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1.
Dis Model Mech ; 15(2)2022 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-33722956

RESUMO

22q11.2 Deletion Syndrome (22q11DS) is a neurodevelopmental disorder associated with cranial nerve anomalies and disordered oropharyngeal function, including pediatric dysphagia. Using the LgDel 22q11DS mouse model, we investigated whether sensory neuron differentiation in the trigeminal ganglion (CNgV), which is essential for normal orofacial function, is disrupted. We did not detect changes in cranial placode cell translocation or neural crest migration at early stages of LgDel CNgV development. However, as the ganglion coalesces, proportions of placode-derived LgDel CNgV cells increase relative to neural crest cells. In addition, local aggregation of placode-derived cells increases and aggregation of neural crest-derived cells decreases in LgDel CNgV. This change in cell-cell relationships was accompanied by altered proliferation of placode-derived cells at embryonic day (E)9.5, and premature neurogenesis from neural crest-derived precursors, reflected by an increased frequency of asymmetric neurogenic divisions for neural crest-derived precursors by E10.5. These early differences in LgDel CNgV genesis prefigure changes in sensory neuron differentiation and gene expression by postnatal day 8, when early signs of cranial nerve dysfunction associated with pediatric dysphagia are observed in LgDel mice. Apparently, 22q11 deletion destabilizes CNgV sensory neuron genesis and differentiation by increasing variability in cell-cell interaction, proliferation and sensory neuron differentiation. This early developmental divergence and its consequences may contribute to oropharyngeal dysfunction, including suckling, feeding and swallowing disruptions at birth, and additional orofacial sensory/motor deficits throughout life.

2.
Genesis ; : e23418, 2021 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-33826226

RESUMO

The left-right (L-R) axis of most bilateral animals is established during gastrulation when a transient ciliated structure creates a directional flow of signaling molecules that establish asymmetric gene expression in the lateral plate mesoderm. However, in some animals, an earlier differential distribution of molecules and cell division patterns initiate or at least influence L-R patterning. Using single-cell high-resolution mass spectrometry, we previously reported a limited number of small molecule (metabolite) concentration differences between left and right dorsal-animal blastomeres of the eight-cell Xenopus embryo. Herein, we examined whether altering the distribution of some of these molecules influenced early events in L-R patterning. Using lineage tracing, we found that injecting right-enriched metabolites into the left cell caused its descendant cells to disperse in patterns that varied from those in control gastrulae; this did not occur when left-enriched metabolites were injected into the right cell. At later stages, injecting left-enriched metabolites into the right cell perturbed the expression of genes known to: (a) be required for the formation of the gastrocoel roof plate (foxj1); (b) lead to the asymmetric expression of Nodal (dand5/coco); or (c) result from asymmetrical nodal expression (pitx2). Despite these perturbations in gene expression, we did not observe heterotaxy in heart or gut looping at tadpole stages. These studies indicate that altering metabolite distribution at cleavage stages at the concentrations tested in this study impacts the earliest steps of L-R gene expression that then can be compensated for during organogenesis.

3.
Am J Med Genet A ; 185(6): 1932-1939, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33660912

RESUMO

The Society for Craniofacial Genetics and Developmental Biology (SCGDB) held its 43rd annual meeting in a virtual format on October 19-20, 2020. The SCGDB meeting included the presentation of the SCGDB Distinguished Scientists in Craniofacial Research Awards to Marilyn Jones and Kerstin Ludwig and four scientific sessions on the molecular regulation of craniofacial development, craniofacial morphogenesis, translational craniofacial biology, and signaling during craniofacial development. The meeting also included workshops on career development, NIH/NIDCR funding, and the utility of the FaceBase database, as well as two poster sessions. Over 190 attendees from 21 states, representing over 50 different scientific institutions, participated. This diverse group of scientists included cell biologists, developmental biologists, and clinical geneticists. While in-person interactions were missed due to the virtual meeting format imposed by the COVID-19 pandemic, the meeting platform provided ample opportunities for participant interactions and discussions, thus strengthening the community.


