bluuebunny/crossref_metadata_2025 huggingface dataset:
Proximal-distal leg development in Drosophila requires the apterous gene and the Lim1 homologue dlim1
Authors: Pueyo José I. , Galindo Máximo I. , Bishop Sarah A. , Couso Juan P.
Published: April 2021

Proximal-distal leg development in Drosophila involves a battery of genes expressed and required in specific proximal-distal (PD) domains of the appendage. Here we report the characterisation of a new gene of this type, dlim1, a member of the Lhx family of genes whose proteins contain two Lim domains and a homeodomain. We show that the Lhx gene apterous (ap) is also required for PD leg development, and we study the functional interactions between ap, dlim1 and other PD genes during leg development. Our results show that a regulatory network formed by ap and dlim1 plus the homeobox genes aristaless and Bar specifies distal leg cell fates in Drosophila.
Opposing effects of bone morphogenetic proteins on neuron production and survival in the olfactory receptor neuron lineage
Authors: Shou Jianyong , Murray Richard C. , Rim Peter C. , Calof Anne L.
Published: April 2021

In olfactory epithelium (OE) cultures, bone morphogenetic proteins (BMPs) can strongly inhibit neurogenesis. Here we provide evidence that BMPs also promote, and indeed are required, for OE neurogenesis. Addition of the BMP antagonist noggin inhibited neurogenesis in OE-stromal cell co-cultures. Bmp2, Bmp4 and Bmp7 were expressed by OE stroma, and low concentrations of BMP4 (below the threshold for inhibition of neurogenesis) stimulated neurogenesis; BMP7 did not exhibit a stimulatory effect at any concentration tested. Stromal cell conditioned medium also stimulated neurogenesis; part of this effect was due to the presence within it of a noggin-binding factor or factors. Studies of the pro-neurogenic effect of BMP4 indicated that it did not increase progenitor cell proliferation, but rather promoted survival of newly generated olfactory receptor neurons. These findings indicate that BMPs exert both positive and negative effects on neurogenesis, depending on ligand identity, ligand concentration and the particular cell in the lineage that is responding. In addition, they reveal the presence of a factor or factors, produced by OE stroma, that can synergize with BMP4 to stimulate OE neurogenesis.
DRacGAP, a novel Drosophila gene, inhibits EGFR/Ras signalling in the developing imaginal wing disc
Authors: Sotillos Sol , Campuzano Sonsoles
Published: April 2021

We have identified a novel Drosophila gene, DRacGAP, which behaves as a negative regulator of Rho-family GTPases DRac1 and DCdc42. Reduced function of DRacGAP or increased expression of DRac1 in the wing imaginal disc cause similar effects on vein and sensory organ development and cell proliferation. These effects result from enhanced activity of the EGFR/Ras signalling pathway. We find that in the wing disc, DRac1 enhances EGFR/Ras-dependent activation of MAP Kinase in the prospective veins. Interestingly, DRacGAP expression is negatively regulated by the EGFR/Ras pathway in these regions. During vein formation, local DRacGAP repression would ensure maximal activity of Rac and, in turn, of Ras pathways in vein territories. Additionally, maximal expression of DRacGAP at the vein/intervein boundaries would help to refine the width of the veins. Hence, control of DRacGAP expression by the EGFR/Ras pathway is a previously undescribed feedback mechanism modulating the intensity and/or duration of its signalling during Drosophila development.
Regulation of the UNC-5 netrin receptor initiates the first reorientation of migrating distal tip cells in Caenorhabditis elegans
Authors: Su Ming-Wan , Merz David C. , Killeen Marie T. , Zhou Youwen , Zheng Hong , Kramer James M. , Hedgecock Edward M. , Culotti Joseph G.
Published: April 2021

Cell migrations play a critical role in animal development and organogenesis. Here, we describe a mechanism by which the migration behaviour of a particular cell type is regulated temporally and coordinated with over-all development of the organism. The hermaphrodite distal tip cells (DTCs) of Caenorhabditis elegans migrate along the body wall in three sequential phases distinguished by the orientation of their movements, which alternate between the anteroposterior and dorsoventral axes. The ventral-to-dorsal second migration phase requires the UNC-6 netrin guidance cue and its receptors UNC-5 and UNC-40, as well as additional, UNC-6-independent guidance systems. We provide evidence that the transcriptional upregulation of unc-5 in the DTCs is coincident with the initiation of the second migration phase, and that premature UNC-5 expression in these cells induces precocious turning in an UNC-6-dependent manner. The DAF-12 steroid hormone receptor, which regulates developmental stage transitions in C. elegans, is required for initiating the first DTC turn and for coincident unc-5 upregulation. We also present evidence for the existence of a mechanism that opposes or inhibits UNC-5 function during the longitudinal first migration phase and for a mechanism that facilitates UNC-5 function during turning. The facilitating mechanism presumably does not involve transcriptional regulation of unc-5 but may represent an inhibition of the phase 1 mechanism that opposes or inhibits UNC-5. These results, therefore, reveal the existence of two mechanisms that regulate the UNC-5 receptor that are critical for responsiveness to the UNC-6 netrin guidance cue and for linking the directional guidance of migrating distal tip cells to developmental stage advancements.
Segmentation of the central nervous system in leech
Authors: Shain Daniel H. , Stuart Duncan K. , Huang Françoise Z. , Weisblat David A.
Published: April 2021

