The Experts below are selected from a list of 213 Experts worldwide ranked by ideXlab platform
Linda Z. Holland - One of the best experts on this subject based on the ideXlab platform.
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nodal and hedgehog synergize in gill slit formation during development of the cephalochordate branchiostoma floridae
Development, 2018Co-Authors: Demian Koop, Linda Z. HollandAbstract:ABSTRACT The larval pharynx of the cephalochordate Branchiostoma (amphioxus) is asymmetrical. The mouth is on the left, and endostyle and gill slits are on the right. At the Neurula, Nodal and Hedgehog (Hh) expression becomes restricted to the left. To dissect their respective roles in gill slit formation, we inhibited each pathway separately for 20 min at intervals during the Neurula stage, before gill slits penetrate, and monitored the effects on morphology and expression of pharyngeal markers. The results pinpoint the short interval spanning the gastrula/Neurula transition as the critical period for specification and positioning of future gill slits. Thus, reduced Nodal signaling shifts the gill slits ventrally, skews the pharyngeal domains of Hh, Pax1/9, Pax2/5/8, Six1/2 and IrxC towards the left, and reduces Hh and Tbx1/10 expression in endoderm and mesoderm, respectively. Nodal auto-regulates. Decreased Hh signaling does not affect gill slit positions or Hh or Nodal expression, but it does reduce the domain of Gli, the Hh target, in the pharyngeal endoderm. Thus, during the Neurula stage, Nodal and Hh cooperate in gill slit development – Hh mediates gill slit formation and Nodal establishes their left-right position.
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developmental expression of the three iroquois genes of amphioxus bfirxa bfirxb and bfirxc with special attention to the gastrula organizer and anteroposterior boundaries in the central nervous system
Gene Expression Patterns, 2009Co-Authors: Stacy L Kaltenbach, Linda Z. Holland, Nicholas D. Holland, Demian KoopAbstract:Abstract Here we describe the developmental expression of the three iroquois genes ( BfIrxA , BfIrxB , and BfIrxC ) of amphioxus. BfIrxB transcription is first detected at the gastrula stage in mesendoderm just within the dorsal lip of the blastopore (a probable homolog of Spemann’s organizer) and in ectoderm. In early Neurulae, expression begins in presumptive pharyngeal endoderm, somitic mesoderm, and neural plate. Mid-Neurulae express BfIrxB throughout the hindbrain, posterior somites, pharyngeal endoderm, and notochord. In early larvae, expression is largely downregulated in the nerve cord, somites and notochord, but remains strong in the pharyngeal endoderm associated with the forming gill slits; also, a late expression domain appears in the ciliary tuft ectoderm. BfIrxA and BfIrxC , are not as widely expressed as BfIrxB . Both are first expressed in the presumptive hindbrain and presumptive pharyngeal endoderm at the early Neurula stages. In the mid-Neurula, additional expression domains appear in the extremities of the notochord. Neural expression is downregulated by late Neurula. In the early larva, expression is chiefly limited to pharyngeal endoderm associated with the forming gill slits, excepting a small new domain of BfIrxC (not BfIrxA ) expression in the ciliary tuft ectoderm. In comparison to developing vertebrates, embryos and larvae of amphioxus express iroquois genes in fewer tissues. Thus, iroquois genes of the proximate ancestor of the vertebrates evidently assumed numerous new roles during vertebrate evolution, including the division of the central nervous system into several sub-regions along its anteroposterior axis.
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Stage- and tissue-specific patterns of cell division in embryonic and larval tissues of amphioxus during normal development.
Evolution & development, 2006Co-Authors: Nicholas D. Holland, Linda Z. HollandAbstract:SUMMARY The distribution of dividing cells is described for embryos and larvae of amphioxus (Branchiostoma floridae) pulse labeled with bromodeoxyuridine. Because cell division is assessed for all of the developing tissues, this is the first comprehensive study of developmental cell proliferation for an animal lacking a stereotyped cell lineage. In amphioxus, cell divisions are virtually synchronous during cleavage, but become asynchronous at the blastula stage. Starting at the Neurula stage, after the origin of the mesoderm, the proportion of dividing cells progressively declines in the somitic mesoderm and notochord. Other tissues, however, deviate from this pattern. For example, in the mid-Neurula, there is a brief, intense burst of mitosis at the anterior end of the neural plate. Also, from the Neurula through the early larval stage, all of the ectoderm cells cease dividing and develop cilia that propel the animal through the water; subsequently, in the epidermis of later larvae, mitosis resumes and the proportion of ciliated cells declines as muscular undulation gradually replaces ciliation for swimming. Finally, in the early larvae, there is a terminal arrest of cell division in three cell types that differentiate early to participate in feeding as soon as the mouth opens—namely the ciliated pharyngeal cells that produce the feeding current and the secretory cells of the club-shaped gland and endostyle that export food-trapping mucus into the pharynx. In sum, these stage- and tissue-specific changes in cell proliferation intensity illustrate how the requirements of embryonic and larval natural history can shape developmental programs.