Assuntos
Anormalidades Craniofaciais/genética , Biologia do Desenvolvimento , Animais , Congressos como Assunto/organização & administração , Anormalidades Craniofaciais/embriologia , Genética Médica , Humanos , Pandemias , Sociedades Médicas/organização & administração , Sociedades Científicas/organização & administração , Comunicação por Videoconferência
4.
Dev Biol ; 467(1-2): 39-50, 2020 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-32891623

RESUMO

The Six1 transcription factor plays a major role in craniofacial development. Mutations in SIX1 and its co-factor, EYA1, are causative for about 50% of Branchio-otic/Branchio-oto-renal syndrome (BOR) patients, who are characterized by variable craniofacial, otic and renal malformations. We previously screened for other proteins that might interact with Six1 to identify additional genes that may play a role in BOR, and herein characterize the developmental role of one of them, Microspherule protein 1 (Mcrs1). We found that in cultured cells, Mcrs1 bound to Six1 and in both cultured cells and embryonic ectoderm reduced Six1-Eya1 transcriptional activation. Knock-down of Mcrs1 in embryos caused an expansion of the domains of neural plate genes and two genes expressed in both the neural plate and neural crest (zic1, zic2). In contrast, two other genes expressed in pre-migratory neural crest (foxd3, sox9) were primarily reduced. Cranial placode genes showed a mixture of expanded and diminished expression domains. At larval stages, loss of Mcrs1 resulted in a significant reduction of otic vesicle gene expression concomitant with a smaller otic vesicle volume. Experimentally increasing Mcrs1 above endogenous levels favored the expansion of neural border and neural crest gene domains over cranial placode genes; it also reduced otic vesicle gene expression but not otic vesicle volume. Co-expression of Mcrs1 and Six1 as well as double knock-down and rescue experiments establish a functional interaction between Mcrs1 and Six1 in the embryo, and demonstrate that this interaction has an important role in the development of craniofacial tissues including the otic vesicle.

5.
Hum Mol Genet ; 29(18): 3081-3093, 2020 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-32901287

RESUMO

We identified divergent modes of initial axon growth that prefigure disrupted differentiation of the trigeminal nerve (CN V), a cranial nerve essential for suckling, feeding and swallowing (S/F/S), a key innate behavior compromised in multiple genetic developmental disorders including DiGeorge/22q11.2 Deletion Syndrome (22q11.2 DS). We combined rapid in vivo labeling of single CN V axons in LgDel+/- mouse embryos, a genomically accurate 22q11.2DS model, and 3D imaging to identify and quantify phenotypes that could not be resolved using existing methods. We assessed these phenotypes in three 22q11.2-related genotypes to determine whether individual CN V motor and sensory axons wander, branch and sprout aberrantly in register with altered anterior-posterior hindbrain patterning and gross morphological disruption of CN V seen in LgDel+/-. In the additional 22q11.2-related genotypes: Tbx1+/-, Ranbp1-/-, Ranbp1+/- and LgDel+/-:Raldh2+/-; axon phenotypes are seen when hindbrain patterning and CN V gross morphology is altered, but not when it is normal or restored toward WT. This disordered growth of CN V sensory and motor axons, whose appropriate targeting is critical for optimal S/F/S, may be an early, critical determinant of imprecise innervation leading to inefficient oropharyngeal function associated with 22q11.2 deletion from birth onward.

6.
Am J Med Genet A ; 182(7): 1555-1561, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32352199

RESUMO

The Society for Craniofacial Genetics and Developmental Biology (SCGDB) 42nd Annual Meeting was held at the MD Anderson Cancer Center in Houston, Texas from October 14-15, 2019. The SCGDB meeting included scientific sessions on the molecular regulation of craniofacial development, cell biology of craniofacial development, signaling during craniofacial development, translational craniofacial biology, and for the first time, a career development workshop. Over a one hundred attendees from 21 states, and representing over 50 different scientific institutions, participated. The diverse group of scientists included cell and developmental biologists and clinical geneticists, promoting excellent discussions about molecular pathways guiding abnormal cell behaviors and the resultant morphological changes to craniofacial development. The results were high-quality science and a welcoming environment for trainees interested in craniofacial biology.

