bluuebunny/crossref_metadata_2025 huggingface dataset:
MAP kinase in situ activation atlas during Drosophila embryogenesis
Authors: Gabay Limor , Seger Rony , Shilo Ben-Zion
Published: April 2021

Receptor tyrosine kinases (RTKs) and the signaling cascades that they trigger play central roles in diverse developmental processes. We describe the capacity to follow the active state of these signaling pathways in situ. This is achieved by monitoring, with a specific monoclonal antibody, the distribution of the active, dual phosphorylated form of MAP kinase (ERK). A dynamic pattern is observed during embryonic and larval phases of Drosophila development, which can be attributed, to a large extent, to the known RTKs. This specific detection has enabled us to determine the time of receptor activation, visualize gradients and boundaries of activation, and postulate the distribution of active ligands. Since the antibody was raised against the phosphorylated form of a conserved ERK peptide containing the TEY motif, this approach is applicable to a wide spectrum of multicellular organisms.
The Drosophila sugarless gene modulates Wingless signaling and encodes an enzyme involved in polysaccharide biosynthesis
Authors: Häcker Udo , Lin Xinhua , Perrimon Norbert
Published: April 2021

We have identified and characterized a Drosophila gene, which we have named sugarless, that encodes a homologue of vertebrate UDP-glucose dehydrogenase. This enzyme is essential for the biosynthesis of various proteoglycans, and we find that in the absence of both maternal and zygotic activities of this gene, mutant embryos develop with segment polarity phenotypes reminiscent to loss of either Wingless or Hedgehog signaling. To analyze the function of Sugarless in cell-cell interaction processes, we have focused our analysis on its requirement for Wingless signaling in different tissues. We report that sugarless mutations impair signaling by Wingless, suggesting that proteoglycans contribute to the reception of Wingless. We demonstrate that overexpression of Wingless can bypass the requirement for sugarless, suggesting that proteoglycans modulate signaling by Wingless, possibly by limiting its diffusion and thereby facilitating the binding of Wingless to its receptor. We discuss the possibility that tissue-specific regulation of proteoglycans may be involved in regulating both Wingless short- or long-range effects.
A forkhead gene related to HNF-3β is required for gastrulation and axis formation in the ascidian embryo
Authors: Olsen Catherine L. , Jeffery William R.
Published: April 2021

We have isolated a member of the HNF-3/forkhead gene family in ascidians as a means to determine the role of winged-helix genes in chordate development. The MocuFH1 gene, isolated from a Molgula oculata cDNA library, exhibits a forkhead DNA-binding domain most similar to zebrafish axial and rodent HNF-3β. MocuFH1 is a single copy gene but there is at least one other related forkhead gene in the M. oculata genome. The MocuFH1 gene is expressed in the presumptive endoderm, mesenchyme and notochord cells beginning during the late cleavage stages. During gastrulation, MocuFH1 expression occurs in the prospective endoderm cells, which invaginate at the vegetal pole, and in the presumptive notochord and mesenchyme cells, which involute over the anterior and lateral lips of the blastopore, respectively. However, this gene is not expressed in the presumptive muscle cells, which involute over the posterior lip of the blastopore. MocuFH1 expression continues in the same cell lineages during neurulation and axis formation, however, during the tailbud stage, MocuFH1 is also expressed in ventral cells of the brain and spinal cord. The functional role of the MocuFH1 gene was studied using antisense oligodeoxynucleotides (ODNs), which transiently reduce MocuFH1 transcript levels during gastrulation. Embryos treated with antisense ODNs cleave normally and initiate gastrulation. However, gastrulation is incomplete, some of the endoderm and notochord cells do not enter the embryo and undergo subsequent movements, and axis formation is abnormal. In contrast, the prospective muscle cells, which do not express MocuFH1, undergo involution and later express muscle actin and acetylcholinesterase, markers of muscle cell differentiation. The results suggest that MocuFH1 is required for morphogenetic movements of the endoderm and notochord precursor cells during gastrulation and axis formation. The effects of inhibiting MocuFH1 expression on embryonic axis formation in ascidians are similar to those reported for knockout mutations of HNF-3β in the mouse, suggesting that HNF-3/forkhead genes have an ancient and fundamental role in organizing the body plan in chordates.
Imprinting of Igf2 and H19 from a 130 kb YAC transgene
Authors: Ainscough Justin F-X. , Koide Tsuyoshi , Tada Masako , Barton Sheila , Surani M. Azim
Published: April 2021

A stringent test for imprint control elements is to examine their function at ectopic loci in transgenic experiments. Igf2 and H19 are part of a larger imprinting region and as a first step, we examined these reciprocally imprinted genes in transgenic experiments using a 130 kb YAC clone. After paternal inheritance, H19 was appropriately repressed and Igf2 was expressed, irrespective of copy number or genetic background. After maternal inheritance H19 was consistently expressed, albeit with some variability. The levels of H19 expression per copy of the transgene inversely correlated with Igf2 (-lacZ) expression in cis. The consistent imprinting of H19 from this YAC contrasts with the previously described imprinting of mini-H19 transgenes, which only occurs at multi-copy loci, is inconsistent, and is prone to genetic background effects. We propose a novel model in which silencing of the H19 gene is the default state and its activation after maternal inheritance is the key mechanistic event for imprinting in this region. In addition, in situ analysis of the Igf2-lacZ reporter indicates that additional mesoderm-specific enhancers are present within the YAC clone. No obvious phenotype was detected from the excess gene dosage of H19.
Macrophages induce apoptosis in normal cells in vivo
Authors: Diez-Roux Graciana , Lang Richard A.
Published: April 2021

