bluuebunny/crossref_metadata_2025 huggingface dataset:
The ALK-2 and ALK-4 activin receptors transduce distinct mesoderm-inducing signals during early Xenopus development but do not co-operate to establish thresholds
Authors: Armes N. A. , Smith J. C.
Published: April 2021

The TGFβ family member activin induces different meso-dermal cell types in a dose-dependent fashion in the Xenopus animal cap assay. High concentrations of activin induce dorsal and anterior cell types such as notochord and muscle, while low concentrations induce ventral and posterior tissues such as mesenchyme and mesothelium. In this paper we investigate whether this threshold phenomenon involves the differential effects of the two type I activin receptors ALK-2 and ALK-4. Injection of RNA encoding constitutively active forms of the receptors (here designated ALK-2* and ALK-4*) reveals that ALK-4* strongly induces the more posterior mesodermal marker Xbra and the dorsoanterior marker goosecoid in animal cap explants. Maximal levels of Xbra expression are attained using lower concentrations of RNA than are required for the strongest activation of goosecoid, and at the highest doses of ALK-4*, levels of Xbra transcription decrease, as is seen with high concentrations of activin. By contrast, the ALK-2* receptor activates Xbra but fails to induce goosecoid to significant levels. Analysis at later stages reveals that ALK-4* signalling induces the formation of a variety of mesodermal derivatives, including dorsal cell types, in a dose-dependent fashion, and that high levels also induce endoderm. By contrast, the ALK-2* receptor induces only ventral mesodermal markers. Consistent with these observations, ALK-4* is capable of inducing a secondary axis when injected into the ventral side of 32-cell stage embryos whilst ALK-2* cannot. Co-injection of RNAs encoding constitutively active forms of both receptors reveals that ventralising signals from ALK-2* antagonise the dorsal mesoderm-inducing signal derived from ALK-4*, suggesting that the two receptors use distinct and interfering signalling pathways. Together, these results show that although ALK-2* and ALK-4* transduce distinct signals, the threshold responses characteristic of activin cannot be due to interactions between these two pathways; rather, thresholds can be established by ALK-4* alone. Furthermore, the effects of ALK-2* signalling are at odds with it behaving as an activin receptor in the early Xenopus embryo.
LEAFY expression and flower initiation in Arabidopsis
Authors: Blázquez Miguel A. , Soowal Lara N. , Lee Ilha , Weigel Detlef
Published: April 2021

During the initial vegetative phase, the Arabidopsis shoot meristem produces leaves with associated lateral shoots at its flanks, while the later reproductive phase is characterized by the formation of flowers. The LEAFY gene is an important element of the transition from the vegetative to the reproductive phase, as LEAFY is both necessary and sufficient for the initiation of individual flowers. We have analyzed in detail the expression of LEAFY during the plant life cycle, and found that LEAFY is extensively expressed during the vegetative phase. In long days, Arabidopsis plants flower soon after germination, and this is paralleled by rapid upregulation of LEAFY. In short days, Arabidopsis plants flower several weeks later than in long days, but LEAFY expression increases gradually before flowering commences. Application of the plant hormone gibberellin, which hastens flowering in short days, enhances the gradual change in LEAFY expression observed in short days. Changes in LEAFY expression before the transition to flowering suggest that the time point of this transition is at least partly controlled by the levels of LEAFY activity that are prevalent at a given time of the life cycle. This assumption is borne out by the finding that increasing the copy number of endogenous LEAFY reduces the number of leaves produced before the first flower is formed. Thus, LEAFY combines properties of flowering-time and flower-meristem-identity genes, indicating that LEAFY is a direct link between the global process of floral induction and the regional events associated with the initiation of individual flowers.
Delta-Notch signalling and the patterning of sensory cell differentiation in the zebrafish ear: evidence from the mind bomb mutant
Authors: Haddon Catherine , Jiang Yun-Jin , Smithers Lucy , Lewis Julian
Published: April 2021

Mechanosensory hair cells in the sensory patches of the vertebrate ear are interspersed among supporting cells, forming a fine-grained pattern of alternating cell types. Analogies with Drosophila mechanosensory bristle development suggest that this pattern could be generated through lateral inhibition mediated by Notch signalling. In the zebrafish ear rudiment, homologues of Notch are widely expressed, while the Delta homologues deltaA, deltaB and deltaD, coding for Notch ligands, are expressed in small numbers of cells in regions where hair cells are soon to differentiate. This suggests that the delta-expressing cells are nascent hair cells, in agreement with findings for Delta1 in the chick. According to the lateral inhibition hypothesis, the nascent hair cells, by expressing Delta protein, would inhibit their neighbours from becoming hair cells, forcing them to be supporting cells instead. The zebrafish mind bomb mutant has abnormalities in the central nervous system, somites, and elsewhere, diagnostic of a failure of Delta-Notch signalling: in the CNS, it shows a neurogenic phenotype accompanied by misregulated delta gene expression. Similar misregulation of delta genes is seen in the ear, along with misregulation of a Serrate homologue, serrateB, coding for an alternative Notch ligand. Most dramatically, the sensory patches in the mind bomb ear consist solely of hair cells, which are produced in great excess and prematurely; at 36 hours post fertilization, there are more than ten times as many as normal, while supporting cells are absent. A twofold increase is seen in the number of otic neurons also. The findings are strong evidence that lateral inhibition mediated by Delta-Notch signalling controls the pattern of sensory cell differentiation in the ear.
Strain-dependent embryonic lethality in mice lacking the retinoblastoma-related p130 gene
Authors: LeCouter Jennifer E. , Kablar Boris , Whyte Peter F. M. , Ying Chuyan , Rudnicki Michael A.
Published: April 2021