8.
Dev Biol ; 462(2): 165-179, 2020 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-32259520

RESUMO

Xenopus laevis frogs from laboratory stocks normally lay eggs exhibiting extensive size variability. We find that these initial size differences subsequently affect the size of the embryos prior to the onset of growth, and the size of tadpoles during the growth period. Even though these tadpoles differ in size, their tissues, organs, and structures always seem to be properly proportioned, i.e. they display static allometry. Initial axial patterning events in Xenopus occur in a spherical embryo, allowing easy documentation of their size-dependent features. We examined the size distribution of early Xenopus laevis embryos and measured diameters that differed by about 38% with a median of about 1.43 â€‹mm. This range of embryo sizes corresponds to about a 1.9-fold difference in surface area and a 2.6-fold difference in volume. We examined the relationship between embryo size and gene expression and observed a significant correlation between diameter and RNA content during gastrula stages. In addition, we investigated the expression levels of genes that pattern the mesoderm, induce the nervous system and mediate the progression of ectodermal cells to neural precursors in large and small embryos. We found that most of these factors were expressed at levels that scaled with the different embryo sizes and total embryo RNA content. In agreement with the changes in transcript levels, the expression domains in larger embryos increased proportionally with the increase in surface area, maintaining their relative expression domain size in relation to the total size of the embryo. Thus, our study identified a mechanism for adapting gene expression domains to embryo size by adjusting the transcript levels of the genes regulating mesoderm induction and patterning. In the neural plate, besides the scaling of the expression domains, we observed similar cell sizes and cell densities in small and large embryos suggesting that additional cell divisions took place in large embryos to compensate for the increased size. Our results show in detail the size variability among Xenopus laevis embryos and the transcriptional adaptation to scale gene expression with size. The observations further support the involvement of BMP/ADMP signaling in the scaling process.

9.
Hum Mol Genet ; 29(6): 1002-1017, 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-32047912

RESUMO

LgDel mice, which model the heterozygous deletion of genes at human chromosome 22q11.2 associated with DiGeorge/22q11.2 deletion syndrome (22q11DS), have cranial nerve and craniofacial dysfunction as well as disrupted suckling, feeding and swallowing, similar to key 22q11DS phenotypes. Divergent trigeminal nerve (CN V) differentiation and altered trigeminal ganglion (CNgV) cellular composition prefigure these disruptions in LgDel embryos. We therefore asked whether a distinct transcriptional state in a specific population of early differentiating LgDel cranial sensory neurons, those in CNgV, a major source of innervation for appropriate oropharyngeal function, underlies this departure from typical development. LgDel versus wild-type (WT) CNgV transcriptomes differ significantly at E10.5 just after the ganglion has coalesced. Some changes parallel altered proportions of cranial placode versus cranial neural crest-derived CNgV cells. Others are consistent with a shift in anterior-posterior patterning associated with divergent LgDel cranial nerve differentiation. The most robust quantitative distinction, however, is statistically verifiable increased variability of expression levels for most of the over 17 000 genes expressed in common in LgDel versus WT CNgV. Thus, quantitative expression changes of functionally relevant genes and increased stochastic variation across the entire CNgV transcriptome at the onset of CN V differentiation prefigure subsequent disruption of cranial nerve differentiation and oropharyngeal function in LgDel mice.

10.
Annu Rev Neurosci ; 43: 315-336, 2020 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-32101484

RESUMO

All mammals must suckle and swallow at birth, and subsequently chew and swallow solid foods, for optimal growth and health. These initially innate behaviors depend critically upon coordinated development of the mouth, tongue, pharynx, and larynx as well as the cranial nerves that control these structures. Disrupted suckling, feeding, and swallowing from birth onward-perinatal dysphagia-is often associated with several neurodevelopmental disorders that subsequently alter complex behaviors. Apparently, a broad range of neurodevelopmental pathologic mechanisms also target oropharyngeal and cranial nerve differentiation. These aberrant mechanisms, including altered patterning, progenitor specification, and neurite growth, prefigure dysphagia and may then compromise circuits for additional behavioral capacities. Thus, perinatal dysphagia may be an early indicator of disrupted genetic and developmental programs that compromise neural circuits and yield a broad range of behavioral deficits in neurodevelopmental disorders.

11.
Dis Model Mech ; 13(3)2020 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-31980437

RESUMO

Single-nucleotide mutations in human SIX1 result in amino acid substitutions in either the protein-protein interaction domain or the homeodomain, and cause ∼4% of branchio-otic (BOS) and branchio-oto-renal (BOR) cases. The phenotypic variation between patients with the same mutation, even within affected members of the same family, make it difficult to functionally distinguish between the different SIX1 mutations. We made four of the BOS/BOR substitutions in the Xenopus Six1 protein (V17E, R110W, W122R, Y129C), which is 100% identical to human in both the protein-protein interaction domain and the homeodomain, and expressed them in embryos to determine whether they cause differential changes in early craniofacial gene expression, otic gene expression or otic morphology. We confirmed that, similar to the human mutants, all four mutant Xenopus Six1 proteins access the nucleus but are transcriptionally deficient. Analysis of craniofacial gene expression showed that each mutant causes specific, often different and highly variable disruptions in the size of the domains of neural border zone, neural crest and pre-placodal ectoderm genes. Each mutant also had differential effects on genes that pattern the otic vesicle. Assessment of the tadpole inner ear demonstrated that while the auditory and vestibular structures formed, the volume of the otic cartilaginous capsule, otoliths, lumen and a subset of the hair cell-containing sensory patches were reduced. This detailed description of the effects of BOS/BOR-associated SIX1 mutations in the embryo indicates that each causes subtle changes in gene expression in the embryonic ectoderm and otocyst, leading to inner ear morphological anomalies.