It is well established that macrophages have a function in scavenging apoptotic bodies from cells undergoing programmed cell death. Here we show that macrophages can also induce apoptosis of normal cells. Using injected toxic liposomes to eliminate macrophages in the anterior chamber of the rat eye, we provide direct evidence that, in vivo, macrophages induce apoptosis in normal vascular endothelial cells during programmed capillary regression. Macrophage elimination resulted in the survival of endothelial cells that normally would die and the persistence of functional capillaries. Furthermore, replacement of eliminated macrophages with bone-marrow-derived macrophages ‘rescued’ lack of capillary regression. Viability of the persistent target cells was demonstrated through their lack of apoptotic morphology, expression of intracellular esterases and synthesis of DNA. These results uncover a new function for macrophages in the remodeling of tissues through the induction of programmed cell death and provide direct evidence of a key role for macrophages in capillary regression.
Ectopic expression of the Drosophila Bam protein eliminates oogenic germline stem cells
Authors: Ohlstein Benjamin , McKearin Dennis
Published: April 2021

The Drosophila germ-cell lineage has emerged as a remark-able system for identifying genes required for changes in cell fate from stem cells into more specialized cells. Previous work indicates that bam expression is necessary for cystoblast differentiation; bam mutant germ cells fail to differentiate, but instead proliferate like stem cells. This paper reports that ectopic expression of bam is sufficient to extinguish stem cell divisions. Heat-induced bam+ expression specifically eliminated oogenic stem cells while somatic stem cell populations were not affected. Together with previous studies of the timing of bam mRNA and protein expression and the state of arrest in bam mutant cells, these data implicate Bam as a direct regulator of the switch from stem cell to cystoblast. Surprisingly, ectopic bam+ had no deleterious consequences for male germline cells suggest-ing that Bam may regulate somewhat different steps of germ-cell development in oogenesis and spermatogenesis. We discuss a model for how bam+ could direct differentiation based on our data (McKearin and Ohlstein, 1995) that Bam protein is essential to assemble part of the germ-cell-specific organelle, the fusome. We propose that fusome biogenesis is an obligate step for cystoblast cell fate and that Bam is the limiting factor for fusome maturation in female germ cells.
cut interacts with Notch and Protein kinase A to regulate egg chamber formation and to maintain germline cyst integrity during Drosophila oogenesis
Authors: Jackson Stephen M. , Blochlinger Karen
Published: April 2021

Communications between the germline and the soma during Drosophila oogenesis have been previously shown to be essential for the formation of egg chambers and to establish polarity in the developing oocyte. In this report, we demonstrate that the function of a somatically expressed gene, cut, is critical for maintaining the structural integrity of germline-derived cells and their arrangement within an egg chamber. Genetic manipulations of cut activity resulted in defective packaging of germline-derived cysts into egg chambers and disintegration of the structural organization of oocyte-nurse cell complexes to generate multinucleate germline-derived cells. We also found that cut interacts genetically with the Notch gene and with the catalytic subunit of Protein kinase A gene during egg chamber morphogenesis. Since cut expression is restricted to the somatic follicle cells and cut mutant germline clones are phenotypically normal, we propose that the defects in the assembly of egg chambers and the changes in germline cell morphology observed in cut mutant egg chambers are the result of altered interactions between follicle cells and germline cells. cut encodes a nuclear protein containing DNA-binding motifs, and we suggest that it participates in inter-cellular communications by regulating the expression of molecules that directly participate in this process.
The Drosophila neurogenic gene big brain, which encodes a membrane-associated protein, acts cell autonomously and can act synergistically with Notch and Delta
Authors: Doherty Dan , Jan Lily Yeh , Jan Yuh Nung
Published: April 2021

In the developing nervous system of Drosophila, cells in each proneural cluster choose between neural and epidermal cell fates. The neurogenic genes mediate the cell-cell communication process whereby one cell adopts the neural cell fate and prevents other cells in the cluster from becoming neural. In the absence of neurogenic gene function, most, if not all of the cells become neural. big brain is a neurogenic gene that encodes a protein with sequence similarity to known channel proteins. It is unique among the neurogenic genes in that previous genetic studies have not revealed any interaction between big brain and the other neurogenic genes. Furthermore, the neural hypertrophy in big brain mutant embryos is less severe than that in embryos mutant for other neurogenic genes.

In this paper, we show by antibody staining that bib is expressed in tissues that give rise to neural precursors and in other tissues that are affected by loss of neurogenic gene function. By immunoelectron microscopy, we found that bib is associated with the plasma membrane and concentrated in apical adherens junctions as well as in small cytoplasmic vesicles. Using mosaic analysis in the adult, we demonstrate that big brain activity is required autonomously in epidermal precursors to prevent neural development. Finally, we demonstrate that ectopically expressed big brain acts synergistically with ectopically expressed Delta and Notch, providing the first evidence that big brain may function by augmenting the activity of the Delta-Notch pathway. These results are consistent with bib acting as a channel protein in proneural cluster cells that adopt the epidermal cell fate, and serving a necessary function in the response of these cells to the lateral inhibition signal.
Cell determination strategies in the Drosophila eye
Authors: Freeman Matthew
Published: April 2021