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Differential mesodermal expression of two amphioxus MyoD family members (AmphiMRF1 and AmphiMRF2)
Gene expression patterns : GEP, 2003Co-Authors: Michael Schubert, Linda Z. Holland, Daniel Meulemans, Marianne Bronner-fraser, Nicholas D. HollandAbstract:To explore the evolution of myogenic regulatory factors in chordates, we isolated two MyoD family genes (AmphiMRF1 and AmphiMRF2) from amphioxus. AmphiMRF1 is first expressed at the late gastrula in the paraxial mesoderm. As the first somites form, expression is restricted to their myotomal region. In the early larva, expression is strongest in the most anterior and most posterior somites. AmphiMRF2 transcription begins at mid/late gastrula in the paraxial mesoderm, but never spreads into its most anterior region. Through much of the Neurula stage, AmphiMRF2 expression is strong in the myotomal region of all somites except the most anterior pair; by late Neurula expression is downregulated except in the most posterior somites forming just rostral to the tail bud. These two MRF genes of amphioxus have partly overlapping patterns of mesodermal expression and evidently duplicated independent of the diversification of the vertebrate MRF family.
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An amphioxus nodal gene (AmphiNodal ) with early symmetrical expression in the organizer and mesoderm and later asymmetrical expression associated with left–right axis formation
Evolution and Development, 2002Co-Authors: Linda Z. Holland, Nicholas D. HollandAbstract:The full-length sequence and zygotic expression of an amphioxus nodal gene are described. Expression is first detected in the early gastrula just within the dorsal lip of the blastopore in a region of hypoblast that is probably comparable with the vertebrate Spemann's organizer. In the late gastrula and early Neurula, expression remains bilaterally symmetrical, limited to paraxial mesoderm and immediately overlying regions of the neural plate. Later in the Neurula stage, all neural expression disappears, and mesodermal expression disappears from the right side. All along the left side of the Neurula, mesodermal expression spreads into the left side of the gut endoderm. Soon thereafter, all expression is down-regulated except near the anterior and posterior ends of the animal, where transcripts are still found in the mesoderm and endoderm on the left side. At this time, expression also begins in the ectoderm on the left side of the head, in the region where the mouth later forms. These results suggest that amphioxus and vertebrate nodal genes play evolutionarily conserved roles in establishing Spemann's organizer, patterning the mesoderm rostrocaudally and setting up the asymmetrical left-right axis of the body.
Ryuji Toyoizumi - One of the best experts on this subject based on the ideXlab platform.
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Subtilisin-like proprotein convertase activity is necessary for left–right axis determination in Xenopus Neurula embryos
Development Genes and Evolution, 2006Co-Authors: Ryuji Toyoizumi, Shigeo Takeuchi, Kazue MogiAbstract:Signaling by members of TGF-β superfamily requires the activity of a family of site-specific endopeptidases, known as Subtilisin-like proprotein convertases (SPCs), which cleave these ligands into mature, active forms. To explore the role of SPCs in lateral plate mesoderm (LPM) differentiation in Xenopus , two SPC inhibitors, decanoyl-Arg-Val-Lys-Arg-chloromethylketone (Dec-RVKR-CMK) and hexa-arginine, were injected into the left and right LPM of Xenopus Neurulae. Left-side injection caused heart-specific left–right reversal, and this phenotype was rescued by co-injection of mature Nodal protein. In contrast, right-side injection caused left–right reversal of both the heart and gut. Tailbud embryos were less sensitive to SPC inhibitors than Neurula embryos. Injection of inhibitors into either side of Neurula embryos completely abolished expression of the left-LPM-specific genes, Xnr-1 , antivin , and pitx2 . SPC1 enzyme (Furin) was injected into the left or right LPM of mid-Neurula embryos to determine the effect of enhancing SPC activity. Left-side injection of SPC1 did not cause a significant left–right reversal of the internal organs. However, right-side injection of SPC1 strongly induced the expression of Xnr-1 and pitx2 in the right LPM, and caused 100% left–right reversal of both the heart and gut. These results suggest that moderate level of SPC activity in the right LPM of the Neurulae is necessary for proper left–right specification. Taken together, SPC enzymatic activity must be present in both LPMs for expression of the left-handed genes and left–right axis determination of the heart and gut in Xenopus embryos.
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Subtilisin-like proprotein convertase activity is necessary for left-right axis determination in Xenopus Neurula embryos.
