorylated Pelle. Importantly, unphosphorylated Pelle becomes autophosphorylated, and activated, by incubation at high concentrations. We discuss these results in the context of Toll-like receptor mediated signaling in both flies and mammals.
Regulation of avian cardiogenesis by Fgf8 signaling
Authors: Alsan Burak H. , Schultheiss Thomas M.
Published: April 2021

The avian heart develops from paired primordia located in the anterior lateral mesoderm of the early embryo. Previous studies have found that the endoderm adjacent to the cardiac primordia plays an important role in heart specification. The current study provides evidence that fibroblast growth factor (Fgf) signaling contributes to the heart-inducing properties of the endoderm. Fgf8 is expressed in the endoderm adjacent to the precardiac mesoderm. Removal of endoderm results in a rapid downregulation of a subset of cardiac markers, including Nkx2.5 and Mef2c. Expression of these markers can be rescued by supplying exogenous Fgf8. In addition, application of ectopic Fgf8 results in ectopic expression of cardiac markers. Expression of cardiac markers is expanded only in regions where bone morphogenetic protein (Bmp) signaling is also present, suggesting that cardiogenesis occurs in regions exposed to both Fgf and Bmp signaling. Finally, evidence is presented that Fgf8 expression is regulated by particular levels of Bmp signaling. Application of low concentrations of Bmp2 results in ectopic expression of Fgf8, while application of higher concentrations of Bmp2 result in repression of Fgf8 expression. Together, these data indicate that Fgf signaling cooperates with Bmp signaling to regulate early cardiogenesis.
Studies on pattern regulation in hydra
Authors: Webster Gerald
Published: April 2021

The dynamic behaviour of the factors (level of inhibition and threshold for inhibition) controlling hypostome formation in hydra has been investigated during regulation using transplantation techniques. The formation of a new hypostome following removal of the original hypostome is accompanied by restoration of the level of inhibition to that of the intact hydra. The maximum time for them to occur is about 9 h. It may be less— about 5 h which is the time taken for a new hypostome to form from the subhypostomal region, as shown in a previous paper. During the formation of a hypostome from the distal peduncle the threshold for inhibition rises in the presumptive hypostomal region. The results suggest that the factors controlling the time for hypostome formation and threshold for inhibition are closely linked. It has not been possible to demonstrate changes in threshold properties in regions other than the presumptive hypostomal region until some time after the new hypostome has formed. The time-threshold properties of a region are determined by its position on the linear axis in relation to the hypostome. The significance of the results in relation to polarized regulation in hydra and other hydroids is discussed, and the concepts developed are used to explain some cases of reversal of polarity following grafting. A model for proportionate regulation in a two-compartment system is presented, based on the concepts of control developed for hydra.
Action of actinomycin and puromycin upon frog oocyte maturation
Authors: Dettlaff T. A.
Published: April 2021

In amphibians, as well as in other vertebrates, occytes start to mature under the action of hypophyseal gonadotropic hormones. (In this paper the term ‘maturation’ implies the transformation into ripe eggs of oocytes that have finished growing.) In the course of maturation the oocytes themselves and the follicle cells surrounding them undergo changes; the changes of these two cell types are not causally connected, their coincidence in time is easily broken in unfavourable conditions (Wright, 1945; Tchou-Su & Wang Yu-lan, 1958). As shown earlier (Dettlaff, Nikitina & Stroeva, 1964), hypophyseal gonadotropic hormones affect amphibian oocytes through the oocyte nucleus, the germinal vesicle. As a result of their action the nuclear sap of the germinal vesicle acquires the ability to induce cytoplasmic maturation (the property revealed by Delage on the oocytes of Asterias glacialis, 1899, 1901, and by Wilson on those of Cerebratulus lacteus, 1903). It is also well known that the presence of gonadotropin is required for oocyte maturation and ovulation only in the first part of maturation period (hormone-dependent maturation period). Shortly before disruption of the germinal vesicle membrane, the maturation processes acquire inertia and do not need further action of hypophyseal hormones (hormone-independent maturation period). It is of importance here that the main changes during maturation (dissolution of the germinal vesicle membrane, changes in the cytoplasmic properties) occur during the hormone-independent period.
An autoradiographic study of RNA synthesis during primary embryonic induction
Authors: Brahma S. K.
Published: April 2021

Although much work has been done on the inducing factors involved in primary embryonic induction, we are far from understanding the mechanism of the phenomenon. Information concerning the changes in the synthetic patterns in the reacting tissue, which may be related to the induction, must be accumulated before we will be able to formulate a hypothesis on the mechanism of embryonic induction at a subcellular level. The work reported in this paper represents an effort to obtain preliminary information concerning the possible changes in RNA synthesis correlated with primary embryonic induction using the autoradiographic technique.