The retinoblastoma-related p130 protein is a member of a conserved family, consisting of Rb, p107 and p130, which are believed to play important roles in cell-cycle control and cellular differentiation. We have generated a null mutation in p130 by gene targeting and crossed the null allele into Balb/cJ and C57BL/6J strains of mice. In an enriched Balb/cJ genetic background, p130−/− embryos displayed arrested growth and died between embryonic days 11 and 13. Histological analysis revealed varying degrees of disorganization in neural and dermamyotomal structures. Immunohistochemistry with antibody reactive with Islet-1 indicated markedly reduced numbers of neurons in the spinal cord and dorsal root ganglia. Immunohistochemistry with antibody reactive with desmin indicated a similar reduction in the number of differentiated myocytes in the myotome. The myocardium of mutant embryos was abnormally thin and resembled an earlier staged two-chambered heart consisting of the bulbus cordis and the ventricular chamber. TUNEL analysis indicated the presence of extensive apoptosis in various tissues including the neural tube, the brain, the dermomyotome, but not the heart. Immunohistochemistry with antibody reactive with PCNA revealed increased cellular proliferation in the neural tube and the brain, and decreased proliferation in the heart. The placentas of p130−/− embryos did not display elevated apoptosis and were indistinguishable from wild type suggesting that the phenotype was not due to placental failure. Following a single cross with the C57BL/6 mice, p130−/− animals were derived that were viable and fertile. These results indicate that p130 in a Balb/cJ genetic background plays an essential role that is required for normal development. Moreover, our experiments establish that second-site modifier genes exist that have an epistatic relationship with p130.
Formation of Rathke’s pouch requires dual induction from the diencephalon
Authors: Takuma Naoyuki , Sheng Hui Z. , Furuta Yasuhide , Ward Jerrold M. , Sharma Kamal , Hogan Brigid L. M. , Pfaff Samuel L. , Westphal Heiner , Kimura Shioko , Mahon Kathleen A.
Published: April 2021

Targeted disruption of the homeobox gene T/ebp (Nkx2.1, Ttf1, Titf1) in mice results in ablation of the pituitary. Paradoxically, while T/ebp is expressed in the ventral diencephalon during forebrain formation, it is not expressed in Rathke’s pouch or in the pituitary gland at any time of embryogenesis. Examination of pituitary development in the T/ebp homozygous null mutant embryos revealed that a pouch rudiment is initially formed but is eliminated by programmed cell death before formation of a definitive pouch. In the diencephalon of the mutant, Bmp4 expression is maintained, whereas Fgf8 expression is not detectable. These data and additional genetic and molecular observations suggest that Rathke’s pouch develops in a two-step process that requires at least two sequential inductive signals from the diencephalon. First, BMP4 is required for induction and formation of the pouch rudiment, a role confirmed by analysis of Bmp4 homozygous null mutant embryos. Second, FGF8 is necessary for activation of the key regulatory gene Lhx3 and subsequent development of the pouch rudiment into a definitive pouch. This study provides firm molecular genetic evidence that morphogenesis of the pituitary primordium is induced in vivo by signals from the adjacent diencephalon.
Dual function of Ras in Raf activation
Authors: Li Willis , Melnick Michael , Perrimon Norbert
Published: April 2021

The small guanine nucleotide binding protein p21Ras plays an important role in the activation of the Raf kinase. However, the precise mechanism by which Raf is activated remains unclear. It has been proposed that the sole function of p21Ras in Raf activation is to recruit Raf to the plasma membrane. We have used Drosophila embryos to examine the mechanism of Raf (Draf) activation in the complete absence of p21Ras (Ras1). We demonstrate that the role of Ras1 in Draf activation is not limited to the translocation of Draf to the membrane through a Ras1-Draf association. In addition, Ras1 is essential for the activation of an additional factor which in turn activates Draf.
A cAMP-phosphodiesterase controls PKA-dependent differentiation
Authors: Shaulsky Gad , Fuller Danny , Loomis William F.
Published: April 2021

A cAMP-specific phosphodiesterase was found that is stimulated by binding to the regulatory subunit of cAMP-dependent protein kinase, PKA-R, from either Dictyostelium or mammals. The phosphodiesterase is encoded by the regA gene of Dictyostelium, which was recovered in a mutant screen for strains that sporulate in the absence of signals from prestalk cells. The sequence of RegA predicts that it will function as a member of a two-component system. Genetic analyses indicate that inhibition of the phosphodiesterase results in an increase in the activity of PKA, which acts at a check point for terminal differentiation. Conserved components known to affect memory, learning and differentiation in flies and vertebrates suggest that a similar circuitry functions in higher eukaryotes.
Fibroblast growth factor receptor 2 (FGFR2)-mediated reciprocal regulation loop between FGF8 and FGF10 is essential for limb induction
Authors: Xu Xiaoling , Weinstein Michael , Li Cuiling , Naski Michael , Cohen Rick I. , Ornitz David M. , Leder Philip , Deng Chuxia
Published: April 2021