Development genes and evolution, 2006Co-Authors: Ryuji Toyoizumi, Shigeo Takeuchi, Kazue MogiAbstract:Signaling by members of TGF-β superfamily requires the activity of a family of site-specific endopeptidases, known as Subtilisin-like proprotein convertases (SPCs), which cleave these ligands into mature, active forms. To explore the role of SPCs in lateral plate mesoderm (LPM) differentiation in Xenopus, two SPC inhibitors, decanoyl-Arg-Val-Lys-Arg-chloromethylketone (Dec-RVKR-CMK) and hexa-arginine, were injected into the left and right LPM of Xenopus Neurulae. Left-side injection caused heart-specific left–right reversal, and this phenotype was rescued by co-injection of mature Nodal protein. In contrast, right-side injection caused left–right reversal of both the heart and gut. Tailbud embryos were less sensitive to SPC inhibitors than Neurula embryos. Injection of inhibitors into either side of Neurula embryos completely abolished expression of the left-LPM-specific genes, Xnr-1, antivin, and pitx2. SPC1 enzyme (Furin) was injected into the left or right LPM of mid-Neurula embryos to determine the effect of enhancing SPC activity. Left-side injection of SPC1 did not cause a significant left–right reversal of the internal organs. However, right-side injection of SPC1 strongly induced the expression of Xnr-1 and pitx2 in the right LPM, and caused 100% left–right reversal of both the heart and gut. These results suggest that moderate level of SPC activity in the right LPM of the Neurulae is necessary for proper left–right specification. Taken together, SPC enzymatic activity must be present in both LPMs for expression of the left-handed genes and left–right axis determination of the heart and gut in Xenopus embryos.
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Xenopus Neurula left-right asymmetry is respeficied by microinjecting TGF-beta5 protein.
The International journal of developmental biology, 2003Co-Authors: Kazue Mogi, Shigeo Takeuchi, Madoka Goto, Eri Ohno, Yoshitaka Azumi, Ryuji ToyoizumiAbstract:A variety of TGF-beta-related ligands regulate the left-right asymmetry of vertebrates but the involvement of TGF-betas in left-right specification has not been reported. We assessed whether TGF-beta signaling is involved in the left-right specification of Xenopus post-gastrula embryos by microinjecting Xenopus TGF-beta5 protein into the left or right flank of Neurula-tailbud embryos. Injection on the right side of Neurulae caused left-right reversal of the internal organs in 93% of the embryos, while injection on the left side caused less than 5% left-right reversal. Expression of Xenopus nodal related-1 (Xnr-1 ), Xenopus antivin and Xenopus Pitx2, which are normally expressed on the left, was unaltered by the left-side injection. In contrast, right-side injection into Neurulae induced the expression of these genes predominantly on the right side. Right-side injection into tailbud embryos caused bilateral expression of these handed genes. Time course analysis of asymmetric gene expression revealed that Xnr-1 could be induced by TGF-beta5 at late Neurula stage, while antivin and Pitx2 could be induced by TGF-beta5 at the latertail bud stage. Injection of the antisense morpholino oligonucleotide against Xenopus TGF-beta5 into the left dorsal blastomere inhibited the normal left-handed expression of Xnr-1 and Pitx2, and caused the organ reversal in the injected embryos. These results suggest that normal left-right balance of endogenous TGF-beta5 signaling in the Neurula embryo may be needed to determine the laterality of the asymmetric genes and to generate the correct left-right axis.
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More than 95% reversal of left-right axis induced by right-sided hypodermic microinjection of activin into Xenopus Neurula embryos.
Developmental biology, 2000Co-Authors: Ryuji Toyoizumi, Kazue Mogi, Shigeo TakeuchiAbstract:Abstract In recent years, genes that show left–right (L-R) asymmetric expression patterns have been identified one after another in vertebrate gastrula–Neurula embryos. However, we still have little information about when the irreversible L-R specification is established in vertebrate embryos. In this report, we show that almost 100% of the embryos develop to be L-R-inverted larvae after microinjection of activin molecules into the right lateral hypodermic space of Xenopus Neurula embryos. After right-side injection of 10–250 pg activin protein, both early Neurulae just after gastrulation movement (stage 13–14) and late Neurulae just before neural tube closure (stage 17–18) showed almost 100% reversal of the heart and gut L-R axes. At higher doses of activin, more than 90% of the L-R-inverted embryos showed L-R reversal of both heart and gut. The survival ratio of the right-injected 4-day embryos was 90% on average. In the left-injected embryos, the occurrence of L-R inversion was less than 2% as observed in normal untreated siblings (1.7%). When the same amount of activin (1–50 pg) was microinjected into both sides of Neurula embryos, the incidence of L-R inversion was reduced to 58%. The injection of activin along the dorsal midline in the trunk region also randomized the visceral L-R axis. Injection of activin into the right side changed normal left-handed expression of Xnr-1 to right-handed or bilateral expression. In contrast, left-handed expression of Pitx2 was switched to the right side by right activin injection. This is the first report of a method that achieves complete inversion of the visceral L-R axis by treatment of embryos at the Neurula stage. Activin not only acts on the Neurulae to cancel the original L-R specification up to the late Neurula stage, but also rebuilds a new L-R axis whose left side coincides with the injection side. It is suggested that the left and right halves of Neurulae have equal potential for L-R differentiation.