Central nervous system (CNS) in leech comprises segmentally iterated progeny derived from five embryonic lineages (M, N, O, P and Q). Segmentation of the leech CNS is characterized by the formation of a series of transverse fissures that subdivide initially continuous columns of segmental founder cells in the N lineage into distinct ganglionic primordia. We have examined the relationship between the N lineage cells that separate to form the fissures and lateral ectodermal and mesodermal derivatives by differentially labeling cells with intracellular lineage tracers and antibodies. Although subsets of both lateral ectoderm and muscle fibers contact N lineage cells at or near the time of fissure formation, ablation experiments suggest that these contacts are not required for initiating fissure formation. It appears, therefore, that this aspect of segmentation occurs autonomously within the N lineage. To support this idea, we present evidence that fundamental differences exist between alternating ganglionic precursor cells (nf and ns primary blast cells) within the N lineage. Specifically, ablation of an nf primary blast cell sometimes resulted in the fusion of ipsilateral hemi-ganglia, while ablation of an ns primary blast cell often caused a ‘slippage’ of blast cells posterior to the lesion. Also, differences in cell behavior were observed in biochemically arrested nf and ns primary blast cells. Collectively, these results lead to a model of segmentation in the leech CNS that is based upon differences in cell adhesion and/or cell motility between the alternating nf and ns primary blast cells. We note that the segmentation processes described here occur well prior to the expression of the leech engrailed-class gene in the N lineage.
Dorsal downregulation of GSK3β by a non-Wnt-like mechanism is an early molecular consequence of cortical rotation in early Xenopus embryos
Authors: Dominguez Isabel , Green Jeremy B. A.
Published: April 2021

Cortical rotation and concomitant dorsal translocation of cytoplasmic determinants are the earliest events known to be necessary for dorsoventral patterning in Xenopus embryos. The earliest known molecular target is β-catenin, which is essential for dorsal development and becomes dorsally enriched shortly after cortical rotation. In mammalian cells cytoplasmic accumulation of β-catenin follows reduction of the specific activity of glycogen synthase kinase 3-beta (GSK3β). In Xenopus embryos, exogenous GSK3β suppresses dorsal development as predicted and GSK3β dominant negative (kinase dead) mutants cause ectopic axis formation. However, endogenous GSK3β regulation is poorly characterized. Here we demonstrate two modes of GSK3β regulation in Xenopus. Endogenous mechanisms cause depletion of GSK3β protein on the dorsal side of the embryo. The timing, location and magnitude of the depletion correspond to those of endogenous β-catenin accumulation. UV and D2O treatments that abolish and enhance dorsal character of the embryo, respectively, correspondingly abolish and enhance GSK3β depletion? A candidate regulator of GSK3β, GSK3-binding protein (GBP), known to be essential for axis formation, also induces depletion of GSK3β. Depletion of GSK3β is a previously undescribed mode of regulation of this signal transducer. The other mode of regulation is observed in response to Wnt and dishevelled expression. Neither Wnt nor dishevelled causes depletion but instead they reduce GSK3β-specific activity? Thus, Wnt/Dsh and GBP appear to effect two biochemically distinct modes of GSK3β regulation.
OVO transcription factors function antagonistically in the Drosophila female germline
Authors: Andrews Justen , Garcia-Estefania David , Delon Isabelle , Lü Jining , Mével-Ninio Maryvonne , Spierer Anne , Payre François , Pauli Daniel , Oliver Brian
Published: April 2021

OVO controls germline and epidermis differentiation in flies and mice. In the Drosophila germline, alternative OVO-B and OVO-A isoforms have a common DNA-binding domain, but different N-termini. We show that these isoforms are transcription factors with opposite regulatory activities. Using yeast one-hybrid assays, we identified a strong activation domain within a common region and a counteracting repression domain within the OVO-A-specific region. In flies, OVO-B positively regulated the ovarian tumor promoter, while OVO-A was a negative regulator of the ovarian tumor and ovo promoters. OVO-B isoforms supplied ovo+ function in the female germline and epidermis, while OVO-A isoforms had dominant-negative activity in both tissues. Moreover, elevated expression of OVO-A resulted in maternal-effect lethality while the absence of OVO-A resulted in maternal-effect sterility. Our data indicate that tight regulation of antagonistic OVO-B and OVO-A isoforms is critical for germline formation and differentiation.
A novel hydra matrix metalloproteinase (HMMP) functions in extracellular matrix degradation, morphogenesis and the maintenance of differentiated cells in the foot process
Authors: Leontovich Alexey A. , Zhang Jinsong , Shimokawa Ken-ichi , Nagase Hideaki , Sarras Michael P.
Published: April 2021