FGFR2 is a membrane-spanning tyrosine kinase that serves as a high affinity receptor for several members of the fibroblast growth factor (FGF) family. To explore functions of FGF/FGFR2 signals in development, we have mutated FGFR2 by deleting the entire immunoglobin-like domain III of the receptor. We showed that murine FGFR2 is essential for chorioallantoic fusion and placenta trophoblast cell proliferation. Fgfr2ΔgIII/ΔIgIII embryos displayed two distinct defects that resulted in failures in formation of a functional placenta. About one third of the mutants failed to form the chorioallantoic fusion junction and the remaining mutants did not have the labyrinthine portion of the placenta. Consequently, all mutants died at 10-11 days of gestation. Interestingly, Fgfr2ΔgIII/ΔIgIII embryos do not form limb buds. Consistent with this defect, the expression of Fgf8, an apical ectodermal factor, is absent in the mutant presumptive limb ectoderm, and the expression of Fgf10, a mesenchymally expressed limb bud initiator, is down regulated in the underlying mesoderm. These findings provide direct genetic evidence that FGF/FGFR2 signals are absolutely required for vertebrate limb induction and that an FGFR2 signal is essential for the reciprocal regulation loop between FGF8 and FGF10 during limb induction.
Control of somite patterning by Sonic hedgehog and its downstream signal response genes
Authors: Borycki Anne-Gaelle , Mendham Lori , Emerson Charles P.
Published: April 2021

In the avian embryo, previous work has demonstrated that the notochord provides inductive signals to activate myoD and pax1 regulatory genes, which are expressed in the dorsal and ventral somite cells that give rise to myotomal and sclerotomal lineages. Here, we present bead implantation and antisense inhibition experiments that show that Sonic hedgehog is both a sufficient and essential notochord signal molecule for myoD and pax1 activation in somites. Furthermore, we show that genes of the Sonic hedgehog signal response pathway, specifically patched, the Sonic hedgehog receptor, and gli and gli2/4, zinc-finger transcription factors, are activated in coordination with somite formation, establishing that Sonic hedgehog response genes play a regulatory role in coordinating the response of somites to the constitutive notochord Sonic hedgehog signal. Furthermore, the expression of patched, gli and gli2/4 is differentially patterned in the somite, providing mechanisms for differentially transducing the Sonic hedgehog signal to the myotomal and sclerotomal lineages. Finally, we show that the activation of gli2/4 is controlled by the process of somite formation and signals from the surface ectoderm, whereas upregulation of patched and activation of gli is controlled by the process of somite formation and a Sonic hedgehog signal. The Sonic hedgehog signal response genes, therefore, have important functions in regulating the initiation of the Sonic hedgehog response in newly forming somites and in regulating the patterned expression of myoD and pax1 in the myotomal and sclerotomal lineages following somite formation.
Localization of genes encoding egg modifiers of paternal genome function to mouse chromosomes one and two
Authors: Latham Keith E. , Sapienza Carmen
Published: April 2021

It is now well established that genomic imprinting effects in mammals require a combination of epigenetic modifications imposed during gametogenesis and additional modifications imposed after fertilization. The earliest post-fertilization modifications to be imposed on the genome are those thought to be mediated by factors in the egg cytoplasm. Strain-dependent differences in the actions of these egg modifiers in mice reveal an important potential for genetic variability in the imprinting process, and also provide valuable genetic systems with which to identify some of the factors that participate in imprinting. Previous studies documented a strain-dependent difference in the modification of paternal genome function between the C57BL/6 and DBA/2 mouse strains. This difference is revealed as a difference in developmental potential of androgenetic embryos produced with eggs from females of the two strains by nuclear transplantation. The specificity of the effect for the paternal genome is consistent with an effect on imprinted genes. The egg phenotype is largely independent of the genotype of the fertilizing sperm, and the C57BL/6 phenotype is dominant in reciprocal F1 hybrids. Genetic studies demonstrated that the difference in egg phenotypes between the two strains is most likely controlled by two independently segregating loci. We now report the results of experiments in which the egg phenotypes of the available BxD recombinant inbred mouse strains have been determined. The results of the analysis are consistent with the two locus model, and we have identified candidate chromosomal locations for the two loci. These data demonstrate clearly that differences in how the egg cytoplasm modifies the incoming paternal genome are indeed genetically determined, and vary accordingly.
Drosophila Goosecoid requires a conserved heptapeptide for repression of Paired-class homeoprotein activators
Authors: Mailhos Carolina , André Sylvain , Mollereau Bertrand , Goriely Anne , Hemmati-Brivanlou Ali , Desplan Claude
Published: April 2021