Cells in the Drosophila eye are determined by inductive signalling. Here I describe a new model of eye development that explains how simple intercellular signals could specify the diverse cell types that constitute the ommatidium. This model arises from the recent observation that the Drosophila homologue of the EGF receptor (DER) is used reiteratively to trigger the differentiation of each of the cell types – successive rounds of DER activation recruit first the photoreceptors, then cone and finally pigment cells. It seems that a cell’s identity is not determined by the specific signal that induces it, but is instead a function of the state of the cell when it receives the signal. DER signalling is activated by the ligand, Spitz, and inhibited by the secreted protein, Argos. Spitz is initially produced by the central cells in the ommatidium and diffuses over a small distance. Argos has a longer range, allowing it to block more distal cells from being activated by low levels of Spitz; I have termed this interplay between a short-range activator and a long-range inhibitor ‘remote inhibition’. Since inductive signalling is common in many organisms and its components have been conserved, it is possible that the logic of signalling may also be conserved.
Regulation of a dpp target gene in the Drosophila embryo
Authors: Rusch Jannette , Levine Michael
Published: April 2021

In Drosophila, two TGF-β growth factors, dpp and screw, function synergistically to subdivide the dorsal ectoderm into two embryonic tissues, the amnioserosa and dorsal epidermis. Previous studies have shown that peak dpp activity is required for the localized expression of zerknüllt (zen), which encodes a homeodomain transcription factor. We present evidence that zen directly activates the amnioserosa-specific expression of a downstream target gene, Race (Related to angiotensin converting enzyme). A 533 bp enhancer from the Race promoter region is shown to mediate selective expression in the amnioserosa, as well as the anterior and posterior midgut rudiments. This enhancer contains three zen protein binding sites, and mutations in these sites virtually abolish the expression of an otherwise normal Race-lacZ fusion gene in the amnioserosa, but not in the gut. Genetic epistasis experiments suggest that zen is not the sole activator of Race, although a hyperactivated form of zen (a zen-VP16 fusion protein) can partially complement reduced levels of dpp activity. These results suggest that dpp regulates multiple transcription factors, which function synergistically to specify the amnioserosa.
Genetic evidence that the retinoid signal is transduced by heterodimeric RXR/RAR functional units during mouse development
Authors: Kastner Philippe , Mark Manuel , Ghyselinck Norbert , Krezel Wojciech , Dupé Valérie , Grondona Jesus M. , Chambon Pierre
Published: April 2021

We describe here the analysis of congenital malformations in compound mutant fetuses bearing null alleles in one RXR (α β or γ) and one RAR (α β or γ) isotype gene. A marked synergy was observed between the effects of mutations in RXRα and RARs, as a large number of developmental defects previously found mainly in RAR single and compound mutants were recapitulated in specific RXRα/RAR compound mutants. Several malformations were seen only in one type of RXRα/RAR mutant combination, whereas others were seen in several types of RXRα/RAR double mutants. No synergy was observed between the effects of mutations of either RXRβ or RXRγ mutations and those of any of the RAR mutations. These genetic data suggest that RXR/RAR heterodimers are the functional units transducing the retinoid signal for a large number of RA-dependent processes, and furthermore, that RXRα is the main RXR implicated in the developmental functions of RARs. The significance of these observations is discussed with respect to the problem of functional specificity and redundancy among retinoid receptors in vivo.
Ectopic application of recombinant BMP-2 and BMP-4 can change patterning of developing chick facial primordia
Authors: Barlow Amanda J. , Francis-West Philippa H.
Published: April 2021

The facial primordia initially consist of buds of undifferentiated mesenchyme, which give rise to a variety of tissues including cartilage, muscle and nerve. These must be arranged in a precise spatial order for correct function. The signals that control facial outgrowth and patterning are largely unknown. The bone morphogenetic proteins Bmp-2 and Bmp-4 are expressed in discrete regions at the distal tips of the early facial primordia suggesting possible roles for BMP-2 and BMP-4 during chick facial development. We show that expression of Bmp-4 and Bmp-2 is correlated with the expression of Msx-1 and Msx-2 and that ectopic application of BMP-2 and BMP-4 can activate Msx-1 and Msx-2 gene expression in the developing facial primordia. We correlate this activation of gene expression with changes in skeletal development. For example, activation of Msx-1 gene expression across the distal tip of the mandibular primordium is associated with an extension of Fgf-4 expression in the epithelium and bifurcation of Meckel’s cartilage. In the maxillary primordium, extension of the normal domain of Msx-1 gene expression is correlated with extended epithelial expression of shh and bifurcation of the palatine bone. We also show that application of BMP-2 can increase cell proliferation of the mandibular primordia. Our data suggest that BMP-2 and BMP-4 are part of a signalling cascade that controls outgrowth and patterning of the facial primordia.
In vivo functional analysis of the Hoxa-1 3′ retinoic acid response element (3′ RARE)
Authors: Dupé Valerie , Davenne Marc , Brocard Jacques , Dollé Pascal , Mark Manuel , Dierich Andrée , Chambon Pierre , Rijli Filippo M.
Published: April 2021