As a member of Cnidaria, the body wall of hydra is structurally reduced to an epithelial bilayer with an intervening extracellular matrix (ECM). Biochemical and cloning studies have shown that the molecular composition of hydra ECM is similar to that seen in vertebrates and functional studies have demonstrated that cell-ECM interactions are important to developmental processes in hydra. Because vertebrate matrix metalloproteinases (MMPs) have been shown to have an important role in cell-ECM interactions, the current study was designed to determine whether hydra has homologues of these proteinases and, if so, what function these enzymes have in morphogenesis and cell differentiation in this simple metazoan. Utilizing a PCR approach, a single hydra matrix metalloproteinase, named HMMP was identified and cloned. The structure of HMMP was similar to that of vertebrate MMPs with an overall identity of about 35%. Detailed structural analysis indicated some unique features in (1) the cysteine-switch region of the prodomain, (2) the hinge region preceding the hemopexin domain, and (3) the hemopexin domain. Using a bacterial system, HMMP protein was expressed and folded to obtain an active enzyme. Substrate analysis studies indicated that recombinant HMMP could digest a number of hydra ECM components such as hydra laminin. Using a fluorogenic MMP substrate assay, it was determined that HMMP was inhibited by peptidyl hydroxamate MMP inhibitors, GM6001 and matlistatin, and by human recombinant TIMP-1. Whole-mount in situ studies indicated that HMMP mRNA was expressed in the endoderm along the entire longitudinal axis of hydra, but at relatively high levels at regions where cell-transdifferentiation occurred (apical and basal poles). Functional studies using GM6001 and TIMP-1 indicated that these MMP inhibitors could reversibly block foot regeneration. Blockage of foot regeneration was also observed using antisense thio-oligo nucleotides to HMMP introduced into the endoderm of the basal pole using a localized electroporation technique. Studies with adult intact hydra found that GM6001 could also cause the reversible de-differentiation or inhibition of transdifferentiation of basal disk cells of the foot process. Basal disk cells are adjacent to those endoderm cells of the foot process that express high levels of HMMP mRNA. In summary, these studies indicate that hydra has at least one MMP that is functionally tied to morphogenesis and cell transdifferentiation in this simple metazoan.
Ca2+ oscillations induced by a cytosolic sperm protein factor are mediated by a maternal machinery that functions only once in mammalian eggs
Authors: Tang Tie-Shan , Dong Jian-Bo , Huang Xiu-Ying , Sun Fang-Zhen
Published: April 2021

At fertilization in mammals, the sperm activates the egg by inducing a series of oscillations in the intracellular free Ca2+ concentration. There is evidence showing that this oscillatory event is triggered by a sperm-derived protein factor which diffuses into egg cytoplasm after gamete membrane fusion. At present the identity of this factor and its precise mechanism of action is unknown. Here, we studied the specificity of action of the sperm factor in triggering Ca2+ oscillations in mammalian eggs. In doing so, we examined the patterns of Ca2+ signaling in mouse eggs, zygotes, parthenogenetic eggs and maturing oocytes following the stimulation of bovine sperm extracts which contain the sperm factor. It is observed that the sperm factor could induce Ca2+ oscillations in metaphase eggs, maturing oocytes and parthenogenetically activated eggs but not in the zygotes. We present evidence that Ca2+ oscillations induced by the sperm factor require a maternal machinery. This machinery functions only once in mammalian oocytes and eggs, and is inactivated by sperm- derived components but not by parthenogenetic activation. In addition, it is found that neither InsP3 receptor sensitivity to InsP3 nor Ca2+ pool size are the determinants that cause the fertilized egg to lose its ability to generate sperm-factor-induced Ca2+ oscillations at metaphase. In conclusion, our study suggests that the orderly sequence of Ca2+ oscillations in mammalian eggs at fertilization is critically dependent upon the presence of a functional maternal machinery that determines whether the sperm- factor-induced Ca2+ oscillations can persist.
GAP-43 mediates retinal axon interaction with lateral diencephalon cells during optic tract formation
Authors: Zhang Fan , Lu Cynthia , Severin Christophe , Sretavan David W.
Published: April 2021

GAP-43 is an abundant intracellular growth cone protein that can serve as a PKC substrate and regulate calmodulin availability. In mice with targeted disruption of the GAP-43 gene, retinal ganglion cell (RGC) axons fail to progress normally from the optic chiasm into the optic tracts. The underlying cause is unknown but, in principle, can result from either the disruption of guidance mechanisms that mediate axon exit from the midline chiasm region or defects in growth cone signaling required for entry into the lateral diencephalic wall to form the optic tracts. Results here show that, compared to wild-type RGC axons, GAP-43-deficient axons exhibit reduced growth in the presence of lateral diencephalon cell membranes. Reduced growth is not observed when GAP-43-deficient axons are cultured with optic chiasm, cortical, or dorsal midbrain cells. Lateral diencephalon cell conditioned medium inhibits growth of both wild-type and GAP-43-deficient axons to a similar extent and does not affect GAP-43-deficient axons more so. Removal or transplant replacement of the lateral diencephalon optic tract entry zone in GAP-43-deficient embryo preparations results in robust RGC axon exit from the chiasm. Together these data show that RGC axon exit from the midline region does not require GAP-43 function. Instead, GAP-43 appears to mediate RGC axon interaction with guidance cues in the lateral diencephalic wall, suggesting possible involvement of PKC and calmodulin signaling during optic tract formation.
BMPs are necessary for stomach gland formation in the chicken embryo: a study using virally induced BMP-2 and Noggin expression
Authors: Narita Tomohiro , Saitoh Kanako , Kameda Takashi , Kuroiwa Atsushi , Mizutani Makoto , Koike Chika , Iba Hideo , Yasugi Sadao
Published: April 2021