Goosecoid (Gsc) is a homeodomain protein expressed in the organizer region of vertebrate embryos. Its Drosophila homologue, D-Gsc, has been implicated in the formation of the Stomatogastric Nervous System. Although there are no apparent similarities between the phenotypes of mutations in the gsc gene in flies and mice, all known Gsc proteins can rescue dorsoanterior structures in ventralized Xenopus embryos. We describe how D-Gsc behaves as a transcriptional repressor in Drosophila cells, acting through specific palindromic HD binding sites (P3K). D-Gsc is a ‘passive repressor’ of activator homeoproteins binding to the same sites and an ‘active repressor’ of activators binding to distinct sites. In addition, D-Gsc is able to strongly repress transcription activated by Paired-class homeoproteins through P3K, via specific protein-protein interactions in what we define as ‘interactive repression’. This form of repression requires the short conserved GEH/eh-1 domain, also present in the Engrailed repressor. Although the GEH/eh-1 domain is necessary for rescue of UV-ventralized Xenopus embryos, it is dispensable for ectopic induction of Xlim-1 expression, demonstrating that this domain is not required for all Gsc functions in vivo. Interactive repression may represent specific interactions among Prd-class homeoproteins, several of which act early during development of invertebrate and vertebrate embryos.
Follistatin regulates the relative proportions of endocrine versus exocrine tissue during pancreatic development
Authors: Miralles Francisco , Czernichow Paul , Scharfmann Raphael
Published: April 2021

In this study, we have investigated the role of the embryonic mesenchyme in the development of the pancreas. We have compared the development in vitro of E12.5 rat pancreatic rudiments grown in the presence or absence of mesenchyme. When the E12.5 pancreatic epithelial rudiment is cultured in the presence of its surrounding mesenchyme, both morphogenesis and cytodifferentiation of the exocrine component of the pancreas are completely achieved, while only a few immature endocrine cells develop. The pancreatic rudiments grown in the absence of mesenchyme develop in a completely different way; the exocrine tissue develops poorly and fails to undergo acinar morphogenesis, while the endocrine tissue develops actively. Four times more insulin-positive cells develop after removal of the mesenchyme than in the cultures performed in the presence of mesenchyme. Moreover, the insulin-expressing cells developed in the mesenchyme-depleted rudiments appear mature since they do not coexpress glucagon, express the glucose transporter Glut-2 and express Rab3A, a molecule associated with the secretory granules. Moreover, these endocrine cells are able to associate and form true islets. Both the inductive effect of the mesenchyme on the proper development of the exocrine tissue and its repressive effect on the development of the endocrine cells are mediated by soluble factors. Follistatin, which is expressed by E12.5 pancreatic mesenchyme, can mimic both inductive and repressive effects of the mesenchyme. Follistatin could thus represent one of the mesenchymal factors required for the development of the exocrine tissue while exerting a repressive role on the differentiation of the endocrine cells.
Bone morphogenetic protein 8A plays a role in the maintenance of spermatogenesis and the integrity of the epididymis
Authors: Zhao Guang-Quan , Liaw Lucy , Hogan Brigid L. M.
Published: April 2021

The murine Bmp8a and Bmp8b genes are tightly linked on mouse chromosome 4 and have similar expression during reproduction. Previous studies have shown that targeted mutagenesis of Bmp8b causes male infertility due to germ cell degeneration. To investigate the function of Bmp8a, we have inactivated the gene by homologous recombination. Heterozygous and homozygous Bmp8a mutants reveal normal embryonic and postnatal development. Despite high levels of Bmp8a expression in the deciduum, homozygous mutant females have normal fertility, suggesting that the gene is not essential for female reproduction. Bmp8a and Bmp8b are expressed in similar patterns in male germ cells. Unlike homozygous Bmp8btm1 mutants, homozygous Bmp8atm1 males do not show obvious germ cell defects during the initiation of spermatogenesis. However, germ cell degeneration is observed in 47% of adult homozygous Bmp8atm1 males, establishing a role of Bmp8a in the maintenance of spermatogenesis. A small proportion of the mating homozygous Bmp8atm1 males also show degeneration of the epididymal epithelium, indicating a novel role for BMPs in the control of epididymal function.
Hepatocyte growth factor (HGF) acts as a mesenchyme-derived morphogenic factor during fetal lung development
Authors: Ohmichi Hidenori , Koshimizu Uichi , Matsumoto Kunio , Nakamura Toshikazu
Published: April 2021

Mesenchymal-epithelial tissue interactions are important for development of various organs, and in many cases, soluble signaling molecules may be involved in this interaction. Hepatocyte growth factor (HGF) is a mesenchyme-derived factor which has mitogenic, motogenic and morphogenic activities on various types of epithelial cells and is considered to be a possible mediator of epithelial-mesenchymal interaction during organogenesis and organ regeneration. In this study, we examined the role of HGF during lung development. In situ hybridization analysis showed HGF and the c-met/HGF receptor gene to be respectively expressed in mesenchyme and epithelium in the developing lung. In organ cultures, exogenously added HGF apparently stimulated branching morphogenesis of the fetal lung. In contrast, HGF translation arrest or neutralization assays resulted in clear inhibition of epithelial branching. These results suggest that HGF is a putative candidate for a mesenchyme-derived morphogen regulating lung organogenesis. We also found that HGF is involved in epithelial branching, in collaboration with fibroblast growth factor (FGF) family molecule(s). In mesenchyme-free culture, HGF alone did not induce epithelial morphogenesis, however, addition of both HGF and acidic FGF (aFGF) or keratinocyte growth factor (KGF), ligands for the KGF receptor, induced epithelial branching more extensively than that was observed in explants treated with aFGF or KGF alone. In addition, the simultaneous inhibition of HGF- and FGF- mediated signaling using neutralizing antibody and antisense oligo-DNA resulted in drastic impairment of epithelial growth and branching. Possible interactions between HGF and FGFs or other growth factors in lung development is given consideration.
Promoting notochord fate and repressing muscle development in zebrafish axial mesoderm
Authors: Amacher Sharon L. , Kimmel Charles B.
Published: April 2021