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more than 95 reversal of left right axis induced by right sided hypodermic microinjection of activin into xenopus Neurula embryos
Developmental Biology, 2000Co-Authors: Ryuji Toyoizumi, Kazue Mogi, Shigeo TakeuchiAbstract:Abstract In recent years, genes that show left–right (L-R) asymmetric expression patterns have been identified one after another in vertebrate gastrula–Neurula embryos. However, we still have little information about when the irreversible L-R specification is established in vertebrate embryos. In this report, we show that almost 100% of the embryos develop to be L-R-inverted larvae after microinjection of activin molecules into the right lateral hypodermic space of Xenopus Neurula embryos. After right-side injection of 10–250 pg activin protein, both early Neurulae just after gastrulation movement (stage 13–14) and late Neurulae just before neural tube closure (stage 17–18) showed almost 100% reversal of the heart and gut L-R axes. At higher doses of activin, more than 90% of the L-R-inverted embryos showed L-R reversal of both heart and gut. The survival ratio of the right-injected 4-day embryos was 90% on average. In the left-injected embryos, the occurrence of L-R inversion was less than 2% as observed in normal untreated siblings (1.7%). When the same amount of activin (1–50 pg) was microinjected into both sides of Neurula embryos, the incidence of L-R inversion was reduced to 58%. The injection of activin along the dorsal midline in the trunk region also randomized the visceral L-R axis. Injection of activin into the right side changed normal left-handed expression of Xnr-1 to right-handed or bilateral expression. In contrast, left-handed expression of Pitx2 was switched to the right side by right activin injection. This is the first report of a method that achieves complete inversion of the visceral L-R axis by treatment of embryos at the Neurula stage. Activin not only acts on the Neurulae to cancel the original L-R specification up to the late Neurula stage, but also rebuilds a new L-R axis whose left side coincides with the injection side. It is suggested that the left and right halves of Neurulae have equal potential for L-R differentiation.
Hiroki Nishida - One of the best experts on this subject based on the ideXlab platform.
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vitelline membrane proteins promote left sided nodal expression after Neurula rotation in the ascidian halocynthia roretzi
Developmental Biology, 2019Co-Authors: Yuka Tanaka, Shiori Yamada, Samantha L Connop, Noritaka Hashii, Hitoshi Sawada, Yu Shih, Hiroki NishidaAbstract:Abstract Stereotyped left–right asymmetry both in external and internal organization is found in various animals. Left-right symmetry is broken by the Neurula rotation in the ascidian, Halocynthia roretzi. Neurula embryos rotate along the anterior–posterior axis in a counterclockwise direction, and the rotation stops when the left side of the embryo is oriented downwards, resulting in contact of the left-side epidermis with the vitelline membrane at the bottom of perivitelline space. Then, such contact induces the expression of nodal and its downstream Pitx2 gene in the left-side epidermis. Vitelline membrane is required for the promotion of nodal expression. Here, we showed that a chemical signal from the vitelline membrane promotes nodal gene expression, but mechanical stimulus at the point of contact is unnecessary since the treatment of devitellinated Neurulae with an extract of the vitelline membrane promoted nodal expression on both sides. The signal molecules are already present in the vitelline membranes of unfertilized eggs. These signal molecules are proteins but not sugars. Specific fractions in gel filtration chromatography had the nodal promoting activity. By mass spectrometry, we selected 48 candidate proteins. Proteins that contain both a zona pellucida (ZP) domain and epidermal growth factor (EGF) repeats were enriched in the candidates of the nodal inducing molecules. Six of the ZP proteins had multiple EGF repeats that are only found in ascidian ZP proteins. These were considered to be the most viable candidates of the nodal-inducing molecules. Signal molecules are anchored to the entire vitelline membrane, and contact sites of signal-receiving cells are spatially and mechanically controlled by the Neurula rotation. In this context, ascidians are unusual with respect to mechanisms for specification of the left-right axis. By suppressing formation of epidermis monocilia, we also showed that epidermal cilia drive the Neurula rotation but are dispensable for sensing the signal from the vitelline membrane.
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wavy movements of epidermis monocilia drive the Neurula rotation that determines left right asymmetry in ascidian embryos
Developmental Biology, 2019Co-Authors: Shiori Yamada, Yuka Tanaka, Motohiko Saigou, Kaoru S Imai, Takeshi A Onuma, Hiroki NishidaAbstract:Abstract Tadpole larvae of the ascidian, Halocynthia roretzi, show morphological left–right asymmetry in the brain structures and the orientation of tail bending within the vitelline membrane. Neurula embryos rotate along the anterior–posterior axis in a counterclockwise direction, and then this rotation stops when the left side of the embryo is oriented downwards. Contact of the left-side epidermis with the vitelline membrane promotes nodal gene expression in the left-side epidermis. This is a novel mechanism in which rotation of whole embryos provides the initial cue for breaking left–right symmetry. Here we show that epidermal monocilia, which appear at the Neurula rotation stage, generate the driving force for rotation. A ciliary protein, Arl13b, fused with Venus YFP was used for live imaging of ciliary movements. Although overexpression of wild-type Arl13b fusion protein resulted in aberrant movements of the cilia and abrogation of Neurula rotation, mutant Arl13b fusion protein, in which the GTPase and coiled-coil domains were removed, did not affect the normal ciliary movements and Neurula rotation. Epidermis cilia moved in a wavy and serpentine way like sperm flagella but not in a rotational way or beating way with effective stroke and recovery stroke. They moved very slowly, at 1/7 Hz, consistent with the low angular velocity of Neurula rotation (ca. 43°/min). The tips of most cilia pointed in the opposite direction of embryonic rotation. Similar motility was also observed in Ciona robusta embryos. When embryos were treated with a dynein inhibitor, Ciliobrevin D, both ciliary movements and Neurula rotation were abrogated, showing that ciliary movements drive Neurula rotation in Halocynthia. The drug also inhibited Ciona Neurula rotation. Our observations suggest that the driving force of rotation is generated using the vitelline membrane as a substrate but not by making a water current around the embryo. It is of evolutionary interest that ascidians use ciliary movements to break embryonic left–right symmetry, like in many vertebrates. Meanwhile, ascidian embryos rotate as a whole, similar to embryos of non-vertebrate deuterostomes, such as echinoderm, hemichordate, and amphioxus, while swimming.