12.
Anal Chem ; 91(7): 4797-4805, 2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30827088

RESUMO

Label-free single-cell proteomics by mass spectrometry (MS) is currently incompatible with complex tissues without requiring cell culturing, single-cell dissection, or tissue dissociation. We here report the first example of label-free single-cell MS-based proteomics directly in single cells in live vertebrate embryos. Our approach integrates optically guided in situ subcellular capillary microsampling, one-pot extraction-digestion of the collected proteins, peptide separation by capillary electrophoresis, ionization by an ultrasensitive electrokinetically pumped nanoelectrospray, and detection by high-resolution MS (Orbitrap). With a 700 zmol (420 000 copies) lower limit of detection, this trace-sensitive technology confidently identified and quantified ∼750-800 protein groups (<1% false-discovery rate) by analyzing just ∼5 ng of protein digest, viz. <0.05% of the total protein content from individual cells in a 16-cell Xenopus laevis (frog) embryo. After validating the approach by recovering animal-vegetal-pole proteomic asymmetry in the frog zygote, the technology was applied to uncover proteomic reorganization as the animal-dorsal (D11) cell of the 16-cell embryo gave rise to its neural-tissue-fated clone in the embryo developing to the 32-, 64-, and 128-cell stages. In addition to enabling proteomics on smaller cells in X. laevis, we also demonstrated this technology to be scalable to single cells in live zebrafish embryos. Microsampling single-cell MS-based proteomics raises exciting opportunities to study cell and developmental processes directly in complex tissues and whole organisms at the level of the building block of life: the cell.


Assuntos
Proteômica , Análise de Célula Única , Proteínas de Xenopus/análise , Proteínas de Peixe-Zebra/análise , Animais , Células Clonais/química , Células Clonais/citologia , Eletroforese Capilar , Embrião não Mamífero/química , Embrião não Mamífero/citologia , Espectrometria de Massas , Xenopus laevis , Peixe-Zebra
13.
Am J Med Genet A ; 179(5): 864-869, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30793834

RESUMO

The mission of the Society for Craniofacial Genetics and Developmental Biology (SCGDB) is to promote education, research, and communication about normal and abnormal development of the tissues and organs of the head. The SCGDB welcomes as members undergraduate students, graduate students, postdoctoral researchers, medical and dental practitioners, scientists, and academicians who possess an interest in craniofacial biology. Each year our members come together to share their novel findings, build upon, and challenge current knowledge of craniofacial biology.


Assuntos
Anormalidades Craniofaciais/diagnóstico , Anormalidades Craniofaciais/etiologia , Anormalidades Craniofaciais/terapia , Biologia do Desenvolvimento , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Modelos Biológicos , Organogênese
14.
Cold Spring Harb Protoc ; 2019(1)2019 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-29769392

RESUMO

The individual blastomeres of Xenopus two- to 32-cell embryos have been fate mapped. This work identified the precursors of most of the embryonic cell types, tissues and organs; however, the maps do not reveal the cell interactions or signaling pathways that are required for establishing cell fates. This protocol describes an explant culture approach for culturing blastomeres in isolation to test whether a cell's fate has been determined. Cleavage blastomeres can be cultured in a simple salt medium without added factors because they contain intracellular yolk platelets, which provide an intrinsic energy source. This method allows one to test whether an isolated blastomere can produce specific cell types or express tissue-specific genes independent of interactions with other cells or specific signaling pathways. The role of cell-cell interactions can be revealed by co-culturing different sets of blastomeres. One can identify the molecules that are required for those cell fates by applying knockdown approaches to the isolated cell. One also can determine the developmental time at which cell fates are committed by explanting blastomere lineages at different stages.