Cell fate decisions in early embryonic cells are controlled by interactions among developmental regulatory genes. Zebrafish floating head mutants lack a notochord; instead, muscle forms under the neural tube. As shown previously, axial mesoderm in floating head mutant gastrulae fails to maintain expression of notochord genes and instead expresses muscle genes. Zebrafish spadetail mutant gastrulae have a nearly opposite phenotype; notochord markers are expressed in a wider domain than in wild-type embryos and muscle marker expression is absent. We examined whether these two phenotypes revealed an antagonistic genetic interaction by constructing the double mutant. Muscle does not form in the spadetail;floating head double mutant midline, indicating that spadetail function is required for floating head mutant axial mesoderm to transfate to muscle. Instead, the midline of spadetail;floating head double mutants is greatly restored compared to that of floating head mutants; the floor plate is almost complete and an anterior notochord develops. In addition, we find that floating head mutant cells can make both anterior and posterior notochord when transplanted into a wild-type host, showing that enviromental signals can override the predisposition of floating head mutant midline cells to make muscle. Taken together, these results suggest that repression of spadetail function by floating head is critical to promote notochord fate and prevent midline muscle development, and that cells can be recruited to the notochord by environmental signals.
Disruption of primary imprinting during oocyte growth leads to the modified expression of imprinted genes during embryogenesis
Authors: Obata Yayoi , Kaneko-Ishino Tomoko , Koide Tsuyoshi , Takai Yasushi , Ueda Takayuki , Domeki Ikuo , Shiroishi Toshihiko , Ishino Fumitoshi , Kono Tomohiro
Published: April 2021

Parthenogenetic embryos, which contained one genome from a neonate-derived non-growing oocyte and the other from a fully grown oocyte, developed to day 13.5 of gestation in mice, 3 days longer than previously recorded for parthenogenetic development. To investigate the hypothesis that disruption of primary imprinting during oocyte growth leads to the modified expression of imprinted genes and this parthenogenetic phenotype, we have examined Peg1/Mest, Igf2, Peg3, Snrpn, H19, Igf2r and excess p57KIP2. We show that paternally expressed genes, Peg1/Mest, Peg3 and Snrpn, are expressed in the parthenotes, presumably due to a lack of maternal epigenetic modifications during oocyte growth. In contrast, the expression of Igf2, which is repressed in a competitive manner by transcription of the H19 gene, was very low. Furthermore, we show that the maternally expressed Igf2r and p57KIP2 genes were repressed in the alleles of the non-growing oocyte indicating maternal modifications during oocyte growth are necessary for its expression. Thus, our results show that primary imprinting during oocyte growth exhibits a crucial effect on both the expression and repression of maternal alleles during embryogenesis.
vasa is required for GURKEN accumulation in the oocyte, and is involved in oocyte differentiation and germline cyst development
Authors: Styhler Sylvia , Nakamura Akira , Swan Andrew , Suter Beat , Lasko Paul
Published: April 2021

The Drosophila gene vasa is required for pole plasm assembly and function, and also for completion of oogenesis. To investigate the role of vasa in oocyte development, we generated a new null mutation of vasa, which deletes the entire coding region. Analysis of vasa-null ovaries revealed that the gene is involved in the growth of germline cysts. In vasa-null ovaries, germaria are atrophied, and contain far fewer developing cysts than do wild-type germaria; a phenotype similar to, but less severe than, that of a null nanos allele. The null mutant also revealed roles for vasa in oocyte differentiation, anterior-posterior egg chamber patterning, and dorsal-ventral follicle patterning, in addition to its better-characterized functions in posterior embryonic patterning and pole cell specification. The anterior-posterior and dorsal-ventral patterning phenotypes resemble those observed in gurken mutants. vasa-null oocytes fail to efficiently accumulate many localized RNAs, such as Bicaudal-D, orb, oskar, and nanos, but still accumulate gurken RNA. However, GRK accumulation in the oocyte is severely reduced in the absence of vasa function, suggesting a function for VASA in activating gurken translation in wild-type ovaries.
Equivalence of the fly orthodenticle gene and the human OTX genes in embryonic brain development of Drosophila
Authors: Leuzinger Sandra , Hirth Frank , Gerlich Daniel , Acampora Dario , Simeone Antonio , Gehring Walter J. , Finkelstein Robert , Furukubo-Tokunaga Katsuo , Reichert Heinrich
Published: April 2021