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Wavy movements of epidermis monocilia drive the Neurula rotation that determines left–right asymmetry in ascidian embryos
Developmental Biology, 2018Co-Authors: Shiori Yamada, Yuka Tanaka, Motohiko Saigou, Kaoru S Imai, Takeshi A Onuma, Hiroki NishidaAbstract:Abstract Tadpole larvae of the ascidian, Halocynthia roretzi, show morphological left–right asymmetry in the brain structures and the orientation of tail bending within the vitelline membrane. Neurula embryos rotate along the anterior–posterior axis in a counterclockwise direction, and then this rotation stops when the left side of the embryo is oriented downwards. Contact of the left-side epidermis with the vitelline membrane promotes nodal gene expression in the left-side epidermis. This is a novel mechanism in which rotation of whole embryos provides the initial cue for breaking left–right symmetry. Here we show that epidermal monocilia, which appear at the Neurula rotation stage, generate the driving force for rotation. A ciliary protein, Arl13b, fused with Venus YFP was used for live imaging of ciliary movements. Although overexpression of wild-type Arl13b fusion protein resulted in aberrant movements of the cilia and abrogation of Neurula rotation, mutant Arl13b fusion protein, in which the GTPase and coiled-coil domains were removed, did not affect the normal ciliary movements and Neurula rotation. Epidermis cilia moved in a wavy and serpentine way like sperm flagella but not in a rotational way or beating way with effective stroke and recovery stroke. They moved very slowly, at 1/7 Hz, consistent with the low angular velocity of Neurula rotation (ca. 43°/min). The tips of most cilia pointed in the opposite direction of embryonic rotation. Similar motility was also observed in Ciona robusta embryos. When embryos were treated with a dynein inhibitor, Ciliobrevin D, both ciliary movements and Neurula rotation were abrogated, showing that ciliary movements drive Neurula rotation in Halocynthia. The drug also inhibited Ciona Neurula rotation. Our observations suggest that the driving force of rotation is generated using the vitelline membrane as a substrate but not by making a water current around the embryo. It is of evolutionary interest that ascidians use ciliary movements to break embryonic left–right symmetry, like in many vertebrates. Meanwhile, ascidian embryos rotate as a whole, similar to embryos of non-vertebrate deuterostomes, such as echinoderm, hemichordate, and amphioxus, while swimming.
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Neurula rotation determines left-right asymmetry in ascidian tadpole larvae
Development (Cambridge England), 2012Co-Authors: Kazuhiko Nishide, Michio Mugitani, Gaku Kumano, Hiroki NishidaAbstract:Tadpole larvae of the ascidian Halocynthia roretzi show morphological left-right asymmetry. The tail invariably bends towards the left side within the vitelline membrane. The structure of the larval brain is remarkably asymmetric. nodal, a conserved gene that shows left-sided expression, is also expressed on the left side in H. roretzi but in the epidermis unlike in vertebrates. We show that nodal signaling at the late Neurula stage is required for stereotypic morphological left-right asymmetry at later stages. We uncover a novel mechanism to break embryonic symmetry, in which rotation of whole embryos provides the initial cue for left-sided expression of nodal. Two hours prior to the onset of nodal expression, the Neurula embryo rotates along the anterior-posterior axis in a counterclockwise direction when seen in posterior view, and then this rotation stops when the left side of the embryo is oriented downwards. It is likely that epidermis monocilia, which appear at the Neurula rotation stage, generate the driving force for the rotation. When the embryo lies on the left side, protrusion of the neural fold physically prevents it from rotating further. Experiments in which Neurula rotation is perturbed by various means, including centrifugation and sandwiching between glass, indicate that contact of the left epidermis with the vitelline membrane as a consequence of Neurula rotation promotes nodal expression in the left epidermis. We suggest that chemical, and not mechanical, signals from the vitelline membrane promote nodal expression. Neurula rotation is also conserved in other ascidian species.
Kazue Mogi - One of the best experts on this subject based on the ideXlab platform.
