bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence that preaxial polydactyly in the Doublefoot mutant is due to ectopic Indian Hedgehog signaling
Authors: Yang Yingzi , Guillot Pascale , Boyd Yvonne , Lyon Mary F. , McMahon Andrew P.
Published: April 2021

Patterning of the vertebrate limb along the anterior-posterior axis is controlled by the zone of polarizing activity (ZPA) located at the posterior limb margin. One of the vertebrate Hh family members, Shh, has been shown to be able to mediate the function of the ZPA. Several naturally occurring mouse mutations with the phenotype of preaxial polydactyly exhibit ectopic Shh expression at the anterior limb margin. In this study, we report the molecular characterization of a spontaneous mouse mutation, Doublefoot (Dbf). Dbf is a dominant mutation which maps to chromosome 1. Heterozygous and homozygous embryos display a severe polydactyly with 6 to 8 digits on each limb. We show here that Shh is expressed normally in Dbf mutants. In contrast, a second Hh family member, Indian hedgehog (Ihh) which maps close to Dbf, is ectopically expressed in the distal limb bud. Ectopic Ihh expression in the distal and anterior limb bud results in the ectopic activation of several genes associated with anterior-posterior and proximal-distal patterning (Fgf4, Hoxd13, Bmp2). In addition, specific components in the Hedgehog pathway are either ectopically activated (Ptc, Ptc-2, Gli1) or repressed (Gli2). We propose that misexpression of Ihh, and not a novel Smoothened ligand as recently suggested (Hayes et al., 1998), is responsible for the Dbf phenotype. We consider that Ihh has a similar activity to Shh when expressed in the early Shh-responsive limb bud. To determine whether Dbf maps to the Ihh locus, which is also on chromosome 1, we performed an interspecific backcross. These results demonstrate that Dbf and Ihh are genetically separated by approximately 1.3 centimorgans, suggesting that Dbf mutation may cause an exceptionally long-range disruption of Ihh regulation. Although this leads to ectopic activation of Ihh, normal expression of Ihh in the cartilaginous elements is retained.
The ich1 gene of the mushroom Coprinus cinereus is essential for pileus formation in fruiting
Authors: Muraguchi Hajime , Kamada Takashi
Published: April 2021

The formation of the pileus in homobasidiomycete fungi is essential for sexual reproduction, because the pileus bears the hymenium, a layer of cells that includes the specialised basidia in which nuclear fusion, meiosis and sporulation occur. The developmental mutant ichijiku of Coprinus cinereus fails to develop a differentiated pileus at the apex of the primordial shaft, which is the basal part of the fruit-body primordia and formed in an early stage of fruit-body differentiation. Genetic analysis indicates that this phenotype is caused by a recessive mutation in a single gene (ich1). The ich1 gene was mapped to chromosome XII using restriction fragment length polymorphism markers and the marker chromosome method, and cloned by complementation using a chromosome-XII-specific cosmid library. The ich1 gene encodes a novel protein of 1,353 amino acids. The Ich1 amino-acid sequence contains nuclear targeting signals, suggesting that the Ich1 protein would function in the nucleus. Northern blot analysis indicates that the ich1 gene is specifically expressed in the pileus of the wild-type fruit-body. No ich1 mRNA was detected in the ichijiku mutant, consistent with loss of the promoter region of ich1 in the mutant genome. These data demonstrate that the ich1 gene product is essential for pileus formation.
Female mouse germ cells form synchronously dividing cysts
Authors: Pepling Melissa E. , C. Spradling Allan
Published: April 2021

Oocytes from many invertebrates initiate development within distinctive cysts of interconnected cells, which are formed through synchronous divisions of a progenitor cell. Recently, processes underlying cyst formation have been extensively characterized at the molecular level in Drosophila. Defects in this process cause sterility in female flies. Early female mouse germ cells are organized as cell clusters as well, but it is uncertain whether these groups are similar to the cysts of invertebrates. We find that mouse germ cells are connected by intercellular bridges in the ovaries of 11.5 to 17.5 days postcoitum embryos; microtubules and organelles have been observed within these bridges. Confocal microscopy shows that cells within mouse clusters divide synchronously and frequently correspond in number to powers of two. Thus, female mouse germ cell clusters exhibit key characteristics of invertebrate germline cysts indicating that the process of germline cyst formation is conserved in the mouse.
slimb coordinates wg and dpp expression in the dorsal-ventral and anterior-posterior axes during limb development
Authors: Theodosiou Nicole A. , Zhang Sheng , Wang Wei-Yi , Xu Tian
Published: April 2021

In the Drosophila leg disc, wingless (wg) and decapentaplegic (dpp) are expressed in a ventral-anterior and dorsal-anterior stripe of cells, respectively. This pattern of expression is essential for proper limb development. While the Hedgehog (Hh) pathway regulates dpp and wg expression in the anterior-posterior (A/P) axis, mechanisms specifying their expression in the dorsal-ventral (D/V) axis are not well understood. We present evidence that slimb mutant clones in the disc deregulate wg and dpp expression in the D/V axis. This suggests for the first time that their expression in the D/V axis is actively regulated during imaginal disc development. Furthermore, slimb is unique in that it also deregulates wg and dpp in the A/P axis. The misexpression phenotypes of slimb− clones indicate that the regulation of wg and dpp expression is coordinated in both axes, and that slimb plays an essential role in integrating A/P and D/V signals for proper patterning during development. Our genetic analysis further reveals that slimb intersects the A/P pathway upstream of smoothened (smo).
The iroquois complex controls the somatotopy of Drosophila notum mechanosensory projectionss
Authors: Grillenzoni Nicola , Helden Jacques van , Dambly-Chaudière Christine , Ghysen Alain
Published: April 2021