Members of the orthodenticle gene family are essential for embryonic brain development in animals as diverse as insects and mammals. In Drosophila, mutational inactivation of the orthodenticle gene results in deletions in anterior parts of the embryonic brain and in defects in the ventral nerve cord. In the mouse, targeted elimination of Overexpression of either orthodenticle or the human OTX the homologous Otx2 or Otx1 genes causes defects in forebrain and/or midbrain development. To determine the morphogenetic properties and the extent of evolutionary conservation of the orthodenticle gene family in embryonic orthodenticle gene homologs indicates that these genes are brain development, genetic rescue experiments were carried out in Drosophila. Ubiquitous overexpression of the orthodenticle gene rescues both the brain defects and the role of the orthodenticle gene family in brain development. ventral nerve cord defects in orthodenticle mutant embryos; morphology and nervous system-speciﬁc gene expression are restored. Two different time windows exist for the rescue of the brain versus the ventral nerve cord. Ubiquitous overexpression of the human OTX1 or OTX2 genes also rescues the brain and ventral nerve cord phenotypes in orthodenticle mutant embryos; in the brain, the efficiency of morphological rescue is lower than that obtained with overexpression of orthodenticle. Overexpression of either orthodenticle or the human OTX gene homologs in the wild-type embryo results in ectopic neural structures. The rescue of highly complex brain structures in Drosophila by either fly or human orthodenticle gene homologs indicates that these genes are interchangeable between vertebrates and invertebrates and provides further evidence for an evolutionarily conserved role of the orthodenticle gene family in brain development.
Discrete spatial and temporal cis-acting elements regulate transcription of the Arabidopsis floral homeotic gene APETALA3
Authors: Hill Theresa A. , Day Christopher D. , Zondlo Susan C. , Thackeray Andrea G. , Irish Vivian F.
Published: April 2021

The APETALA3 floral homeotic gene is required for petal and stamen development in Arabidopsis. APETALA3 transcripts ﬁrst detected in a meristematic region that will give rise to the petal and stamen primordia, and expression is maintained in this region during subsequent development of these organs. To dissect how the APETALA3 gene is expressed in this spatially and temporally restricted domain, various APETALA3 promoter fragments were fused to the uidA reporter gene encoding β -glucuronidase and assayed for the resulting patterns of expression in transgenic Arabidopsis plants. Based on these promoter analyses, we deﬁned cis-acting elements required for distinct phases of APETALA3 expression, as well as for petal-speciﬁc and stamen-speciﬁc expression. By crossing the petal-speciﬁc construct into different mutant backgrounds, we have shown that several floral genes, including APETALA3, PISTILLATA, UNUSUAL are FLORAL ORGANS, and APETALA1, encode trans-acting factors required for second-whorl-speciﬁc APETALA3 expression. We have also shown that the products of the APETALA1, APETALA3, PISTILLATA and AGAMOUS genes bind to several conserved sequence motifs within the APETALA3 promoter. We present a model whereby spatially and temporally restricted APETALA3 transcription is controlled via interactions between proteins binding to different domains of the APETALA3 promoter.
Oocyte polarity depends on regulation of gurken by Vasa
Authors: Tomancak Pavel , Guichet Antoine , Zavorszky Peter , Ephrussi Anne
Published: April 2021

Vasa, a DEAD box mRNA helicase similar to eIF4A, is involved in pole plasm assembly in the Drosophila oocyte and appears to regulate translation of oskar and nanos mRNAs. However, several vasa alleles exhibit a wide range of early oogenesis phenotypes. Here we report a detailed analysis of Vasa function during early oogenesis using novel as well as previously identified hypomorphic vasa alleles. We find that vasa is required for the establishment of both anterior-posterior and dorsal-ventral polarity of the oocyte. The polarity defects of vasa mutants appear to be caused by a reduction in the amount of Gurken protein at stages of oogenesis critical for the establishment of polarity. Vasa is required for translation of gurken mRNA during early oogenesis and for achieving wild-type levels of gurken mRNA expression later in oogenesis. A variety of early oogenesis phenotypes observed in vasa ovaries, which cannot be attributed to the defect in gurken expression, suggest that vasa also affects expression of other mRNAs.
A genetic screen for modiﬁers of Drosophila decapentaplegic signaling identiﬁes mutations in punt, Mothers against dpp and the BMP-7 homologue, 60A
Authors: Chen Yijing , Riese Matthew J. , Killinger Mark A. , Hoffmann F. Michael
Published: April 2021

decapentaplegic (dpp) is a Transforming Growth Factor beta (TGF-β) -related growth factor that controls multiple developmental processes in Drosophila. To identify components involved in dpp signaling, we carried out a genetic screen for dominant enhancer mutations of a hypomorphic allele of thick veins (tkv), a type I receptor for dpp. We recovered new alleles of tkv, punt, Mothers against dpp (Mad) and Medea (Med), all of which are known to mediate dpp signaling. We also recovered mutations in the 60A gene which encodes another TGF-β -related factor in Drosophila. DNA sequence analysis established that all three 60A alleles were nonsense mutations in the prodomain of the 60A polypeptide. These mutations in 60A caused defects in midgut morphogenesis and fat body differentiation. We present evidence that when dpp signaling is compromised, lowering the level of 60A impairs several dpp-dependent developmental processes examined, including the patterning of the visceral mesoderm, the embryonic ectoderm and the imaginal discs. These results provide the ﬁrst in vivo evidence for the involvement of 60A in the dpp pathway. We propose that 60A activity is required to maintain optimal signaling capacity of the dpp pathway, possibly by forming biologically active heterodimers with Dpp proteins.
Nerve-muscle interactions during flight muscle development in Drosophila
Authors: Fernandes Joyce J. , Keshishian Haig
Published: April 2021