Retinoids are essential for normal development and both deficiency and excess of retinoic acid (RA) are teratogenic. Retinoic acid response elements (RAREs) have been identified in Hox gene promoters suggesting that endogenous retinoids may be involved in the direct control of Hox gene patterning functions. In order to test this hypothesis, we have mutated the Hoxa-1 3′ RARE using the Cre-loxP targeting strategy, and studied its functional role during mouse development. We find that this enhancer plays an important role in the early establishment of the Hoxa-1 anterior expression boundary in the neural plate. This early disturbance in Hoxa-1 activation results in rhombomere and cranial nerve abnormalities reminiscent of those obtained in the Hoxa-1 total knockout, although their severity and penetrance are lower, thus providing strong evidence for direct control of Hox gene function by retinoids during normal development. Interestingly, we also find that the Hoxa-1 expression response to RA treatment is not entirely controlled by the RARE, suggesting the existence of other retinoid-induced factors mediating the Hoxa-1 response to RA and/or the presence of additional RAREs. Interestingly, although the RARE is not required for the spatiotemporal control of colinear expression of the Hoxa genes, it is absolutely required for correct Hoxa-2 expression in rhombomere 5.
In vitro lens transdifferentiation of Xenopus laevis outer cornea induced by Fibroblast Growth Factor (FGF)
Authors: Bosco Luigi , Venturini Giorgio , Willems Daniela
Published: April 2021

It has been shown that lens regeneration from outer cornea of larval Xenopus laevisis dependent on neural retina both in vivo and in tissue culture. The isolated outer cornea cultured in the presence of bovine brain-derived acidic Fibroblast Growth Factor (aFGF) is able to reprogram the differentiation into lens fibers, although this transdifferentiative process is not coupled with the formation of a normally organized lens. The capacity of aFGF to promote lens differentiation from cornea is not linked to its mitogenic activity. The cultured corneal cells can transdifferentiate into lens fibers in the presence of aFGF when DNA replication and cell proliferation are prevented by addition of aphidicolin, a specific inhibitor of DNA polymerase in eukaryotes, to the culture medium.
Induction of the primary dorsalizing center in Xenopus by the Wnt/GSK/β-catenin signaling pathway, but not by Vg1, Activin or Noggin
Authors: Fagotto François , Guger Kathleen , Gumbiner Barry M.
Published: April 2021

The molecular nature of the primary dorsalizing inducing event in Xenopus is controversial and several secreted factors have been proposed as potential candidates: Wnts, Vg1, Activin and Noggin. Recent studies, however, have provided new insight into the activity of the dorsalizing region, called the Nieuwkoop Center. (1) The activity of this dorsalizing center involves an entire signal transduction pathway that requires maternal β-catenin (Heasman, J., Crawford, A., Goldstone, K., Garner-Hamrick, P., Gumbiner, B., McCrea, P., Kintner, C., Noro, C. Y. and Wylie, C. (1994)Cell 79, 791-803). (2) A transcription factor with potent dorsalizing activity, Siamois, is expressed within the Nieuwkoop Center (Lemaire, P., Garrett, N. and Gurdon, J. B. (1995)Cell 81, 85-94). We have used these two properties of the Nieuwkoop Center to evaluate the dorsalizing activity of the four secreted factors Wnt8, Vg1, Activin and Noggin. The requirement for β-catenin was tested by coexpressing a cadherin, which sequesters β-catenin at the cell membrane and specifically blocks its intracellular signaling activity (Fagotto, F., Funayama, N., Glück, U. and Gumbiner, B. M. (1996)J. Cell Biol. 132, 1105-1114). Induction of Siamois expression was detected by RT-PCR. Of the four growth factors, only Wnt was sensitive to inhibition of β-catenin activity and only Wnt could induce Siamois expression. Therefore, Wnt is able to induce a bonafide Nieuwkoop Center, while Vg1, Activin and Noggin probably induce dorsal structures by a different mechanism.

To order the steps in the Nieuwkoop Center signaling cascade, we have tested the relationship between β-catenin and GSK, a serine-threonine kinase that has been implicated in axis formation in a step downstream of Wnt. We found that GSK acts upstream of β-catenin, similar to the order of these components in the Wingless pathway in Drosophila. We have also examined the relationship between the Wnt/β-catenin pathway and Siamois. We show that β-catenin induces expression of Siamois and that the free signaling pool of β-catenin is required for normal expression of endogenous Siamois. We conclude that the sequence of steps in the signaling pathway is Wnt ⊣ GSK ⊣ β-catenin→ Siamois.
Direct neural induction and selective inhibition of mesoderm and epidermis inducers by Xnr3
Authors: Hansen Carin S. , Marion Christopher D. , Steele Kirsten , George Sean , Smith William C.
Published: April 2021

During gastrulation in amphibians, secreted factors from Spemann’s organizer act on dorsal ectoderm to induce the central nervous system. A number of secreted factors produced by Spemann’s organizer have recently been identified. The TGFβ family member Xnr3 is similar in amino acid sequence to the mouse factor nodal and is expressed in a restricted group of cells in the superficial layer of Spemann’s organizer. Xnr3, unlike the related factors nodal, Xnr1 and Xnr2, lacks mesoderm-inducing activity. We report here that Xnr3 can directly induce neural tissue in Xenopus ectoderm explants (animal caps). Injection of animal caps with either Xnr3 RNA or plasmids induces the expression of the pan-neural genes NCAM and nrp1, as well as the anterior neural marker Cpl1. A growing body of evidence suggests that neural induction in Xenopus proceeds as the default in the absence of epidermis inducers. The best candidates for the endogenous epidermis inducers are BMP-4 and BMP-7. The neural inducing activity of Xnr3 can be inhibited by overexpression of BMP-4, as has been observed with the neural inducers noggin, chordin and follistatin. Furthermore, Xnr3 can block mesoderm induction by BMP-4 and activin, but not by Xnr2. The structural basis underlying the divergent activities of Xnr2 and Xnr3 was analyzed using site-directed mutagenesis. Mutations introduced to the conserved cysteine residues characteristic of the TGFβ family were found to inactivate Xnr2, but not Xnr3. The most unique feature of Xnr3 is the absence of a conserved cysteine at the C terminus of the protein. This feature distinguishes Xnr3 from other TGFβ family members, including Xnr2. However, we observed that changing the C terminus of Xnr3 to more closely resemble other TGFβ family members did not significantly alter its activity, suggesting that other structural features of Xnr3 distinguish its biological activity from Xnr2.
Coordinate actions of BMPs, Wnts, Shh and Noggin mediate patterning of the dorsal somite
Authors: Marcelle Christophe , Stark Michael R. , Bronner-Fraser Marianne
Published: April 2021