Sensory neurons can establish topologically ordered projections in the central nervous system, thereby building an internal representation of the external world. We analyze how this ordering is genetically controlled in Drosophila, using as a model system the neurons that innervate the mechanosensory bristles on the back of the fly (the notum). Sensory neurons innervating the medially located bristles send an axonal branch that crosses the central nervous system midline, defining a ‘medial’ identity, while the ones that innervate the lateral bristles send no such branch, defining a ‘lateral’ identity. We analyze the role of the proneural genes achaete and scute, which are involved in the formation of the medial and lateral bristles, and we show that they have no effect on the ‘medial’ and ‘lateral’ identities of the neurons. We also analyze the role of the prepattern genes araucan and caupolican, two members of the iroquois gene complex which are required for the expression of achaete and scute in the lateral region of the notum, and we show that their expression is responsible for the ‘lateral’ identity of the projection.
A Drosophila RNA helicase gene, pitchoune, is required for cell growth and proliferation and is a potential target of d-Myc
Authors: Zaffran S. , Chartier A. , Gallant P. , Astier M. , Arquier N. , Doherty D. , Gratecos D. , Sémériva M.
Published: April 2021

This article describes the characterization of a new Drosophila gene that we have called pitchoune (pit) (meaning small in Provence) because mutations in this gene produce larvae that cannot grow beyond the first instar larval stage although they can live as long as 7-10 days. All the tissues are equally affected and the perfectly shaped larvae are indistinguishable from first instar wild-type animals. Analysis of mutant somatic clones suggests a function in cell growth and proliferation, which is supported by the fact that cell proliferation is promoted by pit overexpression. Tagged-Pit, when transfected in S2 cells, localizes mainly to the nucleolus, pointing towards a possible role in ribosome biogenesis and, consequently, in protein biosynthesis. pit encodes a DEAD-box RNA helicase, a family of proteins involved in the control of RNA structure in many cellular processes and its closest homologue is a human DEAD-box RNA helicase, MrDb, whose corresponding gene transcription is directly activated by Myc-Max heterodimers (Grandori, C., Mac, J., Siëbelt, F., Ayer, D. E. and Eisenman, R. N. (1996) EMBO J. 15, 4344-4357). The patterns of expression of d-myc and pit are superimposable. Ectopic expression of myc in the nervous system drives an ectopic expression of pit in this tissue indicating that in Drosophila as well, pit is a potential target of d-Myc. These results suggest that myc might promote cell proliferation by activating genes that are required in protein biosynthesis, thus linking cell growth and cell proliferation.
A Serum Response Factor homolog is required for spore differentiation in Dictyostelium
Authors: Escalante Ricardo , Sastre Leandro
Published: April 2021

A homolog of the Serum Response Factor (SRF) has been isolated from Dictyostelium discoideum and its function studied by analyzing the consequences of its gene disruption. The MADS-box region of Dictyostelium SRF (DdSRF) is highly conserved with those of the human, Drosophila and yeast homologs. srfA is a developmentally regulated gene expressed in prespore and spore cells. This gene plays an essential role in sporulation as its disruption leads to abnormal spore morphology and loss of viability. The mutant spores were round and cellulose deposition seemed to be partially affected. Initial prestalk and prespore cell differentiation did not seem to be compromised in the mutant since the expression of several cell-type-specific markers were found to be unaffected. However, the mRNA level of the spore marker spiA was greatly reduced. Activation of the cAMP-dependent protein kinase (PKA) by 8-Br-cAMP was not able to fully bypass the morphological defects of srfA− mutant spores, although this treatment induced spiA mRNA expression. Our results suggest that DdSRF is required for full maturation of spores and participates in the regulation of the expression of the spore-coat marker spiA and probably other maturation genes necessary for proper spore cell differentiation.
Developmental remodeling and shortening of the cardiac outflow tract involves myocyte programmed cell death
Authors: Watanabe Michiko , Choudhry Aleem , Berlan Michael , Singal Amishi , Siwik Ernest , Mohr Susanne , Fisher Steven A.
Published: April 2021

The embryonic outflow tract is a simple tubular structure that connects the single primitive ventricle with the aortic sac and aortic arch arteries. This structure undergoes a complex sequence of morphogenetic processes to become the portion of the heart that aligns the right and left ventricles with the pulmonary artery and aorta. Abnormalities of the outflow tract are involved in many clinically significant congenital cardiac defects; however, the cellular and molecular processes governing the development of this important structure are incompletely understood. Histologic and tissue-tagging studies indicate that the outflow tract tissues compact and are incorporated predominantly into a region of the right ventricle. The hypothesis tested in the current study was that cell death or apoptosis in the muscular portion of the outflow tract is an important cellular mechanism for outflow tract shortening. The tubular outflow tract myocardium was specifically marked by infecting myocytes of the chicken embryo heart with a recombinant replication-defective adenovirus expressing β-galactosidase (β-gal) under the control of the cytomegalovirus promoter. Histochemical detection of the β-gal-labeled outflow tract myocytes revealed that the tubular structure shortened to become a compact ring at the level of the pulmonic infundibulum over several days of development (stages 25-32, embryonic days 4-8). The appearance of apoptotic cardiomyocytes was correlated with OFT shortening by two histologic assays, TUNEL labeling of DNA fragments and AnnexinV binding. The rise and fall in the number of apoptotic myocytes detected by histologic analyses paralleled the change in activity levels of Caspase-3, a protease in the apoptotic cascade, measured in outflow tract homogenates. These results suggest that the elimination of myocytes by programmed cell death is one mechanism by which the outflow tract myocardium remodels to form the proper connection between the ventricular chambers and the appropriate arterial trunks.
Generation of multiple antagonistic domains along the proximodistal axis during Drosophila leg development
Authors: Abu-Shaar Muna , Mann Richard S.
Published: April 2021