Epithelial-mesenchymal interactions are necessary for the normal development of various digestive organs. In chicken proventriculus (glandular stomach), morphogenesis and differentiation of the epithelium depend upon the inductive signals coming from underlying mesenchyme. However, the nature of such signals is still unclear despite extensive analyses carried out using experimental tissue recombinations. In this study we have examined the possible involvement of bone morphogenetic proteins (BMPs) in the formation of stomach glands in the chicken embryo. Analysis of the expression patterns of BMP-2, -4 and -7 showed that these BMPs were present in the proventricular mesenchyme prior to the initiation of the proventricular gland formation. BMP-2 expression, in particular, was restricted to the proventriculus among anterior digestive organs. Virus-mediated BMP-2 overexpression resulted in an increase in the number of glands formed. Moreover, ectopic expression of Noggin, which antagonizes the effect of BMPs, in the proventricular mesenchyme or epithelium, led to the complete inhibition of gland formation, indicating that BMP signals are necessary for the proventricular gland formation. These findings suggest that BMPs are of prime importance as mesenchymal signals for inducing proventricular glands.
Fragile skeletal muscle attachments in dystrophic mutants of Caenorhabditis elegans: isolation and characterization of the mua genes
Authors: Plenefisch John D. , Zhu Xiaoping , Hedgecock Edward M.
Published: April 2021

Over 30 Caenorhabditis elegans mutants were identified with normal muscle differentiation and initial locomotion followed by catastrophic detachment of skeletal muscles from the body wall. Reducing the strength of muscle contraction in these mutants with a myosin gene mutation suppresses muscle detachment. These dystrophic mutants identify a novel class of genes required for growth and maintenance of functional muscle attachments, not exceptional alleles of genes required for muscle differentiation and contractility. Nine new genes, named mua, and two previously published loci, unc-23 and vab-10, cause fragile muscle attachments. The primary sites of muscle detachment, including the plane of tissue separation, are characteristic for each gene. We suggest these genes identify feedback mechanisms whereby local strain regulates the extent of myofibril contraction and the placement of new muscle attachments in functioning muscles. Finally, we draw some comparisons to vertebrate skin fragility diseases and muscular dystrophies.
A retrograde signal is involved in activity-dependent remodeling at a C. elegans neuromuscular junction
Authors: Zhao Hongjuan , Nonet Michael L.
Published: April 2021

We have characterized how perturbations of normal synaptic activity influence the morphology of cholinergic SAB motor neurons that innervate head muscle in C. elegans. Mutations disrupting components of the presynaptic release apparatus, acetylcholine (ACh) synthesis or ACh loading into synaptic vesicles each induced sprouting of SAB axonal processes. These sprouts usually arose in the middle of the normal innervation zone and terminated with a single presynaptic varicosity. Sprouting SAB neurons with a similar morphology were also observed upon reducing activity in muscle, either by using mutants lacking a functional nicotinic ACh receptor subunit or through muscle-specific expression of a gain-of-function potassium channel. Analysis of temperature-sensitive mutants in the choline acetyltransferase gene revealed that the sprouting response to inactivity was developmentally regulated; reduction of synaptic activity in early larval stages, but not in late larval stages, induced both sprouting and addition of varicosities. Our results indicate that activity levels regulate the structure of certain synaptic connections between nerve and muscle in C. elegans. One component of this regulatory machinery is a retrograde signal from the postsynaptic cell that mediates the formation of synaptic connections.
The Neurofibromatosis-2 homologue, Merlin, and the tumor suppressor expanded function together in Drosophila to regulate cell proliferation and differentiation
Authors: McCartney Brooke M. , Kulikauskas Rima M. , LaJeunesse Dennis R. , Fehon Richard G.
Published: April 2021

Neurofibromatosis-2 is an inherited disorder characterized by the development of benign schwannomas and other Schwann-cell-derived tumors associated with the central nervous system. The Neurofibromatosis-2 tumor suppressor gene encodes Merlin, a member of the Protein 4.1 superfamily most closely related to Ezrin, Radixin and Moesin. This discovery suggested a novel function for Protein 4.1 family members in the regulation of cell proliferation; proteins in this family were previously thought to function primarily to link transmembrane proteins to underlying cortical actin. To understand the basic cellular functions of Merlin, we are investigating a Drosophila Neurofibromatosis-2 homologue, Merlin. Loss of Merlin function in Drosophila results in hyperplasia of the affected tissue without significant disruptions in differentiation. Similar phenotypes have been observed for mutations in another Protein 4.1 superfamily member in Drosophila, expanded. Because of the phenotypic and structural similarities between Merlin and expanded, we asked whether Merlin and Expanded function together to regulate cell proliferation. In this study, we demonstrate that recessive loss of function of either Merlin or expanded can dominantly enhance the phenotypes associated with mutations in the other. Consistent with this genetic interaction, we determined that Merlin and Expanded colocalize in Drosophila tissues and cells, and physically interact through a conserved N-terminal region of Expanded, characteristic of the Protein 4.1 family, and the C-terminal domain of Merlin. Loss of function of both Merlin and expanded in clones revealed that these proteins function to regulate differentiation in addition to proliferation in Drosophila. Further genetic analyses suggest a role for Merlin and Expanded specifically in Decapentaplegic-mediated differentiation events. These results indicate that Merlin and Expanded function together to regulate proliferation and differentiation, and have implications for understanding the functions of other Protein 4.1 superfamily members.
Zebrafish Mesp family genes, mesp-a and mesp-b are segmentally expressed in the presomitic mesoderm, and Mesp-b confers the anterior identity to the developing somites
Authors: Sawada Atsushi , Fritz Andreas , Jiang Yun-Jin , Yamamoto Akihito , Yamasu Kyo , Kuroiwa Atsushi , Saga Yumiko , Takeda Hiroyuki
Published: April 2021