Shortly after their formation, somites of vertebrate embryos differentiate along the dorsoventral axis into sclerotome, myotome and dermomyotome. The dermomyotome is then patterned along its mediolateral axis into medial, central and lateral compartments, which contain progenitors of epaxial muscle, dermis and hypaxial muscle, respectively. Here, we used Wnt-11 as a molecular marker for the medial compartment of dermomyotome (the ‘medial lip’) to demonstrate that BMP in the dorsal neural tube indirectly induces formation of the medial lip by up-regulating Wnt-1 and Wnt-3a (but not Wnt-4) expression in the neural tube. Noggin in the dorsal somite may inhibit the direct action of BMP on this tissue. Wnt-11 induction is antagonized by Sonic Hedgehog, secreted by the notochord and the floor plate. Together, our results show that the coordinated actions of the dorsal neural tube (via BMP and Wnts), the ventral neural tube/notochord (via Shh) and the somite itself (via noggin) mediates patterning of the dorsal compartment of the somite.
Overexpression of FGF-2 modulates fiber cell differentiation and survival in the mouse lens
Authors: Stolen Craig M. , Jackson Mark W. , Griep Anne E.
Published: April 2021

During mammalian embryogenesis, the ocular lens forms through a temporally and spatially regulated pattern of differentiation which is thought to be coordinated at least in part by the FGF-1 and FGF-2 members of the fibroblast growth factor (FGF) family. Previous transgenic experiments in which FGF-1 or dominant negative FGF receptors were overexpressed in the lens indicated that FGF-1 could induce differentiation while differentiated lens cells rely upon FGF signaling for their survival. In this study, we asked if the 17.5 kDa FGF-2 protein was capable of inducing differentiation of lens cells in transgenic mice. Unexpectedly, differentiation was inhibited by lens-specific expression of a transgene encoding a secreted form of the 17.5 kDa bovine FGF-2 protein under the transcriptional control of the murine αA-crystallin promoter (αAIgFGF-2 transgenic mice). To address the possibility that FGF-2 functions as a modulator of fiber cell survival, αAIgFGF-2 transgenic mice were crossed to transgenic mice exhibiting extensive apoptosis in the lens due to the functional inactivation of the retinoblastoma protein (αAE7 transgenic mice). The level of apoptosis in the lenses of double trans-genic mice was substantially reduced as compared to the level in lenses from αAE7 only mice. These studies indicate that FGF-2 can act as a modulator of the later stages of differentiation including fiber cell survival. Additionally, they imply that control of lens development by FGFs is a complex process in which FGF-1 and FGF-2 play distinct roles.
The Danforth’s short tail mutation acts cell autonomously in notochord cells and ventral hindgut endoderm
Authors: Maatman Ronald , Zachgo Jochen , Gossler Achim
Published: April 2021

Danforth’s short tail (Sd) is a semidominant mutation in mouse affecting the axial skeleton and urogenital system. The notochord is the first visibly abnormal structure in mutant embryos, and disintegrates beginning around embryonic day 9.5 along its entire length, suggesting an essential role for Sd in notochord development and main-tenance.

Here, we report on the fate of Sd/+ and Sd/Sd cells in chimeric embryos. Up to day 9-9.5, Sd cells contributed effi-ciently to the notochord of chimeric embryos. In advanced day 9.5 embryos, Sd cells were less abundant in the posterior-most region of the notochord and in the noto-chordal plate. During subsequent development, Sd cells were specifically lost from the notochord and replaced by wild-type cells. In Sd/+↔+/+ chimeras, the notochord appeared histologically and functionally normal, leading to a rescue of the mutant phenotype. However, strong Sd/Sd↔+/+ chimeras showed malformations of the axial skeleton and urogenital system. All Sd/Sd↔+/+ chimeras with malformations of the axial skeleton also had kidney defects, whereas chimeras without vertebral column defects had highly chimeric kidneys that appeared normal, suggesting that the urogenital malformations arise secon-darily to impaired posterior development caused by the degenerating notochord. Sd mutant cells also were specifi-cally absent from the ventral portion of the hindgut, whereas they contributed efficiently to the dorsal region, implying the existence of distinct cell populations in the dorsal and ventral hindgut.