homothorax (hth) is a Drosophila member of the Meis family of homeobox genes. hth function is required for the nuclear localization of the Hox cofactor Extradenticle (EXD). We show here that there is also a post-transcriptional control of HTH by exd: exd activity is required for the apparent stability of the HTH protein. In leg imaginal discs, hth expression is limited to the domain of exd function and this domain is complementary to the domain in which the Wingless (WG) and Decapentaplegic (DPP) signals are active. We demonstrate that WG and DPP act together through their targets Distal-less (Dll) and dachshund (dac) to restrict hth expression, and therefore EXD’s nuclear localization, to the most proximal regions of the leg disc. Furthermore, there is a reciprocal repression exerted by HTH on these and other DPP and WG downstream targets that restricts their expression to non-hth-expressing cells. Thus, there exists in the leg disc a set of mutually antagonistic interactions between proximal cells, which we define as those that express hth, and distal cells, or those that do not express hth. In addition, we show that dac negatively regulates Dll. We suggest that these antagonistic relationships help to convert the WG and DPP activity gradients into discreet domains of gene expression along the proximodistal axis.
Sperm-induced local[Ca2+]i rise separated from the Ca2+ wave in sea urchin eggs in the presence of a gamete fusion inhibitor, jaspisin
Authors: Mohri Tatsuma , Miyazaki Shunichi , Shirakawa Hideki , Ikegami Susumu
Published: April 2021

An increase in intracellular Ca2+ concentration ([Ca2+]i) at a focal plane was recorded simultaneously with sperm-egg binding and membrane current upon insemination of sea urchin Hemicentrotus pulcherrimus eggs. No change in current and [Ca2+]i occurred in the presence of jaspisin, a novel substance that inhibits metallo-endoproteinase and sperm-egg membrane fusion (S. Ikegami, H. Kobayashi, Y. Myotoishi, S. Ohta and K. H. Kato (1994) J. Biol. Chem. 269, 23262-23267). With low doses of jaspisin, a spermatozoon first produced a step inward current (Ion) as an indication of gamete membrane fusion and then induced a local [Ca2+]i rise at the site of sperm attachment 6-10 seconds after Ion. The sperm, however, soon detached from the egg. Increasing inward current was abruptly cut off (Ioff) within 9-15 seconds and the local [Ca2+]i rise began to decline 1-3 seconds after Ioff. In most cases, no further responses or an elevation of fertilization envelope (FE) occurred. In some cases, [Ca2+]i at the sperm attachment site increased again even after the sperm detached and triggered a Ca2+ wave which caused an activation current and FE formation. This recording of a gamete membrane- fusion-induced local [Ca2+]i rise, separated from the Ca2+ wave, is a key phenomenon for elucidating the initial sperm stimulation of the egg at fertilization.
Lhx2, a vertebrate homologue of apterous, regulates vertebrate limb outgrowth
Authors: Rodriguez-Esteban Concepción , Schwabe John W. R. , Peña Jennifer De La , Rincon-Limas Diego E. , Magallón Jorge , Botas Juan , Izpisúa Belmonte Juan Carlos
Published: April 2021

apterous specifies dorsal cell fate and directs outgrowth of the wing during Drosophila wing development. Here we show that, in vertebrates, these functions appear to be performed by two separate proteins. Lmx-1 is necessary and sufficient to specify dorsal identity and Lhx2 regulates limb outgrowth. Our results suggest that Lhx2 is closer to apterous than Lmx-1, yet, in vertebrates, Lhx2 does not specify dorsal cell fate. This implies that in vertebrates, unlike Drosophila, limb outgrowth can be dissociated from the establishment of the dorsoventral axis.
Requirement for Brn-3c in maturation and survival, but not in fate determination of inner ear hair cells
Authors: Xiang Mengqing , Gao Wei-Qiang , Hasson Tama , Shin Joyce J.
Published: April 2021

Mutations in the POU domain gene Brn-3c causes hearing impairment in both the human and mouse as a result of inner ear hair cell loss. We show here that during murine embryogenesis, Brn-3c is expressed in postmitotic cells committed to hair cell phenotype but not in mitotic progenitors in the inner ear sensory epithelium. In developing auditory and vestibular sensory epithelia of Brn-3c−/− mice, hair cells are found to be generated and undergo initial differentiation as indicated by their morphology, laminar position and expression of hair cell markers, including myosins VI and VIIa, calretinin and parvalbumin. However, a small number of hair cells are anomalously retained in the supporting cell layer in the vestibular sensory epithelia. Furthermore, the initially differentiated hair cells fail to form stereociliary bundles and degenerate by apoptosis in the Brn-3c−/− mice. These data indicate a crucial role for Brn-3c in maturation, survival and migration of hair cells, but not in proliferation or commitment of hair cell progenitors.
The Drosophila trithorax group proteins BRM, ASH1 and ASH2 are subunits of distinct protein complexes
Authors: Papoulas O. , Beek S.J. , Moseley S.L. , McCallum C.M. , Sarte M. , Shearn A. , Tamkun J.W.
Published: April 2021