Segmentation of a vertebrate embryo begins with the subdivision of the paraxial mesoderm into somites through a not-well-understood process. Recent studies provided evidence that the Notch-Delta and the FGFR (fibroblast growth factor receptor) signalling pathways are required for segmentation. In addition, the Mesp family of bHLH transcription factors have been implicated in establishing a segmental prepattern in the presomitic mesoderm. In this study, we have characterized zebrafish mesp-a and mesp-b genes that are closely related to Mesp family genes in other vertebrates. During gastrulation, only mesp-a is expressed in the paraxial mesoderm at the blastoderm margin. During the segmentation period, both genes are segmentally expressed in one to three stripes in the anterior parts of somite primordia. In fused somites (fss) embryos, in which all early somite boundary formation is blocked, initial mesp-a expression at the gastrula stage remains intact, but the expression of mesp-a and mesp-b is not detected during the segmentation period. This suggests that these genes are downstream targets of fss at the segmentation stage. Comparison with her1 expression (Müller, M., von Weizsäcker, E. and Campos-Ortega, J. A. (1996) Development 122, 2071-2078) suggests that, like her1, mesp genes are not expressed in primordia of the first several somites. Furthermore, we found that zebrafish her1 expression oscillates in the presomitic mesoderm. The her1 stripe, which first appears in the tailbud region, moves in a caudal to rostral direction, and it finally overlaps the most rostral mesp stripe. Thus, in the trunk region, both her1 and mesp transcripts are detected in every somite primordium posterior to the forming somites.

Ectopic expression of Mesp-b in embryos causes a loss of the posterior identity within the somite primordium, leading to a segmentation defect. These embryos show a reduction in expression of the posterior genes, myoD and notch5, with uniform expression of the anterior genes, FGFR1, papc and notch6. These observations suggest that zebrafish mesp genes are involved in anteroposterior specification within the presumptive somites, by regulating the essential signalling pathways mediated by Notch-Delta and FGFR.
The branchial arches and HGF are growth-promoting and chemoattractant for cranial motor axons
Authors: Caton Adele , Hacker Adam , Naeem Arifa , Livet Jean , Maina Flavio , Bladt Friedhelm , Klein Rüdiger , Birchmeier Carmen , Guthrie Sarah
Published: April 2021

During development, cranial motor neurons extend their axons along distinct pathways into the periphery. For example, branchiomotor axons extend dorsally to leave the hindbrain via large dorsal exit points. They then grow in association with sensory ganglia, to their targets, the muscles of the branchial arches. We have investigated the possibility that pathway tissues might secrete diffusible chemorepellents or chemoattractants that guide cranial motor axons, using co-cultures in collagen gels. We found that explants of dorsal neural tube or hindbrain roof plate chemorepelled cranial motor axons, while explants of cranial sensory ganglia were weakly chemoattractive. Explants of branchial arch mesenchyme were strongly growth-promoting and chemoattractive for cranial motor axons. Enhanced and oriented axon outgrowth was also elicited by beads loaded with Hepatocyte Growth Factor (HGF); antibodies to this protein largely blocked the outgrowth and orientation effects of the branchial arch on motor axons. HGF was expressed in the branchial arches, whilst Met, which encodes an HGF receptor, was expressed by subpopulations of cranial motor neurons. Mice with targetted disruptions of HGF or Met showed defects in the navigation of hypoglossal motor axons into the branchial region. Branchial arch tissue may thus act as a target-derived factor that guides motor axons during development. This influence is likely to be mediated partly by Hepatocyte Growth Factor, although a component of branchial arch-mediated growth promotion and chemoattraction was not blocked by anti-HGF antibodies.
Microtubules and mitotic cycle phase modulate spatiotemporal distributions of F-actin and myosin II in Drosophila syncytial blastoderm embryos.
Authors: Foe Victoria E. , Field Christine M. , Odell Garrett M.
Published: April 2021

We studied cyclic reorganizations of filamentous actin, myosin II and microtubules in syncytial Drosophila blastoderms using drug treatments, time-lapse movies and laser scanning confocal microscopy of fixed stained embryos (including multiprobe three-dimensional reconstructions). Our observations imply interactions between microtubules and the actomyosin cytoskeleton. They provide evidence that filamentous actin and cytoplasmic myosin II are transported along microtubules towards microtubule plus ends, with actin and myosin exhibiting different affinities for the cell’s cortex. Our studies further reveal that cell cycle phase modulates the amounts of both polymerized actin and myosin II associated with the cortex. We analogize pseudocleavage furrow formation in the Drosophila blastoderm with how the mitotic apparatus positions the cleavage furrow for standard cytokinesis, and relate our findings to polar relaxation/global contraction mechanisms for furrow formation.

Movies available on-line: http://www.biologists.com/Development/movies/dev7765.html http://raven.zoology.washington.edu/downTheTubes/
Temporal and spatial gradients of Fgf8 and Fgf17 regulate proliferation and differentiation of midline cerebellar structures
Authors: Liu Jingsong Xu, Zhonghao , Ornitz David M.
Published: April 2021