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Subtilisin-like proprotein convertase activity is necessary for left–right axis determination in Xenopus Neurula embryos
Development Genes and Evolution, 2006Co-Authors: Ryuji Toyoizumi, Shigeo Takeuchi, Kazue MogiAbstract:Signaling by members of TGF-β superfamily requires the activity of a family of site-specific endopeptidases, known as Subtilisin-like proprotein convertases (SPCs), which cleave these ligands into mature, active forms. To explore the role of SPCs in lateral plate mesoderm (LPM) differentiation in Xenopus , two SPC inhibitors, decanoyl-Arg-Val-Lys-Arg-chloromethylketone (Dec-RVKR-CMK) and hexa-arginine, were injected into the left and right LPM of Xenopus Neurulae. Left-side injection caused heart-specific left–right reversal, and this phenotype was rescued by co-injection of mature Nodal protein. In contrast, right-side injection caused left–right reversal of both the heart and gut. Tailbud embryos were less sensitive to SPC inhibitors than Neurula embryos. Injection of inhibitors into either side of Neurula embryos completely abolished expression of the left-LPM-specific genes, Xnr-1 , antivin , and pitx2 . SPC1 enzyme (Furin) was injected into the left or right LPM of mid-Neurula embryos to determine the effect of enhancing SPC activity. Left-side injection of SPC1 did not cause a significant left–right reversal of the internal organs. However, right-side injection of SPC1 strongly induced the expression of Xnr-1 and pitx2 in the right LPM, and caused 100% left–right reversal of both the heart and gut. These results suggest that moderate level of SPC activity in the right LPM of the Neurulae is necessary for proper left–right specification. Taken together, SPC enzymatic activity must be present in both LPMs for expression of the left-handed genes and left–right axis determination of the heart and gut in Xenopus embryos.
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Subtilisin-like proprotein convertase activity is necessary for left-right axis determination in Xenopus Neurula embryos.
Development genes and evolution, 2006Co-Authors: Ryuji Toyoizumi, Shigeo Takeuchi, Kazue MogiAbstract:Signaling by members of TGF-β superfamily requires the activity of a family of site-specific endopeptidases, known as Subtilisin-like proprotein convertases (SPCs), which cleave these ligands into mature, active forms. To explore the role of SPCs in lateral plate mesoderm (LPM) differentiation in Xenopus, two SPC inhibitors, decanoyl-Arg-Val-Lys-Arg-chloromethylketone (Dec-RVKR-CMK) and hexa-arginine, were injected into the left and right LPM of Xenopus Neurulae. Left-side injection caused heart-specific left–right reversal, and this phenotype was rescued by co-injection of mature Nodal protein. In contrast, right-side injection caused left–right reversal of both the heart and gut. Tailbud embryos were less sensitive to SPC inhibitors than Neurula embryos. Injection of inhibitors into either side of Neurula embryos completely abolished expression of the left-LPM-specific genes, Xnr-1, antivin, and pitx2. SPC1 enzyme (Furin) was injected into the left or right LPM of mid-Neurula embryos to determine the effect of enhancing SPC activity. Left-side injection of SPC1 did not cause a significant left–right reversal of the internal organs. However, right-side injection of SPC1 strongly induced the expression of Xnr-1 and pitx2 in the right LPM, and caused 100% left–right reversal of both the heart and gut. These results suggest that moderate level of SPC activity in the right LPM of the Neurulae is necessary for proper left–right specification. Taken together, SPC enzymatic activity must be present in both LPMs for expression of the left-handed genes and left–right axis determination of the heart and gut in Xenopus embryos.
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Xenopus Neurula left-right asymmetry is respeficied by microinjecting TGF-beta5 protein.
The International journal of developmental biology, 2003Co-Authors: Kazue Mogi, Shigeo Takeuchi, Madoka Goto, Eri Ohno, Yoshitaka Azumi, Ryuji ToyoizumiAbstract:A variety of TGF-beta-related ligands regulate the left-right asymmetry of vertebrates but the involvement of TGF-betas in left-right specification has not been reported. We assessed whether TGF-beta signaling is involved in the left-right specification of Xenopus post-gastrula embryos by microinjecting Xenopus TGF-beta5 protein into the left or right flank of Neurula-tailbud embryos. Injection on the right side of Neurulae caused left-right reversal of the internal organs in 93% of the embryos, while injection on the left side caused less than 5% left-right reversal. Expression of Xenopus nodal related-1 (Xnr-1 ), Xenopus antivin and Xenopus Pitx2, which are normally expressed on the left, was unaltered by the left-side injection. In contrast, right-side injection into Neurulae induced the expression of these genes predominantly on the right side. Right-side injection into tailbud embryos caused bilateral expression of these handed genes. Time course analysis of asymmetric gene expression revealed that Xnr-1 could be induced by TGF-beta5 at late Neurula stage, while antivin and Pitx2 could be induced by TGF-beta5 at the latertail bud stage. Injection of the antisense morpholino oligonucleotide against Xenopus TGF-beta5 into the left dorsal blastomere inhibited the normal left-handed expression of Xnr-1 and Pitx2, and caused the organ reversal in the injected embryos. These results suggest that normal left-right balance of endogenous TGF-beta5 signaling in the Neurula embryo may be needed to determine the laterality of the asymmetric genes and to generate the correct left-right axis.