The trithorax group gene brahma (brm) encodes an activator of Drosophila homeotic genes that functions as the ATPase subunit of a large protein complex. To determine if BRM physically interacts with other trithorax group proteins, we purified the BRM complex from Drosophila embryos and analyzed its subunit composition. The BRM complex contains at least seven major polypeptides. Surprisingly, the majority of the subunits of the BRM complex are not encoded by trithorax group genes. Furthermore, a screen for enhancers of a dominant-negative brm mutation identified only one trithorax group gene, moira (mor), that appears to be essential for brm function in vivo. Four of the subunits of the BRM complex are related to subunits of the yeast chromatin remodeling complexes SWI/SNF and RSC. The BRM complex is even more highly related to the human BRG1 and hBRM complexes, but lacks the subunit heterogeneity characteristic of these complexes. We present biochemical evidence for the existence of two additional complexes containing trithorax group proteins: a 2 MDa ASH1 complex and a 500 kDa ASH2 complex. These findings suggest that BRM plays a role in chromatin remodeling that is distinct from the function of most other trithorax group proteins.
The knirps and knirps-related genes organize development of the second wing vein in Drosophila
Authors: Lunde Karen , Biehs Brian , Nauber Ulrich , Bier Ethan
Published: April 2021

The neighboring homologous knirps (kni) and knirpsrelated (knrl) genes in Drosophila encode transcription factors in the steroid hormone receptor superfamily. During early embryogenesis, kni functions as a gap gene to control expression of segmentation genes within the abdominal region of the embryo. In this study, we present evidence that kni and knrl link A/P positional information in larval wing imaginal discs to morphogenesis of the second longitudinal wing vein (L2). We show that kni and knrl are expressed in similar narrow stripes corresponding to the position of the L2 primordium. The kni and knrl L2 stripes abut the anterior border of the broad central expression domain of the Dpp target gene spalt major (salm). We provide evidence that radius incompletus (ri), a well-known viable mutant lacking the L2 vein, is a regulatory mutant of the kni/knrl locus. In ri mutant wing discs, kni and knrl fail to be expressed in the L2 primordium. In addition, the positions of molecular breakpoints in the kni/knrl locus indicate that the ri function is provided by cis-acting sequences upstream of the kni transcription unit. Epistasis tests reveal that the kni/knrl locus functions downstream of spalt major (salm) and upstream of genes required to initiate vein-versusintervein differentiation. Mis-expression experiments suggest that kni and knrl expressing cells inhibit neighboring cells from becoming vein cells. Finally, kni and knrl are likely to refine the L2 position by positively auto-regulating their own expression and by providing negative feedback to repress salm expression. We propose a model in which the combined activities of kni and knrl organize development of the L2 vein in the appropriate position.
The Drosophila Pax gene eye gone is required for embryonic salivary duct development
Authors: Jones Nikolas A. , Kuo Yien Ming , Sun Y. Henry , Beckendorf Steven K.
Published: April 2021

What are the developmental mechanisms required for conversion of an undifferentiated, two-dimensional field of cells into a patterned, tubular organ? In this report, we describe the contribution of the Drosophila Pax gene eye gone to the development of the embryonic salivary glands and ducts. eye gone expression in salivary tissues is controlled by several known regulators of salivary fate. After the initial establishment of the salivary primordium by Sex combs reduced, fork head excludes eye gone expression from the pregland cells so that its salivary expression is restricted to the posterior preduct cells. trachealess, in contrast, activates eye gone expression in the posterior preduct cells. We have previously described the process by which fork head and the EGF receptor pathway define the border between the gland and duct primordia. Here we show that eye gone is required for the subdivision of the duct primordium itself into the posterior individual duct and the anterior common duct domains. In the absence of eye gone, individual ducts as well as the precursor of the adult salivary glands, the imaginal ring, are absent. We took advantage of this ductless phenotype to show that Drosophila larvae do not have an obligate requirement for salivary glands and ducts. In addition to its role in the salivary duct, eye gone is required in the embryo for the development of the eye-antennal imaginal disc and the chemosensory antennal organ.
Role of Dpp signalling in prepattern formation of the dorsocentral mechanosensory organ in Drosophila melanogaster
Authors: Tomoyasu Yoshinori , Nakamura Makoto , Ueno Naoto
Published: April 2021