The midbrain-hindbrain (MHB) junction has the properties of an organizer that patterns the MHB region early in vertebrate development. Fgf8 is thought to mediate this organizer function. In addition to Fgf8, Fgf17 and Fgf18 are also expressed in the MHB junction. Fgf17 is expressed later and broader than either Fgf8 or Fgf18. Disrupting the Fgf17 gene in the mouse decreased precursor cell proliferation in the medial cerebellar (vermis) anlage after E11.5. Loss of an additional copy of Fgf8 enhanced the phenotype and accelerated its onset, demonstrating that both molecules cooperate to regulate the size of the precursor pool of cells that develop into the cerebellar vermis. However, expression patterns of Wnt1, En2, Pax5 and Otx2 were not altered suggesting that specification and patterning of MHB tissue was not perturbed and that these FGFs are not required to pattern the vermis at this stage of development. The consequence of this developmental defect is a progressive, dose-dependent loss of the most anterior lobe of the vermis in mice lacking Fgf17 and in mice lacking Fgf17 and one copy of Fgf8. Significantly, the differentiation of anterior vermis neuroepithelium was shifted rostrally and medially demonstrating that FGF also regulates the polarized progression of differentiation in the vermis anlage. Finally, this developmental defect results in an ataxic gait in some mice.
Calmodulin and Son of sevenless dependent signaling pathways regulate midline crossing of axons in the Drosophila CNS
Authors: Fritz Janice L. , VanBerkum Mark F. A.
Published: April 2021

The establishment of axon trajectories is ultimately determined by the integration of intracellular signaling pathways. Here, a genetic approach in Drosophila has demonstrated that both Calmodulin and Son of sevenless signaling pathways are used to regulate which axons cross the midline. A loss in either signaling pathway leads to abnormal projection of axons across the midline and these increase with roundabout or slit mutations. When both Calmodulin and Son of sevenless are disrupted, the midline crossing of axons mimics that seen in roundabout mutants, although Roundabout remains expressed on crossing axons. Calmodulin and Son of sevenless also regulate axon crossing in a commissureless mutant. These data suggest that Calmodulin and Son of sevenless signaling pathways function to interpret midline repulsive cues which prevent axons crossing the midline.
The autoregulation of retinal ganglion cell number
Authors: González-Hoyuela Maritza , Barbas Julio A. , Rodríguez-Tébar Alfredo
Published: April 2021

The development of the nervous system is dependent on a complex set of signals whose precise co-ordination ensures that the correct number of neurones are generated. This regulation is achieved through a variety of cues that influence both the generation and the maintenance of neurones during development. We show that in the chick embryo, stratified retinal ganglion cells (RGCs) are themselves responsible for providing the signals that control the number of RGCs that are generated, both by inhibiting the generation of new ganglion cells and by killing incoming migratory ganglion cells. Selective toxicological ablation of RGCs in the chick embryo resulted in the achronic generation of ganglion cells, which eventually led to the repopulation of the ganglion cell layer and a large decrease in the physiological cell death affecting postmitotic migratory neurones. Interestingly, the application of exogenous NGF reversed the effects of ganglion cell ablation on ganglion cell death. Because the only source of NGF in the retina is that produced by the stratified ganglion cells, we infer that these differentiated neurones regulate their own cell number by secreting NGF, a neurotrophin that has previously been shown to be responsible for the death of migrating ganglion cells.
DNA damage-induced neural precursor cell apoptosis requires p53 and caspase 9 but neither Bax nor caspase 3
Authors: D’Sa-Eipper Cleta , Leonard Jeffrey R. , Putcha Girish , Zheng Timothy S. , Flavell Richard A. , Rakic Pasko , Kuida Keisuke , Roth Kevin A.
Published: April 2021

Programmed cell death (apoptosis) is critical for normal brain morphogenesis and may be triggered by neurotrophic factor deprivation or irreparable DNA damage. Members of the Bcl2 and caspase families regulate neuronal responsiveness to trophic factor withdrawal; however, their involvement in DNA damage-induced neuronal apoptosis is less clear. To define the molecular pathway regulating DNA damage-induced neural precursor cell apoptosis, we have examined the effects of drug and γ-irradiation-induced DNA damage on telencephalic neural precursor cells derived from wild-type embryos and mice with targeted disruptions of apoptosis- associated genes. We found that DNA damage-induced neural precursor cell apoptosis, both in vitro and in vivo, was critically dependent on p53 and caspase 9, but neither Bax nor caspase 3 expression. Neural precursor cell apoptosis was also unaffected by targeted disruptions of Bclx and Bcl2, and unlike neurotrophic factor-deprivation- induced neuronal apoptosis, was not associated with a detectable loss of cytochrome c from mitochondria. The apoptotic pathway regulating DNA damage-induced neural precursor cell death is different from that required for normal brain morphogenesis, which involves both caspase 9 and caspase 3 but not p53, indicating that additional apoptotic stimuli regulate neural precursor cell numbers during telencephalic development.
Axial skeletal patterning in mice lacking all paralogous group 8 Hox genes
Authors: Akker Eric van den , Fromental-Ramain Catherine , Graaff Wim de , Le Mouellic Hervé , Brûlet Philippe , Chambon Pierre , Deschamps Jacqueline
Published: April 2021