During Drosophila pupal metamorphosis, the motoneurons and muscles differentiate synchronously, providing an opportunity for extensive intercellular regulation during synapse formation. We examined the existence of such interactions by developmentally delaying or permanently eliminating synaptic partners during the formation of indirect flight muscles. When we experimentally delayed muscle development, we found that although adult-specific primary motoneuron branching still occurred, the higher order (synaptic) branching was suspended until the delayed muscle fibers reached a favourable developmental state. In reciprocal experiments we found that denervation caused a decrease in the myoblast pool. Furthermore, the formation of certain muscle fibers (dorsoventral muscles) was specifically blocked. Exceptions were the adult muscles that use larval muscle fibers as myoblast fusion targets (dorsal longitudinal muscles). However, when these muscles were experimentally compelled to develop without their larval precursors, they showed an absolute dependence on the motoneurons for their formation. These data show that the size of the myoblast pool and early events in fiber formation depend on the presence of the nerve, and that,
Analysis of molecular marker expression reveals neuronal homology in distantly related arthropods
Authors: Duman-Scheel Molly , Patel Nipam H.
Published: April 2021

Morphological studies suggest that insects and crustaceans of the Class Malacostraca (such as crayfish) share a set of homologous neurons. However, expression of molecular markers in these neurons has not been investigated, and the homology of insect and malacostracan neuroblasts, the neural stem cells that produce these neurons, has been questioned. Furthermore, it is not known whether crustaceans of the Class Branchiopoda (such as brine shrimp) or arthropods of the Order Collembola (springtails) possess neurons that are homologous to those of other arthropods. Assaying expression of molecular markers in the developing nervous systems of various arthropods could resolve some of these issues. Here, we examine expression of Even-skipped and Engrailed, two transcription factors that serve as insect embryonic CNS markers, across a number of arthropod species. This molecular analysis allows us to verify the homology of previously identified malacostracan neurons and to identify additional homologous neurons in malacostracans, collembolans and branchiopods. Engrailed expression in the neural stem cells of a number of crustaceans was also found to be conserved. We conclude that despite their distant phylogenetic relationships and divergent mechanisms of neurogenesis, insects, malacostracans, branchiopods and collembolans share many common CNS components.
Analysis of an even-skipped rescue transgene reveals both composite and discrete neuronal and early blastoderm enhancers, and multi-stripe positioning by gap gene repressor gradients*
Authors: Fujioka Miki , Emi-Sarker Yukiko , Yusibova Galina L. , Goto Tadaatsu , Jaynes James B.
Published: April 2021

The entire functional even-skipped locus of Drosophila melanogaster is contained within a 16 kilobase region. As a transgene, this region is capable of rescuing even-skipped mutant flies to fertile adulthood. Detailed analysis of the 7.7 kb of regulatory DNA 3′ of the transcription unit revealed ten novel, independently regulated patterns. Most of these patterns are driven by non-overlapping regulatory elements, including ones for syncytial blastoderm stage stripes 1 and 5, while a single element specifies both stripes 4 and 6. Expression analysis in gap gene mutants showed that stripe 5 is restricted anteriorly by Krüppel and posteriorly by giant, the same repressors that regulate stripe 2. Consistent with the coregulation of stripes 4 and 6 by a single cis-element, both the anterior border of stripe 4 and the posterior border of stripe 6 are set by zygotic hunchback, and the region between the two stripes is ‘carved out’ by knirps. Thus the boundaries of stripes 4 and 6 are set through negative regulation by the same gap gene domains that regulate stripes 3 and 7 (Small, S., Blair, A. and Levine, M. (1996) Dev. Biol. 175, 314-24), but at different concentrations. The 3′ region also contains a single element for neurogenic expression in ganglion mother cells 4-2a and 1-1a, and neurons derived from them (RP2, a/pCC), suggesting common regulators in these lineages. In contrast, separable elements were found for expression in EL neurons, U/CQ neurons and the mesoderm. The even-skipped 3′ untranslated region is required to maintain late stage protein expression in RP2 and a/pCC neurons, and appears to affect protein levels rather than mRNA levels. Additionally, a strong pairing-sensitive repression element was localized to the 3′ end of the locus, but was not found to contribute to efficient functional rescue.
A retinoic acid-inducible transgenic marker of sino-atrial development in the mouse heart
Authors: Xavier-Neto José , Neville Craig M. , Shapiro Michael D. , Houghton Leslie , Wang Gang Feng , Nikovits William , Stockdale Frank E. , Rosenthal Nadia
Published: April 2021

To study the specification of inflow structures in the heart we generated transgenic animals harboring the human alkaline phosphatase (HAP) gene driven by the proximal 840 bp of a quail SMyHC3 promoter. In transgenic mice, the SMyHC3-HAP reporter was expressed in posterior heart precursors at 8.25 dpc, in sinus venosa and in the atrium at 8.5 and 9.0 dpc, and in the atria from 10.5 dpc onwards. SMyHC3-HAP transgene expression overlapped synthesis and endogenous response to retinoic acid (RA) in the heart, as determined by antibodies directed against a key RA synthetic enzyme and by staining of RAREhsplacZ transgenic animals. A single pulse of all-trans RA administered to pregnant mice at 7.5, but not after 8.5, dpc induced cardiac dismorphology, ranging from complete absence of outflow tract and ventricles to hearts with reduced ventricles expressing both SMyHC3-HAP and ventricular markers. Blockade of RA synthesis with disulfiram inhibited RA-induced transcription and produced hearts lacking the atrial chamber. This study defines a novel marker for atrial-restricted transcription in the developing mouse heart. It also suggests that atrial-specific gene expression is controlled by localized synthesis of RA, and that exclusion of RA from ventricular precursors is essential for correct specification of the ventricles.
Zebrafish tbx-c functions during formation of midline structures
Authors: Dheen Thameem , Sleptsova-Friedrich Inna , Xu Yanfei , Clark Matthew , Lehrach Hans , Gong Zhiyuan , Korzh Vladimir
Published: April 2021

Several genes containing the conserved T-box region in invertebrates and vertebrates have been reported recently. Here, we describe three novel members of the T-box gene family in zebrafish. One of these genes, tbx-c, is studied in detail. It is expressed in the axial mesoderm, notably, in the notochordal precursor cells immediately before formation of the notochord and in the chordoneural hinge of the tail bud, after the notochord is formed. In addition, its expression is detected in the ventral forebrain, sensory neurons, fin buds and excretory system. The expression pattern of tbx-c differs from that of the other two related genes, tbx-a and tbx-b.