A proneural cluster of dorsocentral bristles forms adjacent to the dorsal side of wg-expressing cells in the notum region of the wing imaginal disc. It has been shown that wg activity is required for these structures to form. However, the restriction of this proneural cluster to the dorsal posterior side of the wg expression domain in the anterior compartment of the wing imaginal disc has suggested that Wg signalling itself is insufficient to establish the dorsocentral proneural cluster. Some factor(s) from the posterior side must participate in this action in cooperation with Wg signalling. We have examined the role of Dpp signalling in dorsocentral bristle formation by either ectopically activating or conditionally reducing Dpp signalling. Ubiquitous activation of Dpp signalling in the notum region of the wing imaginal disc induced additional dorsocentral proneural cluster all along the dorsal side of the wg expression domain, and altered wg expression. Conditional loss-of-function of Dpp signalling during disc development resulted in the inhibition of dorsocentral proneural cluster formation and expansion of the wg expression domain. These results suggest that Dpp signalling has two indispensable roles in dorsocentral bristle formation: induction of the dorsocentral proneural cluster in cooperation with Wg signalling and restriction of the wg expression domain in the notum region of the wing imaginal disc.
Semaphorins III and IV repel hippocampal axons via two distinct receptors
Authors: Chédotal Alain , Del Rio Jose A. , Ruiz Monica , He Zhigang , Borrell Victor , de Castro Fernando , Ezan Frédéric , Goodman Corey S. , Tessier-Lavigne Marc , Sotelo Constantino , Soriano Eduardo
Published: April 2021

The semaphorins are the largest family of repulsive axon guidance molecules. Secreted semaphorins bind neuropilin receptors and repel sensory, sympathetic and motor axons. Here we show that CA1, CA3 and dentate gyrus axons from E15-E17 mouse embryo explants are selectively repelled by entorhinal cortex and neocortex. The secreted semaphorins Sema III and Sema IV and their receptors Neuropilin-1 and -2 are expressed in the hippocampal formation during appropriate stages. Sema III and Sema IV strongly repel CA1, CA3 and dentate gyrus axons; entorhinal axons are only repelled by Sema III. An antibody against Neuropilin-1 blocks the repulsive action of Sema III and the entorhinal cortex, but has no effect on Sema IV-induced repulsion. Thus, chemorepulsion plays a role in axon guidance in the hippocampus, secreted semaphorins are likely to be responsible for this action, and the same axons can be repelled by two distinct semaphorins via two different receptors.
not really finished is crucial for development of the zebrafish outer retina and encodes a transcription factor highly homologous to human Nuclear Respiratory Factor-1 and avian Initiation Binding Repressor
Authors: Becker Thomas S. , Burgess Shawn M. , Amsterdam Adam H. , Allende Miguel L. , Hopkins Nancy
Published: April 2021

Not really finished (nrf), a larval-lethal mutation in zebrafish generated by retroviral insertion, causes specific retinal defects. Analysis of mutant retinae reveals an extensive loss of photoreceptors and their precursors around the onset of visual function. These neurons undergo apoptosis during differentiation, affecting all classes of photoreceptors, suggesting an essential function of nrf for the development of all types of photoreceptors. In the mutant, some photoreceptors escape cell death, are functional and, as judged by opsin expression, belong to at least three classes of cones and one class of rods. The protein encoded by nrf is a close homologue of human Nuclear Respiratory Factor 1 and avian Initiation Binding Repressor, transcriptional regulators binding the upstream consensus sequence RCGCRYGCGY. At 24 hours of development, prior to neuronal differentiation, nrf is expressed ubiquitously throughout the developing retina and central nervous system. At 48 hours of development, expression of nrf is detected in the ganglion cell layer, in the neurons of the inner nuclear layer, and in the optic nerve and optic tracts, and, at 72 hours of development, is no longer detectable by in situ hybridization. Mutants contain no detectable nrf mRNA and die within 2 weeks postfertilization as larvae with reduced brain size. On the basis of its similarity with NRF-1 and IBR, nrf is likely involved in transcriptional regulation of multiple target genes, including those that encode mitochondrial proteins, growth factor receptors and other transcription factors. This demonstrates the power of insertional mutagenesis as a means for characterizing novel genes necessary for vertebrate retinal development.
Heartbroken is a specific downstream mediator of FGF receptor signalling in Drosophila
Authors: Michelson Alan M. , Gisselbrecht Stephen , Buff Eugene , Skeath James B.
Published: April 2021

Drosophila possesses two FGF receptors which are encoded by the heartless and breathless genes. HEARTLESS is essential for early migration and patterning of the embryonic mesoderm, while BREATHLESS is required for proper branching of the tracheal system. We have identified a new gene, heartbroken, that participates in the signalling pathways of both FGF receptors. Mutations in heartbroken are associated with defects in the migration and later specification of mesodermal and tracheal cells. Genetic interaction and epistasis experiments indicate that heartbroken acts downstream of the two FGF receptors but either upstream of or parallel to RAS1. Furthermore, heartbroken is involved in both the HEARTLESS- and BREATHLESS-dependent activation of MAPK. In contrast, EGF receptor-dependent embryonic functions and MAPK activation are not perturbed in heartbroken mutant embryos. A strong heartbroken allele also suppresses the effects of hyperactivated FGF but not EGF receptors. Thus, heartbroken may contribute to the specificity of developmental responses elicited by FGF receptor signalling.
A highly conserved centrosomal kinase, AIR-1, is required for accurate cell cycle progression and segregation of developmental factors in Caenorhabditis elegans embryos
Authors: Schumacher Jill M. , Ashcroft Neville , Donovan Peter J. , Golden Andy
Published: April 2021