Our findings demonstrate that the Sd mutation acts cell autonomously in cells of the notochord and ventral hind gut. Sd leads to the degeneration of notochord cells and the number or allocation of notochord precursors from the tail bud to the notochordal plate seems impaired, whereas notochord formation from the node appears to be unaffected.
Drosophila tissue polarity requires the cell-autonomous activity of the fuzzy gene, which encodes a novel transmembrane protein
Authors: Collier Simon , Gubb David
Published: April 2021

The tissue polarity gene fuzzy (fy) has two roles in the devel-opment of Drosophila wing hairs. One is to specify the correct orientation of the hair by limiting the site of prehair initiation to the distal vertex of the wing cell. The other is to control wing cell hair number by maintaining the integrity of the cytoskeletal components that direct hair development. The requirement for fy in these processes is temperature dependent, as the amorphic fy phenotype is cold sensitive. Analysis of mosaic wings has shown that the fy gene product functions cell autonomously. We have cloned the fy transcript, which encodes a novel four-pass transmembrane protein that shares significant homology with proteins encoded by vertebrate cDNAs. The fourth putative transmembrane domain does not appear to play a significant role in tissue polarity as it is deleted in a weak fy hypomorph. Expression of the fy transcript is develop-mentally regulated and peaks sharply at the time of wing cell pre-hair initiation.
Glial-cell-line-derived neurotrophic factor is required for bud initiation from ureteric epithelium
Authors: Sainio Kirsi , Suvanto Petro , Davies Jamie , Wartiovaara Jorma , Wartiovaara Kirmo , Saarma Mart , Arumäe Urmas , Meng Xiaojuan , Lindahl Maria , Pachnis Vassilis , Sariola Hannu
Published: April 2021

The shapes of different organs can be explained largely by two fundamental characteristics of their epithelial rudiments – the pattern of branching and the rate of pro-liferation. Glial-cell-line-derived neurotrophic factor (GDNF) has recently been implicated in the development of metanephric ureteric epithelium (Pichel, J. G., Shen, L., Sheng, H. Z., Granholm, A.-C., Drago, J., Grinberg, A., Lee, E. J., Huang, S. P., Saarma, M., Hoffer, B.J., Sariola, H. and Westphal, H. (1996). Nature 382, 73-76; Sánchez, M.P., Silos-Santiago, I., Frisén, J., He, B., Lira, S.A. and Barbacid, M. (1996). Nature 382, 70-73; Vega, Q.C., Worby, C.A., Lechner, M.S., Dixon, J.E. and Dressler, G.R. (1996). Proc. Nat. Acad. Sci. USA 93, 10657-10661). We have analysed the target cells of GDNF and the manner in which it controls ureteric development, and have compared it with other growth factors that have been associated with the regulation of branching morphogenesis, namely hepatocyte growth factor (HGF) and transforming growth factor-β1 (TGFβ1). We show that GDNF binds directly to the tips of ureteric bud branches, and that it has the ability to promote primary ureteric buds from various segments of Wolffian duct and to attract ureteric branches towards the source of GDNF. It increases cell adhesion, but is not obviously mitogenic for ureteric cells. The data indicate that GDNF is required primarily for bud initiation. Com-parison of GDNF, HGF and TGFβ1 suggests that the latter act later than GDNF, and may represent a partially redundant set of mesenchyme-derived growth factors that control ureteric development. Thus, GDNF is the first defined inducer in the embryonic metanephric kidney.
dally, a Drosophila glypican, controls cellular responses to the TGF-β-related morphogen, Dpp
Authors: Jackson Shari M. , Nakato Hiroshi , Sugiura Motoko , Jannuzi Alison , Oakes Robert , Kaluza Vesna , Golden Catherine , Selleck Scott B.
Published: April 2021

Decapentaplegic (Dpp) is a Drosophila member of the Transforming Growth Factor-β (TGF-β)/Bone Morpho-genetic Protein (BMP) superfamily of growth factors. Dpp serves as a classical morphogen, where concentration gradients of this secreted factor control patterning over many cell dimensions. Regulating the level of Dpp signaling is therefore critical to its function during development. One type of molecule proposed to modulate growth factor signaling at the cell surface are integral membrane proteo-glycans. We show here that division abnormally delayed (dally), a Drosophila member of the glypican family of integral membrane proteoglycans is required for normal Dpp signaling during development, affecting cellular responses to this morphogen. Ectopic expression of dally+ can alter the patterning activity of Dpp, suggesting a role for dally+ in modulating Dpp signaling strength. These findings support a role for members of the glypican family in controlling TGF-β/BMP activity in vivo by affecting signaling at the cell surface.
Animal and vegetal pole cells of early Xenopus embryos respond differently to maternal dorsal determinants: implications for the patterning of the organiser
Authors: Darras Sébastien , Marikawa Yusuke , Elinson Richard P. , Lemaire Patrick
Published: April 2021

The maternal dorsal determinants required for the specification of the dorsal territories of Xenopus early gastrulae are located at the vegetal pole of unfertilised eggs and are moved towards the prospective dorsal region of the fertilised egg during cortical rotation. While the molecular identity of the determinants is unknown, there are dorsal factors in the vegetal cortical cytoplasm (VCC). Here, we show that the VCC factors, when injected into animal cells activate the zygotic genes Siamoisand Xnr3, suggesting that they act along the Wnt/β-catenin pathway.