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More than 95% reversal of left-right axis induced by right-sided hypodermic microinjection of activin into Xenopus Neurula embryos.
Developmental biology, 2000Co-Authors: Ryuji Toyoizumi, Kazue Mogi, Shigeo TakeuchiAbstract:Abstract In recent years, genes that show left–right (L-R) asymmetric expression patterns have been identified one after another in vertebrate gastrula–Neurula embryos. However, we still have little information about when the irreversible L-R specification is established in vertebrate embryos. In this report, we show that almost 100% of the embryos develop to be L-R-inverted larvae after microinjection of activin molecules into the right lateral hypodermic space of Xenopus Neurula embryos. After right-side injection of 10–250 pg activin protein, both early Neurulae just after gastrulation movement (stage 13–14) and late Neurulae just before neural tube closure (stage 17–18) showed almost 100% reversal of the heart and gut L-R axes. At higher doses of activin, more than 90% of the L-R-inverted embryos showed L-R reversal of both heart and gut. The survival ratio of the right-injected 4-day embryos was 90% on average. In the left-injected embryos, the occurrence of L-R inversion was less than 2% as observed in normal untreated siblings (1.7%). When the same amount of activin (1–50 pg) was microinjected into both sides of Neurula embryos, the incidence of L-R inversion was reduced to 58%. The injection of activin along the dorsal midline in the trunk region also randomized the visceral L-R axis. Injection of activin into the right side changed normal left-handed expression of Xnr-1 to right-handed or bilateral expression. In contrast, left-handed expression of Pitx2 was switched to the right side by right activin injection. This is the first report of a method that achieves complete inversion of the visceral L-R axis by treatment of embryos at the Neurula stage. Activin not only acts on the Neurulae to cancel the original L-R specification up to the late Neurula stage, but also rebuilds a new L-R axis whose left side coincides with the injection side. It is suggested that the left and right halves of Neurulae have equal potential for L-R differentiation.
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more than 95 reversal of left right axis induced by right sided hypodermic microinjection of activin into xenopus Neurula embryos
Developmental Biology, 2000Co-Authors: Ryuji Toyoizumi, Kazue Mogi, Shigeo TakeuchiAbstract:Abstract In recent years, genes that show left–right (L-R) asymmetric expression patterns have been identified one after another in vertebrate gastrula–Neurula embryos. However, we still have little information about when the irreversible L-R specification is established in vertebrate embryos. In this report, we show that almost 100% of the embryos develop to be L-R-inverted larvae after microinjection of activin molecules into the right lateral hypodermic space of Xenopus Neurula embryos. After right-side injection of 10–250 pg activin protein, both early Neurulae just after gastrulation movement (stage 13–14) and late Neurulae just before neural tube closure (stage 17–18) showed almost 100% reversal of the heart and gut L-R axes. At higher doses of activin, more than 90% of the L-R-inverted embryos showed L-R reversal of both heart and gut. The survival ratio of the right-injected 4-day embryos was 90% on average. In the left-injected embryos, the occurrence of L-R inversion was less than 2% as observed in normal untreated siblings (1.7%). When the same amount of activin (1–50 pg) was microinjected into both sides of Neurula embryos, the incidence of L-R inversion was reduced to 58%. The injection of activin along the dorsal midline in the trunk region also randomized the visceral L-R axis. Injection of activin into the right side changed normal left-handed expression of Xnr-1 to right-handed or bilateral expression. In contrast, left-handed expression of Pitx2 was switched to the right side by right activin injection. This is the first report of a method that achieves complete inversion of the visceral L-R axis by treatment of embryos at the Neurula stage. Activin not only acts on the Neurulae to cancel the original L-R specification up to the late Neurula stage, but also rebuilds a new L-R axis whose left side coincides with the injection side. It is suggested that the left and right halves of Neurulae have equal potential for L-R differentiation.
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Ran expression pattern in amphioxus Branchiostoma lanceolatum embryos and larvae.
2018Co-Authors: Ugo Coppola, Nicholas D. Holland, Filomena Caccavale, Marta Scelzo, Filomena Ristoratore, Salvatore D’anielloAbstract:A) middle Neurula; B) late Neurula; C) pre-mouth larva; D) 3 dpf larva; D’) enlargement of the 3 dpf larva’s tail. E) section of the late Neurula in B at the level “e”. F) section of the late Neurula in B at the level “f”. G) section of the late Neurula in B at the level “g”. H) section of the pre-mouth in C at the level “h”. I) section of the pre-mouth in C at the level “i”. Abbreviation: ng, neural grove; bv, brain vesicle; o-en, oral endoderm; op, pre-oral pit; mo-gs, mouth and gill slits. White arrowheads indicate the anterior and posterior limits of Ran expression in gut. In all images the anterior is to the left and dorsal to the top. Scale bars: 60 μm in A-B-C-D; 15 μm in E.
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PHH3 antibody immunostaining in amphioxus Branchiostoma lanceolatum embryos and larvae.