S. cerevisiae Ipl1, Drosophila Aurora, and the mammalian centrosomal protein IAK-1 define a new subfamily of serine/threonine kinases that regulate chromosome segregation and mitotic spindle dynamics. Mutations in ipl1 and aurora result in the generation of severely aneuploid cells and, in the case of aurora, monopolar spindles arising from a failure in centrosome separation. Here we show that a related, essential protein from C. elegans, AIR-1 (Aurora/Ipl1 related), is localized to mitotic centrosomes. Disruption of AIR-1 protein expression in C. elegans embryos results in severe aneuploidy and embryonic lethality. Unlike aurora mutants, this aneuploidy does not arise from a failure in centrosome separation. Bipolar spindles are formed in the absence of AIR-1, but they appear to be disorganized and are nucleated by abnormal-looking centrosomes. In addition to its requirement during mitosis, AIR-1 may regulate microtubule-based developmental processes as well. Our data suggests AIR-1 plays a role in P-granule segregation and the association of the germline factor PIE-1 with centrosomes.
Sperm-triggered calcium oscillations during meiosis in ascidian oocytes first pause, restart, then stop: correlations with cell cycle kinase activity
Authors: McDougall Alex , Levasseur Mark
Published: April 2021

We have investigated the relationship between the sperm-triggered Ca2+ oscillations and the activities of two cell cycle kinases (MPF activity and MAP kinase activity) at fertilisation of Ascidiella aspersa oocytes. Maturation Promoting Factor (MPF) activity is elevated in the metaphase I (MI)-arrested unfertilised oocyte (as measured by phosphorylation of exogenous histone H1) and falls 5 minutes after fertilisation to remain at low levels for 5 minutes. The first polar body (pb1) is extruded when the MPF activity is low. The MPF activity is elevated again 15 minutes after fertilisation and finally becomes inactivated 25 minutes after fertilisation when the pb2 is extruded. MAP kinase activity increases from an initially elevated level to reach maximal activity 10 minutes after fertilisation and subsequently falls to reach low levels 25 minutes after fertilisation. Sperm trigger a series of Ca2+ oscillations that pause for 5 minutes while only the MPF activity is low and are present when both MPF and MAP kinase activity are elevated.

We next attempted to determine whether the second phase of calcium oscillations is required to reactivate the MPF activity that precedes extrusion of the second polar body. To do this, we triggered a monotonic Ca2+ signal. This leads to the inactivation of MPF followed by MPF reactivation. The MPF activity then remains elevated for an extended period of time. During this period, the chromatin remains condensed and a metaphase II (MII) spindle forms. Fertilisation of these MII oocytes triggers extrusion of pb2 in 7 minutes. Interestingly, the second phase of Ca2+ oscillations is completely absent when MII oocytes are fertilised. Thus, in both MI and MII oocytes, the sperm-triggered Ca2+ oscillations follow the MPF activity.

Finally we discuss our finding that the Ca2+ release system remains sensitive during the metaphase-like state (including the period when the Ca2+ oscillations pause).
A GATA-dependent nkx-2.5 regulatory element activates early cardiac gene expression in transgenic mice
Authors: Searcy Robin D. , Vincent Eric B. , Liberatore Christine M. , Yutzey Katherine E.
Published: April 2021

nkx-2.5 is one of the first genes expressed in the developing heart of early stage vertebrate embryos. Cardiac expression of nkx-2.5 is maintained throughout development and nkx-2.5 also is expressed in the developing pharyngeal arches, spleen, thyroid and tongue. Genomic sequences flanking the mouse nkx-2.5 gene were analyzed for early developmental regulatory activity in transgenic mice. Approximately 3 kb of 5′ flanking sequence is sufficient to activate gene expression in the cardiac crescent as early as E7.25 and in limited regions of the developing heart at later stages. Expression also was detected in the developing spleen anlage at least 24 hours before the earliest reported spleen marker and in the pharyngeal pouches and their derivatives including the thyroid. The observed expression pattern from the −3 kb construct represents a subset of the endogenous nkx-2.5 expression pattern which is evidence for compartment-specific nkx-2.5 regulatory modules. A 505 bp regulatory element was identified that contains multiple GATA, NKE, bHLH, HMG and HOX consensus binding sites. This element is sufficient for gene activation in the cardiac crescent and in the heart outflow tract, pharynx and spleen when linked directly to lacZ or when positioned adjacent to the hsp68 promoter. Mutation of paired GATA sites within this element eliminates gene activation in the heart, pharynx and spleen primordia of transgenic embryos. The dependence of this nkx-2.5 regulatory element on GATA sites for gene activity is evidence for a GATA-dependent regulatory mechanism controlling nkx-2.5 gene expression. The presence of consensus binding sites for other developmentally important regulatory factors within the 505 bp distal element suggests that combinatorial interactions between multiple regulatory factors are responsible for the initial activation of nkx-2.5 in the cardiac, thyroid and spleen primordia.
Stage-specific expression of the Kit receptor and its ligand (KL) during male gametogenesis in the mouse: a Kit-KL interaction critical for meiosis
Authors: Vincent Stéphane , Segretain Dominique , Nishikawa Satomi , Nishikawa Shin-Ichi , Sage Julien , Cuzin François , Rassoulzadegan Minoo
Published: April 2021