In addition, Siamoisand Xnr3 are activated at the vegetal pole of UV-irradiated embryos, indicating that these two genes are targets of the VCC factors in all embryonic cells. However, the consequences of their activation in cells that occupy different positions along the animal-vegetal axis differ. Dorsal vegetal cells of normal embryos or VCC-treated injected animal cells are able to dorsalise ventral mesoderm in conjugate experiments but UV-treated vegetal caps do not have this property. This difference is unlikely to reflect different levels of activation of FGF or activilike signal transduction pathways but may reflect the activation of different targets of Siamois. Chordin, a marker of the head and axial mesoderm, is activated by the VCC/Siamois pathway in animal cells but not in vegetal cells whereas cerberus, a marker of the anterior mesendoderm which lacks dorsalising activity, can only be activated by the VCC/Siamois pathway in vegetal cells. We propose that the regionalisation of the organiser during gastrulation proceeds from the differential interpretation along the animal-vegetal axis of the activation of the VCCβ-catenin/Siamois pathway.
Neural tube-ectoderm interactions are required for trigeminal placode formation
Authors: Stark Michael R. , Sechrist John , Bronner-Fraser Marianne , Marcelle Christophe
Published: April 2021

Cranial sensory ganglia in vertebrates develop from the ectodermal placodes, the neural crest, or both. Although much is known about the neural crest contribution to cranial ganglia, relatively little is known about how placode cells form, invaginate and migrate to their targets. Here, we identify Pax-3 as a molecular marker for placode cells that contribute to the ophthalmic branch of the trigeminal ganglion and use it, in conjunction with DiI labeling of the surface ectoderm, to analyze some of the mechanisms underlying placode development. Pax-3 expression in the ophthalmic placode is observed as early as the 4-somite stage in a narrow band of ectoderm contiguous to the midbrain neural folds. Its expression broadens to a patch of ectoderm adjacent to the midbrain and the rostral hindbrain at the 8-to 10-somite stage. Invagination of the first Pax-3-positive cells begins at the 13-somite stage. Placodal invagination continues through the 35-somite stage, by which time condensation of the trigeminal ganglion has begun. To challenge the normal tissue interactions leading to placode formation, we ablated the cranial neural crest cells or implanted barriers between the neural tube and the ectoderm. Our results demonstrate that, although the presence of neural crest cells is not mandatory for Pax-3 expression in the forming placode, a diffusible signal from the neuroectoderm is required for induction and/or maintenance of the ophthalmic placode.
Mesodermal defects and cranial neural crest apoptosis in α5 integrin-null embryos
Authors: Goh Keow Lin , Yang Joy T. , Hynes Richard O.
Published: April 2021

α5β1 integrin is a cell surface receptor that mediates cell-extracellular matrix adhesions by interacting with fibronectin. α5 subunit-deficient mice die early in gestation and display mesodermal defects; most notably, embryos have a truncated posterior and fail to produce posterior somites. In this study, we report on the in vivo effects of the α5-null mutation on cell proliferation and survival, and on mesodermal development. We found no significant differences in the numbers of apoptotic cells or in cell proliferation in the mesoderm of α5-null embryos compared to wild-type controls. These results suggest that changes in overall cell death or cell proliferation rates are unlikely to be responsible for the mesodermal deficits seen in theα5-null embryos. No increases in cell death were seen inα5-null embryonic yolk sac, amnion and allantois compared with wild-type, indicating that the mutant phenotype is not due to changes in apoptosis rates in these extraembryonic tissues. Increased numbers of dying cells were, however, seen in migrating cranial neural crest cells of the hyoid arch and in endodermal cells surrounding the omphalomesenteric artery in α5-null embryos, indicating that these sub-populations of cells are dependent on α5 integrin function for their survival. Mesodermal markers mox-1, Notch-1, Brachyury (T) and Sonic hedgehog (Shh)were expressed in the mutant embryos in a regionally appropriate fashion. Both T and Shh, however, showed discontinuous expression in the notochords of α5-null embryos due to (1) degeneration of the notochordal tissue structure, and (2) non-maintenance of gene expression. Consistent with the disorganization of notochordal signals in the α5-null embryos, reduced Pax-1 expression and misexpression of Pax-3 were observed. Anteriorly expressed HoxB genes were expressed normally in the α5-null embryos. However, expression of the posteriormost HoxB gene, Hoxb-9, was reduced in α5-null embryos. These results suggest that α5β1-fibronectin interactions are not essential for the initial commitment of mesodermal cells, but are crucial for maintenance of meso-dermal derivatives during postgastrulation stages and also for the survival of some neural crest cells.
Forced expression of the homeodomain protein Gax inhibits cardiomyocyte proliferation and perturbs heart morphogenesis
Authors: Fisher Steven A. , Siwik Ernest , Branellec Didier , Walsh Kenneth , Watanabe Michiko
Published: April 2021

The development of the tubular heart into a complex four-chambered organ requires precise temporal and region-specific regulation of cell proliferation, migration, death and differentiation. While the regulatory mechanisms in heart morphogenesis are not well understood, increasing attention has focused on the homeodomain proteins, which are generally linked to morphogenetic processes. The homeodomain containing gene Gax has been shown to be expressed in heart and smooth muscle tissues. In this study, the Gax protein was detected in the nuclei of myocardial cells relatively late in chicken heart development, at a time when myocyte proliferation is declining. To test the hypothesis that the Gax protein functions as a negative regulator of cardiomyocyte proliferation, a replication-defective adenovirus was used to force its precocious nuclear expression during chicken heart morphogenesis. In experiments in which Gax- and β-galactosidase-expressing adenoviruses were co-injected, clonal expansion of myocytes was reduced, consistent with inhibition of myocyte proliferation. This effect on proliferation was corroborated by the finding that the percentage of exogenous Gax-expressing myocytes that were positive for the cell cycle marker PCNA decreased over time and was lower than in control myocytes. The precocious nuclear expression of Gax in tubular hearts resulted in abnormal heart morphology, including small ventricles with rounded apices, a thinned compact zone and coarse trabeculae. These results suggest a role for the Gax protein in heart morphogenesis causing proliferating cardiomyocytes to withdraw from the cell cycle, thus influencing the size and shape that the heart ultimately attains.
The metalloprotease-disintegrin Kuzbanian participates in Notch activation during growth and patterning of Drosophila imaginal discs
Authors: Sotillos Sol , Roch Fernando , Campuzano Sonsoles
Published: April 2021