We present a detailed study of the genetic basis of mesodermal axial patterning by paralogous group 8 Hox genes in the mouse. The phenotype of Hoxd8 loss-of- function mutants is presented, and compared with that of Hoxb8- and Hoxc8-null mice. Our analysis of single mutants reveals common features for the Hoxc8 and Hoxd8 genes in patterning lower thoracic and lumbar vertebrae. In the Hoxb8 mutant, more anterior axial regions are affected. The three paralogous Hox genes are expressed up to similar rostral boundaries in the mesoderm, but at levels that strongly vary with the axial position. We find that the axial region affected in each of the single mutants mostly corresponds to the area with the highest level of gene expression. However, analysis of double and triple mutants reveals that lower expression of the other two paralogous genes also plays a patterning role when the mainly expressed gene is defective. We therefore conclude that paralogous group 8 Hox genes are involved in patterning quite an extensive anteroposterior (AP) axial region. Phenotypes of double and triple mutants reveal that Hoxb8, Hoxc8 and Hoxd8 have redundant functions at upper thoracic and sacral levels, including positioning of the hindlimbs. Interestingly, loss of functional Hoxb8 alleles partially rescues the phenotype of Hoxc8- and Hoxc8/Hoxd8-null mutants at lower thoracic and lumbar levels. This suggests that Hoxb8 affects patterning at these axial positions differently from the other paralogous gene products. We conclude that paralogous Hox genes can have a unique role in patterning specific axial regions in addition to their redundant function at other AP levels.
A characterization of the effects of Dpp signaling on cell growth and proliferation in theDrosophilawing
Authors: Martín-Castellanos Cristina , Edgar Bruce A.
Published: April 2021

Cell proliferation and patterning must be coordinated for the development of properly proportioned organs. If the same molecules were to control both processes, such coordination would be ensured. Here we address this possibility in the Drosophila wing using the Dpp signaling pathway. Previous studies have shown that Dpp forms a gradient along the AP axis that patterns the wing, that Dpp receptors are autonomously required for wing cell proliferation, and that ectopic expression of either Dpp or an activated Dpp receptor, TkvQ253D, causes overgrowth. We extend these findings with a detailed analysis of the effects of Dpp signaling on wing cell growth and proliferation. Increasing Dpp signaling by expressing TkvQ253D accelerated wing cell growth and cell cycle progression in a coordinate and cell-autonomous manner. Conversely, autonomously inhibiting Dpp signaling using a pathway specific inhibitor, Dad, or a mutation in tkv, slowed wing cell growth and division, also in a coordinate fashion. Stimulation of cell cycle progression by TkvQ253D was blocked by the cell cycle inhibitor RBF, and required normal activity of the growth effector, PI3K. Among the known Dpp targets, vestigial was the only one tested that was required for TkvQ253D-induced growth. The growth response to altering Dpp signaling varied regionally and temporally in the wing disc, indicating that other patterned factors modify the response.
Patterning of angiogenesis in the zebrafish embryo
Authors: Childs Sarah , Chen Jau-Nian , Garrity Deborah M. , Fishman Mark C.
Published: April 2021

Little is known about how vascular patterns are generated in the embryo. The vasculature of the zebrafish trunk has an extremely regular pattern. One intersegmental vessel (ISV) sprouts from the aorta, runs between each pair of somites, and connects to the dorsal longitudinal anastomotic vessel (DLAV). We now define the cellular origins, migratory paths and cell fates that generate these metameric vessels of the trunk. Additionally, by a genetic screen we define one gene, out of bounds (obd), that constrains this angiogenic growth to a specific path.

We have performed lineage analysis, using laser activation of a caged dye and mosaic construction to determine the origin of cells that constitute the ISV. Individual angioblasts destined for the ISVs arise from the lateral posterior mesoderm (LPM), and migrate to the dorsal aorta, from where they migrate between somites to their final position in the ISVs and dorsal longitudinal anastomotic vessel (DLAV). Cells of each ISV leave the aorta only between the ventral regions of two adjacent somites, and migrate dorsally to assume one of three ISV cell fates. Most dorsal is a T-shaped cell, based in the DLAV and branching ventrally; the second constitutes a connecting cell; and the third an inverted T-shaped cell, based in the aorta and branching dorsally. The ISV remains between somites during its ventral course, but changes to run mid-somite dorsally. This suggests that the pattern of ISV growth ventrally and dorsally is guided by different cues.

We have also performed an ENU mutagenesis screen of 750 mutagenized genomes and identified one mutation, obd that disrupts this pattern. In obd mutant embryos, ISVs sprout precociously at abnormal sites and migrate anomalously in the vicinity of ventral somite. The dorsal extent of the ISV is less perturbed. Precocious sprouting can be inhibited in a VEGF morphant, but the anomalous site of origin of obd ISVs remains. In mosaic embryos, obd somite causes adjacent wild-type endothelial cells to assume the anomalous ISV pattern of obd embryos.

Thus, the launching position of the new sprout and its initial trajectory are directed by inhibitory signals from ventral somites. Zebrafish ISVs are a tractable system for defining the origins and fates of vessels, and for dissecting elements that govern patterns of vessel growth.
A pea seed mutant affected in the differentiation of the embryonic epidermis is impaired in embryo growth and seed maturation
Authors: Borisjuk Ljudmilla , Wang Trevor L. , Rolletschek Hardy , Wobus Ulrich , Weber Hans
Published: April 2021

During legume seed development the epidermis of the embryos differentiates into a transfer cell layer which mediates nutrient uptake during the storage phase. This specific function of the epidermal cells is acquired at the onset of embryo maturation. We investigated this process in the pea seed mutant E2748. The epidermal cells of the mutant embryo, instead of turning into transfer cells, enlarge considerably and become vacuolated and tightly associated with adjacent seed tissues. Expression of a sucrose transporter gene that is upregulated in wild-type transfer cells decreases in the mutant and changes its spatial pattern. This indicates that the outermost cell layer of mutant cotyledons cannot acquire transfer cell morphology but loses epidermal cell identity and does not function as a sucrose uptake system. Seed coat growth as well as composition, concentration and dynamics of sugars within the endospermal vacuole are unchanged. The loss of epidermal identity has severe consequences for further embryo development and is followed by disruption of the symplast within the parenchyma, the breach of the developmental gradient, lower sucrose and starch levels and initiation of callus-like growth. It is concluded that the E2748 gene controls differentiation of the cotyledonary epidermis into transfer cells and thus is required for the regional specialisation with a function in embryo nutrition.
The Snail repressor positions Notch signaling in theDrosophilaembryo
Authors: Cowden John , Levine Michael
Published: April 2021