The Kit receptor and its ligand KL, which together constitute an essential effector at various stages of embryonic development, are both present during adult gametogenesis. In the testis, KL is expressed in Sertoli cells, and Kit in germ cells, starting at the premeiotic stages. A series of observations indicated previously a role in spermatogonia survival, without excluding a possible function at later stages. We identified a complex pattern of expression of the two components in the adult murine testis, suggestive of a role in the meiotic progression of spermatocytes. At stages VII-VIII of the cycle of the seminiferous epithelium, the time when spermatocytes enter meiosis, the membrane-associated form of KL extends on the Sertoli cell from the peripheral to the adluminal compartment of the tubule. We also found that the receptor is present on the surface of germ cells up to the pachytene stage. The availability of differentiated Sertoli cell lines, which express the KL protein and support part of the maturation of germ cells in coculture, allowed us to ask whether, in the in vitro reconstructed system, transit of spermatocytes through meiosis requires the Kit-KL interaction. Addition of a blocking monoclonal antibody against the Kit receptor (ACK2) inhibited extensively the appearance of haploid cells and the expression of a haploid-phase-specific gene (Prm1). Recognition of the supporting Sertoli cell by germ cells was not affected, indicating a requirement for the activity of the receptor for either entering or completing meiosis. Involvement of the membrane-associated form of the ligand was suggested by the observation that addition of the soluble form of KL was equally inhibitory.
Hematopoietic induction and respecification of A-P identity by visceral endoderm signaling in the mouse embryo
Authors: Belaoussoff Maria , Farrington Sarah M. , Baron Margaret H.
Published: April 2021

The anteroposterior axis of the developing embryo becomes morphologically apparent at the onset of gastrulation with the formation of the primitive streak. This structure, where the first mesodermal cells arise, marks the posterior aspect of the embryo. To examine the potential role of non-mesodermal signals in specifying posterior (hematopoietic and endothelial) cell fates in the mouse embryo, we have devised a transgenic explant culture system. We show that interactions between primitive endoderm and adjacent embryonic ectoderm or nascent mesoderm are required early in gastrulation for initiation of hematopoiesis and vasculogenesis. Surprisingly, primitive endoderm signals can respecify anterior (prospective neural) ectoderm to a posterior mesodermal fate, resulting in formation of blood and activation of endothelial markers. Reprogramming of anterior ectoderm does not require cell contact and is effected by stage-dependent, short-range, diffusible signal(s). Therefore, primitive endoderm signaling is a critical early determinant of hematopoietic and vascular development and plays a decisive role in anterior-posterior patterning during mouse embryogenesis.
Nod factor internalization and microtubular cytoskeleton changes occur concomitantly during nodule differentiation in alfalfa
Authors: Timmers Antonius C. J. , Auriac Marie-Christine , de Billy Françoise , Truchet Georges
Published: April 2021

Reorganization of the plant cytoskeleton is thought to play an important role during nodule ontogeny. In situ immunolocalisation of tubulin reveals that important cytoskeletal changes, implying a transient disorganization followed by a newly patterned reorganization, occur in indeterminate and determinate nodules. In alfalfa nodules, cytoskeletal changes closely parallel the symbiotic differentiation features related to cell infection, bacterial release, endopolyploidization, cell enlargement, cell spatial organization and organelle ultrastructure and positioning. Moreover, the fact that microtubule disorganization can be correlated with Nod factor internalization in central infected cells suggests that Nod factors are possibly involved in the control of cytoskeletal changes which direct the differentiation of bacteria-containing cells.
Early posterior neural tissue is induced by FGF in the chick embryo
Authors: Storey Kate G. , Goriely Anne , Sargent Catherine M. , Brown Jennifer M. , Burns Helen D. , Abud Helen M. , Heath John K.
Published: April 2021

Signals that induce neural cell fate in amniote embryos emanate from a unique cell population found at the anterior end of the primitive streak. Cells in this region express a number of fibroblast growth factors (FGFs), a group of secreted proteins implicated in the induction and patterning of neural tissue in the amphibian embryo. Here we exploit the large size and accessibility of the early chick embryo to analyse the function of FGF signalling specifically during neural induction. Our results demonstrate that extraembryonic epiblast cells previously shown to be responsive to endogenous neural-inducing signals express early posterior neural genes in response to local, physiological levels of FGF signal. This neural tissue does not express anterior neural markers or undergo neuronal differentiation and forms in the absence of axial mesoderm. Prospective mesodermal tissue is, however, induced and we present evidence for both the direct and indirect action of FGFs on prospective posterior neural tissue. These findings suggest that FGF signalling underlies a specific aspect of neural induction, the initiation of the programme that leads to the generation of the posterior central nervous system.
Retinal axon guidance by region-specific cues in diencephalon
Authors: Tuttle Rebecca , Braisted Janet E. , Richards Linda J. , O’Leary Dennis D. M.
Published: April 2021

Retinal axons show region-specific patterning along the dorsal-ventral axis of diencephalon: retinal axons grow in a compact bundle over hypothalamus, dramatically splay out over thalamus, and circumvent epithalamus as they continue toward the dorsal midbrain. In vitro, retinal axons are repulsed by substrate-bound and soluble activities in hypothalamus and epithalamus, but invade thalamus. The repulsion is mimicked by a soluble floor plate activity. Tenascin and neurocan, extracellular matrix molecules that inhibit retinal axon growth in vitro, are enriched in hypothalamus and epithalamus. Within thalamus, a stimulatory activity is specifically upregulated in target nuclei at the time that retinal axons invade them. These findings suggest that region-specific, axon repulsive and stimulatory activities control retinal axon patterning in the embryonic diencephalon.
Dual genetic pathways of endothelin-mediated intercellular signaling revealed by targeted disruption of endothelin converting enzyme-1 gene
Authors: Yanagisawa Hiromi , Yanagisawa Masashi , Kapur Raj P. , Richardson James A. , Williams S. Clay , Clouthier David E. , Wit Damiane de , Emoto Noriaki , Hammer Robert E.
Published: April 2021