The developmental role of tbx-c has been analysed by overexpression of the full-length tbx-c mRNA and a truncated form of tbx-c mRNA, which encodes the dominant-negative Tbx-c. Overexpression of tbx-c causes expansion of the midline mesoderm and formation of ectopic midline structures at the expense of lateral mesodermal cells. In dominant-negative experiments, the midline mesoderm is reduced with the expansion of lateral mesoderm to the midline. These results suggest that tbx-c plays a role in formation of the midline mesoderm, particularly, the notochord. Moreover, modulation of tbx-c activity alters the development of primary motor neurons.

Results of in vitro analysis in zebrafish animal caps suggest that tbx-c acts downstream of early mesodermal inducers (activin and ntl) and reveal an autoregulatory feedback loop between ntl and tbx-c. These data and analysis of midline (ntl−/− and flh−/−) and lateral mesoderm (spt−/−) mutants suggest that tbx-c may function during formation of the notochord.
Specification of distinct motor neuron identities by the singular activities of individual Hox genes
Authors: Jungbluth Stefan , Bell Esther , Lumsden Andrew
Published: April 2021

Hox genes have been implicated in specifying positional values along the anteroposterior axis of the caudal central nervous system, but their nested and overlapping expression has complicated the understanding of how they confer specific neural identity. We have employed a direct gain-of-function approach using retroviral vectors to misexpress Hoxa2 and Hoxb1 outside of the normal Hox expression domains, thereby avoiding complications resulting from possible interactions with endogenous Hox genes. Misexpression of either Hoxa2 or Hoxb1 in the anteriormost hindbrain (rhombomere1, r1) leads to the generation of motor neurons in this territory, even though it is normally devoid of this cell type. These ectopic neurons have the specific identity of branchiomotor neurons and, in the case of Hoxb1-induced cells, their axons leave the hindbrain either by fasciculating with the resident cranial motor axons at isthmic (trochlear) or r2 (trigeminal) levels of the axis or via novel ectopic exit points in r1. Next, we have attempted to identify the precise branchiomotor subtypes that are generated after misexpression and our results suggest that the ectopic motor neurons generated following Hoxa2 misexpression are trigeminal-like, while those generated following Hoxb1 misexpression are facial-like. Our data demonstrate, therefore, that at least to a certain extent and for certain cell types, the singular activities of individual Hox genes (compared to a combinatorial mode of action, for example) are sufficient to impose on neuronal precursor cells the competence to generate distinctly specified cell types. Moreover, as these particular motor neuron subtypes are normally generated in the most anterior domains of Hoxa2 and Hoxb1 expression, respectively, our data support the idea that the main site of individual Hox gene action is in the anteriormost subdomain of their expression, consistent with the phenomenon of posterior dominance.
Cell division genes promote asymmetric interaction between Numb and Notch in the Drosophila CNS
Authors: Wai Philip , Truong Bidong , Bhat Krishna Moorthi
Published: April 2021

Cell intrinsic and cell extrinsic factors mediate asymmetric cell divisions during neurogenesis in the Drosophila embryo. In the NB4-2→GMC-1→RP2/sib lineage, one of the well-studied neuronal lineages in the ventral nerve cord, the Notch (N) signaling interacts with the asymmetrically localized Numb (Nb) to specify sibling neuronal fates to daughter cells of GMC-1. In this current study, we have investigated asymmetric cell fate specifications by N and Nb in the context of cell cycle. We have used loss-of-function mutations in N and nb, cell division mutants cyclinA (cycA), regulator of cyclin A1 (rca1) and string/cdc25 phosphatase (stg), and the microtubule destabilizing agent, nocodazole, to investigate this issue. We report that the loss of cycA, rca1 or stg leads to a block in the division of GMC-1, however, this GMC-1 exclusively adopts an RP2 identity. While the loss of N leads to the specification of RP2 fates to both progeny of GMC-1 and loss of nb results in the specification of sib fates to these daughter cells, the GMC-1 in the double mutant between nb and cycA assumes a sib fate. These epistasis results indicate that both N and nb function downstream of cell division genes and that progression through cell cycle is required for the asymmetric localization of Nb. In the absence of entry to metaphase, the Nb protein prevents the N signaling from specifying sib fate to the RP2/sib precursor. These results are also consistent with our finding that the sib cell is specified as RP2 in N; nb double mutants. Finally, our results show that nocodazole-arrested GMC-1 in wild-type embryos randomly assumes either an RP2 fate or a sib fate. This suggests that microtubules are involved in mediating the antagonistic interaction between Nb and N during RP2 and sib fate specification.