2018Co-Authors: Ugo Coppola, Nicholas D. Holland, Filomena Caccavale, Marta Scelzo, Filomena Ristoratore, Salvatore D’anielloAbstract:A) middle Neurula; B) late Neurula; C) pre-mouth larva; D) 3 dpf larva. Abbreviation: ng, neural grove; nt, neural tube; bv, brain vesicle; o-en, oral endoderm; mo-gs, mouth and gill slits; tb, tailbud. In all images the anterior is to the left and dorsal to the top. Scale bars: 60 μm.
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developmental expression of the three iroquois genes of amphioxus bfirxa bfirxb and bfirxc with special attention to the gastrula organizer and anteroposterior boundaries in the central nervous system
Gene Expression Patterns, 2009Co-Authors: Stacy L Kaltenbach, Linda Z. Holland, Nicholas D. Holland, Demian KoopAbstract:Abstract Here we describe the developmental expression of the three iroquois genes ( BfIrxA , BfIrxB , and BfIrxC ) of amphioxus. BfIrxB transcription is first detected at the gastrula stage in mesendoderm just within the dorsal lip of the blastopore (a probable homolog of Spemann’s organizer) and in ectoderm. In early Neurulae, expression begins in presumptive pharyngeal endoderm, somitic mesoderm, and neural plate. Mid-Neurulae express BfIrxB throughout the hindbrain, posterior somites, pharyngeal endoderm, and notochord. In early larvae, expression is largely downregulated in the nerve cord, somites and notochord, but remains strong in the pharyngeal endoderm associated with the forming gill slits; also, a late expression domain appears in the ciliary tuft ectoderm. BfIrxA and BfIrxC , are not as widely expressed as BfIrxB . Both are first expressed in the presumptive hindbrain and presumptive pharyngeal endoderm at the early Neurula stages. In the mid-Neurula, additional expression domains appear in the extremities of the notochord. Neural expression is downregulated by late Neurula. In the early larva, expression is chiefly limited to pharyngeal endoderm associated with the forming gill slits, excepting a small new domain of BfIrxC (not BfIrxA ) expression in the ciliary tuft ectoderm. In comparison to developing vertebrates, embryos and larvae of amphioxus express iroquois genes in fewer tissues. Thus, iroquois genes of the proximate ancestor of the vertebrates evidently assumed numerous new roles during vertebrate evolution, including the division of the central nervous system into several sub-regions along its anteroposterior axis.
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Stage- and tissue-specific patterns of cell division in embryonic and larval tissues of amphioxus during normal development.
Evolution & development, 2006Co-Authors: Nicholas D. Holland, Linda Z. HollandAbstract:SUMMARY The distribution of dividing cells is described for embryos and larvae of amphioxus (Branchiostoma floridae) pulse labeled with bromodeoxyuridine. Because cell division is assessed for all of the developing tissues, this is the first comprehensive study of developmental cell proliferation for an animal lacking a stereotyped cell lineage. In amphioxus, cell divisions are virtually synchronous during cleavage, but become asynchronous at the blastula stage. Starting at the Neurula stage, after the origin of the mesoderm, the proportion of dividing cells progressively declines in the somitic mesoderm and notochord. Other tissues, however, deviate from this pattern. For example, in the mid-Neurula, there is a brief, intense burst of mitosis at the anterior end of the neural plate. Also, from the Neurula through the early larval stage, all of the ectoderm cells cease dividing and develop cilia that propel the animal through the water; subsequently, in the epidermis of later larvae, mitosis resumes and the proportion of ciliated cells declines as muscular undulation gradually replaces ciliation for swimming. Finally, in the early larvae, there is a terminal arrest of cell division in three cell types that differentiate early to participate in feeding as soon as the mouth opens—namely the ciliated pharyngeal cells that produce the feeding current and the secretory cells of the club-shaped gland and endostyle that export food-trapping mucus into the pharynx. In sum, these stage- and tissue-specific changes in cell proliferation intensity illustrate how the requirements of embryonic and larval natural history can shape developmental programs.
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Differential mesodermal expression of two amphioxus MyoD family members (AmphiMRF1 and AmphiMRF2)
Gene expression patterns : GEP, 2003Co-Authors: Michael Schubert, Linda Z. Holland, Daniel Meulemans, Marianne Bronner-fraser, Nicholas D. HollandAbstract:To explore the evolution of myogenic regulatory factors in chordates, we isolated two MyoD family genes (AmphiMRF1 and AmphiMRF2) from amphioxus. AmphiMRF1 is first expressed at the late gastrula in the paraxial mesoderm. As the first somites form, expression is restricted to their myotomal region. In the early larva, expression is strongest in the most anterior and most posterior somites. AmphiMRF2 transcription begins at mid/late gastrula in the paraxial mesoderm, but never spreads into its most anterior region. Through much of the Neurula stage, AmphiMRF2 expression is strong in the myotomal region of all somites except the most anterior pair; by late Neurula expression is downregulated except in the most posterior somites forming just rostral to the tail bud. These two MRF genes of amphioxus have partly overlapping patterns of mesodermal expression and evidently duplicated independent of the diversification of the vertebrate MRF family.