The Notch transmembrane protein is the receptor of an evolutionary conserved pathway that mediates intercellular signalling leading to the specification of different cell types during development. In this pathway, many aspects of the signal transduction mechanism remain poorly understood, especially the role of proteolytic processing of Notch. We present genetic evidence indicating that the met-alloprotease-disintegrin kuzbanian (J. Rooke, D. Pan, T. Xu and G. M. Rubin (1996) Science 273, 1227–1231) is a new component of the Notch signalling pathway and is involved in Notch activation. kuzbanian genetic mosaics demonstrate that, during neurogenesis, wing margin formation and vein width specification kuzbanian is autonomously required in the cell where Notch is activated. Genetic interactions between kuzbanian and different genes of the Notch pathway indicate that kuzbanian is required upstream of Suppressor of Hairless. Moreover, the requirement of kuzbanian for signalling by a ligand-dependent Abruptex receptor, but not by a constitutively activated form of Notch, suggests that kuzbanian is involved in the generation of a Notch functional receptor and/or in its activation. However, differences in the phenotypes of loss-of-function Notch and kuzbanian mutations suggest the existence of alternative Kuzbanian-independent mechanisms that generate Notch functional receptors.
Gene dosage-dependent effects of the Hoxa-13 and Hoxd-13 mutations on morphogenesis of the terminal parts of the digestive and urogenital tracts
Authors: Warot Xavier , Fromental-Ramain Catherine , Fraulob Valérie , Chambon Pierre , Dollé Pascal
Published: April 2021

Gene targeting experiments have shown that the murine Hoxa-13 and Hoxd-13 paralogous genes control skeletal patterning in the distal region of the developing limbs. However, both genes are also expressed in the terminal part of the digestive and urogenital tracts during embryogenesis and postnatal development. Here, we report the abnormalities occuring in these systems in Hoxa-13−/− and Hoxa-13/Hoxd-13 compound mutant mice. Hoxa-13−/− mutant fetuses show agenesis of the caudal portion of the Müllerian ducts, lack of development of the presumptive urinary bladder and premature stenosis of the umbilical arteries, which could account for the lethality of this mutation at mid-gestational stages. Due to such lethality, only Hoxa-13+/−/Hoxd-13−/− compound mutants can reach adulthood. These compound mutants display: (i) agenesis or hypoplasia of some of the male accessory sex glands, (ii) malpositioning of the vaginal, urethral and anal openings, and improper separation of the vagina from the urogenital sinus, (iii) hydronephrosis and (iv) anomalies of the muscular and epithelial layers of the rectum. Thus, Hoxa-13 and Hoxd-13 play important roles in the morphogenesis of the terminal part of the gut and urogenital tract. While Hoxa-13−/−/Hoxd-13+/− fetuses show severely impaired development of the urogenital sinus, double null (Hoxa-13−/−/Hoxd-13−/−) fetuses display no separation of the terminal (cloacal) hindgut cavity into a urogenital sinus and presumptive rectum, and no development of the genital bud, thereby demonstrating that both genes act, in a partly redundant manner, during early morphogenesis of posterior trunk structures.
The Bearded box, a novel 3′ UTR sequence motif, mediates negative post-transcriptional regulation of Bearded and Enhancer of split Complex gene expression
Authors: Lai Eric C. , Posakony James W.
Published: April 2021

During the development of the Drosophila adult peripheral nervous system (PNS), inhibitory cell-cell interactions mediated by the Notch receptor are essential for proper specification of sensory organ cell fates. We have reported previously (M. W. Leviten, E. C. Lai and J. W. Posakony (1997) Development 124, 4039-4051) that the 3′ untranslated regions (UTRs) of many genes involved in Notch signalling, including Bearded (Brd) and the genes of the Enhancer of split Complex (E(spl)-C), contain (often in multiple copies) two novel heptanucleotide sequence motifs, the Brd box (AGCTTTA) and the GY box (GTCTTCC). Moreover, the molecular lesion associated with a strong gain-of-function mutant of Brd suggested that the loss of these sequence elements from its 3′ UTR might be responsible for the hyperactivity of the mutant gene. We show here that the wild-type Brd 3′ UTR confers negative regulatory activity on heterologous transcripts in vivo and that this activity requires its three Brd box elements and, to a lesser extent, its GY box. We find that Brd box-mediated regulation decreases both transcript and protein levels, and our results suggest that deadenylation or inhibition of polyadenylation is a component of this regulation. Though Brd and the E(spl)-C genes are expressed in spatially restricted patterns in both embryos and imaginal discs, we find that the regulatory activity that functions through the Brd box is both temporally and spatially general. A Brd genomic DNA transgene with specific mutations in its Brd and GY boxes exhibits hypermorphic activity that results in characteristic defects in PNS development, demonstrating that Brd is normally regulated by these motifs. Finally, we show that Brd boxes and GY boxes in the E(spl)m4 gene are specifically conserved between two distantly related Drosophila species, strongly suggesting that E(spl)-C genes are regulated by these elements as well.