The maternal Dorsal nuclear gradient initiates the differentiation of the mesoderm, neurogenic ectoderm and dorsal ectoderm in the precellular Drosophila embryo. Each tissue is subsequently subdivided into multiple cell types during gastrulation. We have investigated the formation of the mesectoderm within the ventral-most region of the neurogenic ectoderm. Previous studies suggest that the Dorsal gradient works in concert with Notch signaling to specify the mesectoderm through the activation of the regulatory gene sim within single lines of cells that straddle the presumptive mesoderm. This model was confirmed by misexpressing a constitutively activated form of the Notch receptor, NotchIC, in transgenic embryos using the eve stripe2 enhancer. The NotchIC stripe induces ectopic expression of sim in the neurogenic ectoderm where there are low levels of the Dorsal gradient. sim is not activated in the ventral mesoderm, due to inhibition by the localized zinc-finger Snail repressor, which is selectively expressed in the ventral mesoderm. Additional studies suggest that the Snail repressor can also stimulate Notch signaling. A stripe2-snail transgene appears to induce Notch signaling in ‘naïve’ embryos that contain low uniform levels of Dorsal. We suggest that these dual activities of Snail, repression of Notch target genes and stimulation of Notch signaling, help define precise lines of sim expression within the neurogenic ectoderm.
An essential role for connexin43 gap junctions in mouse coronary artery development
Authors: Li W. E. I. , Waldo K. , Linask K. L. , Chen T. , Wessels A. , Parmacek M. S. , Kirby M. L. , Lo C. W.
Published: April 2021

Connexin43 knockout mice die neonatally from conotruncal heart malformation and outflow obstruction. Previous studies have indicated the involvement of neural crest perturbations in these cardiac anomalies. We provide evidence for the involvement of another extracardiac cell population, the proepicardial cells. These cells give rise to the vascular smooth muscle cells of the coronary arteries and cardiac fibroblasts in the heart. We have observed the abnormal presence of fibroblast and vascular smooth muscle cells in the infundibular pouches of the connexin43 knockout mouse heart. In addition, the connexin43 knockout mice exhibit a variety of coronary artery patterning defects previously described for neural crest-ablated chick embryos, such as anomalous origin of the coronary arteries, absent left or right coronary artery, and accessory coronary arteries. However, we show that proepicardial cells also express connexin43 gap junctions abundantly. The proepicardial cells are functionally well coupled, and this coupling is significantly reduced with the loss of connexin43 function. Further analysis revealed an elevation in the speed of cell locomotion and cell proliferation rate in the connexin43-deficient proepicardial cells. A parallel analysis of proepicardial cells in transgenic mice with dominant negative inhibition of connexin43 targeted only to neural crest cells showed none of these coupling, proliferation or migration changes. These mice exhibit outflow obstruction, but no infundibular pouches. Together these findings indicate an important role for connexin43 in coronary artery patterning, a role that probably involves the proepicardial and cardiac neural crest cells. We discuss the potential involvement of connexin43 in human cardiovascular anomalies involving the coronary arteries.
Studies on the functional activity of organotypically cultured mouse ovary
Authors: Chapekar T. N. , Nayak G. V. , Ranadive Kamal J.
Published: April 2021

Short-term maintenance of mouse and rat ovary in organotypic culture system is no longer a problem (Martinovitch, 1938; Gaillard, 1953; Trowell, 1959). Gaillard (1953) cultivated ovaries from 7-to 8-day-old and 21-day-old mice for a week on the plasma clot. Trowell (1959) maintained ovaries of 8-day-old mice on a synthetic medium in an O2-CO2 atmosphere for 9 days. He observed no histological differentiation in the tissues of the ovary. What needs confirmation and further investigation is the possibility of maintenance of functional activity of the ovary under culture conditions. A study was therefore undertaken to investigate if an ovary, cultivated in vitro for some time, shows hormonal activity when transplanted in vivo. In the present work cultured ovaries were grafted in the anterior eye-chamber of spayed female mice and the development of secondary sex organs such as mammary glands and uterus was studied.
Congenital malformations induced by dimethyl sulphoxide in the golden hamster
Authors: Ferm Vergil H.
Published: April 2021

Dimethyl sulphoxide (DMSO) is a relatively simple organic compound with a molecular weight of 78. It has been reported to be exceptionally non-toxic and has been used extensively in biological systems as a cell preservative, including freeze storage (Porterfield & Ashwood-Smith, 1962; Dougherty, 1962), and as a radioprotective agent against X-irradiation in mice (Ashwood-Smith, 1961). Recent clinical investigations in man have suggested that DMSO has a remarkable array of effects, including anti-inflammatory activity, bacteriostatic effects, and analgesic activity (Jacob, Bischel & Herschler, 1964). It has been used extensively as a vehicle for the topical application of various drugs because of its dramatic effect on enhancement of penetration of various drugs through the skin (Kligman, 1965).