Recent gene targeting studies have revealed unexpected roles for endothelins in the development of neural crestderived tissues. Endothelin converting enzyme-1 (ECE-1) catalyzes the proteolytic activation of big endothelin-1 to endothelin-1(ET-1) in vitro. However, the importance of ECE-1 cleavage in the multiple endothelin pathways in vivo is unknown. Here we generated a targeted null mutation in the mouse ECE-1 gene. ECE-1−/− term embryos exhibited craniofacial and cardiac abnormalities virtually identical to the defects seen in ET-1 and endothelin A receptor (ETA)deficient embryos. Epidermal melanocytes as well as enteric neurons of the distal gut were also absent in ECE-1−/− embryos, reproducing the developmental phenotype seen in ET-3−/− and endothelin B receptor (ETB)−/− mice. Surprisingly, large amounts of mature ET-1 peptide are found in ECE-1−/− embryos, indicating that non-ECE-1 protease(s) can activate ET-1 at certain sites. However, these enzymes cannot produce sufficient mature endothelin at the locations crucial for normal embryonic development. These findings reveal that ECE-1 is a bona fide activating protease for both big ET-1 and big ET-3 in vivo, and that the cell-cell communication pathways represented by the ET-1/ECE-1/ETA axis and the ET-3/ECE-1/ETB axis are each involved in the development of distinct subsets of neural crest cell lineages. Mutations in ECE-1 may cause developmental defects in humans, such as Hirschsprung disease, velocardiofacial syndrome and related neurocristopathies.
Xenopus Smad8 acts downstream of BMP-4 to modulate its activity during vertebrate embryonic patterning
Authors: Nakayama Takuya , Snyder Mark A. , Grewal Savraj S. , Tsuneizumi Kazuhide , Tabata Tetsuya , Christian Jan L.
Published: April 2021

Bone morphogenetic proteins (BMPs) participate in the development of nearly all organs and tissues. BMP signaling is mediated by specific Smad proteins, Smad1 and/or Smad5, which undergo serine phosphorylation in response to BMP-receptor activation and are then translocated to the nucleus where they modulate transcription of target genes. We have identified a distantly related member of the Xenopus Smad family, Smad8, which lacks the C-terminal SSXS phosphorylation motif present in other Smads, and which appears to function in the BMP signaling pathway. During embryonic development, the spatial pattern of expression of Smad8 mirrors that of BMP-4. We show that an intact BMP signaling pathway is required for its expression. Overexpression of Smad8 in Xenopus embryos phenocopies the effect of blocking BMP- 4 signaling, leading to induction of a secondary axis on the ventral side of intact embryos and to direct neural induction in ectodermal explants. Furthermore, Smad8 can block BMP-4-mediated induction of ventral mesoderm- specific gene expression in ectodermal explants. Overexpression of Smad8 within dorsal cells, however, causes patterning defects that are distinct from those reported in BMP-4-deficient embryos, suggesting that Smad8 may interact with additional signaling pathways. Indeed, overexpression of Smad8 blocks expression of Xbra in whole animals, and partially blocks activin signaling in animal caps. In addition, Smad8 inhibits involution of mesodermal cells during gastrulation, a phenotype that is not observed following blockade of activin or BMPs in Xenopus. Together, these results are consistent with the hypothesis that Smad8 participates in a negative feedback loop in which BMP signaling induces the expression of Smad8, which then functions to negatively modulate the amplitude or duration of signaling downstream of BMPs and, possibly, downstream of other transforming growth factor-β (TGF-β) family ligands.
Auxin-induced developmental patterns in Brassica juncea embryos
Authors: Hadfi Katalin , Speth Volker , Neuhaus Gunther
Published: April 2021

To investigate the mechanism of auxin action during pattern formation in dicot embryos, we tested the effects of the natural auxin indole-3-acetic acid (IAA), the auxin transport inhibitor N-(1-naphthyl)thalamic acid (NPA) and the antiauxin p-chlorophenoxyisobutyric acid (PCIB). In vitro treatments of isolated zygotic Brassica juncea embryos with these substances led to a wide range of morphogenetic alterations. Treatment of globular embryos with exogenous auxin (10-40 µM) either completely inhibited morphogenesis, resulting in ball-shaped embryos, or caused the development of egg- and cucumber-shaped embryos, which only consisted of a shortened hypocotyl without any apical structures. Axis duplication was observed sometimes after inhibition of auxin transport in globular embryos, and led to the development of twin embryos. During the transition from globular to heart stage, changes in auxin distribution or activity frequently caused the development of either split-collar or collar-cotyledons. Antiauxin inhibited cotyledon growth, leading to embryos with single or no cotyledons, or inhibited the development of the hypocotyl and the radicle. Inhibition of auxin transport in transition embryos sometimes led to axis broadening, which resulted in the development of two radicles. The described changes in embryo shapes represent arrests in different auxin-regulated developmental steps and phenocopy some Arabidopsis morphogenetic mutants.