Assuntos
Blastômeros/citologia , Blástula/citologia , Blástula/crescimento & desenvolvimento , Técnicas de Cultura de Órgãos/métodos , Xenopus/embriologia , Animais , Meios de Cultura/química
15.
Cold Spring Harb Protoc ; 2019(1)2019 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-29769394

RESUMO

The fates of individual cleavage-stage blastomeres and of groups of cells at the blastula through gastrula stages of Xenopus embryos have been mapped in great detail. These studies identified the major contributors of the three germ layers as well as a variety of tissues and organs and several specific cell types. One can use these fate maps to test the commitment of single cells or groups of cells to produce their normal repertoire of descendants, to identify the genes that regulate fate commitment, and to modulate the levels of gene expression in specific lineages to determine gene function in a variety of developmental processes. Here we introduce methods that include how to identify specific blastomeres, inject them with lineage tracers, and alter gene expression levels. We also discuss methods for assaying protein and mRNA expression in situ and for providing novel embryonic environments to test fate commitment. These techniques draw upon classical approaches that are quite easy to perform in the versatile Xenopus embryo.


Assuntos
Blástula/citologia , Diferenciação Celular , Gástrula/citologia , Xenopus/embriologia , Animais , Blástula/crescimento & desenvolvimento , Gástrula/crescimento & desenvolvimento , Coloração e Rotulagem/métodos
16.
Cold Spring Harb Protoc ; 2019(1)2019 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-29769398

RESUMO

Fate maps identify the precursors of an organ, and tracing the members of a blastomere lineage over time shows how its descendants come to populate that organ. The fates of the individual blastomeres of the two- to 32-cell Xenopus embryo have been fully mapped to reveal which cells are the major contributors to various cell types, tissues, and organs. However, because these fate maps were produced in the normal embryo, they do not reveal whether a precursor blastomere is competent to give rise to additional tissues or is already committed to its fate-mapped repertoire of descendants. To identify the mechanisms by which a cell's fate is committed, one needs to expose the cell to different experimental environments. If the cell's fate is determined, it will express its normal fate or gene expression profile in novel environments, whereas if it is not yet determined it will express different fates or gene expression profiles when exposed to novel external factors or neighboring cells. This protocol describes two techniques for testing cell fate commitment: single cell deletion and single cell transplantation. Deleting a blastomere allows one to test whether the deleted cell is required for the remaining cells to produce their normal, specific cell fates. Transplanting a blastomere to a novel location in a host embryo allows one to test whether the transplanted cell is committed to produce its normal fate-mapped repertoire, or whether it is still competent to respond to novel cell-cell interactions.


Assuntos
Blastômeros/citologia , Blástula/citologia , Blástula/crescimento & desenvolvimento , Diferenciação Celular , Técnicas de Cultura de Órgãos/métodos , Transplante/métodos , Xenopus/embriologia , Animais , Meios de Cultura/química
17.
Dev Biol ; 446(1): 68-79, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30529252

RESUMO

The specialized sensory organs of the vertebrate head are derived from thickened patches of cells in the ectoderm called cranial sensory placodes. The developmental program that generates these placodes and the genes that are expressed during the process have been studied extensively in a number of animals, yet very little is known about how these genes regulate one another. We previously found via a microarray screen that Six1, a known transcriptional regulator of cranial placode fate, up-regulates Irx1 in ectodermal explants. In this study, we investigated the transcriptional relationship between Six1 and Irx1 and found that they reciprocally regulate each other throughout cranial placode and otic vesicle formation. Although Irx1 expression precedes that of Six1 in the neural border zone, its continued and appropriately patterned expression in the pre-placodal region (PPR) and otic vesicle requires Six1. At early PPR stages, Six1 expands the Irx1 domain, but this activity subsides over time and changes to a predominantly repressive effect. Likewise, Irx1 initially expands Six1 expression in the PPR, but later represses it. We also found that Irx1 and Sox11, a known direct target of Six1, reciprocally affect each other. This work demonstrates that the interactions between Six1 and Irx1 are continuous during PPR and placode development and their transcriptional effects on one another change over developmental time.


Assuntos
Orelha Interna/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Proteínas do Tecido Nervoso/genética , Placa Neural/metabolismo , Fatores de Transcrição/genética , Proteínas de Xenopus/genética , Animais , Orelha Interna/citologia , Orelha Interna/embriologia , Ectoderma/citologia , Ectoderma/embriologia , Ectoderma/metabolismo , Embrião não Mamífero/citologia , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Cabeça/embriologia , Proteínas de Homeodomínio/metabolismo , Hibridização In Situ , Proteínas do Tecido Nervoso/metabolismo , Placa Neural/citologia , Placa Neural/embriologia , Fatores de Transcrição SOXC/genética , Fatores de Transcrição SOXC/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis
18.
Cold Spring Harb Protoc ; 2018(12)2018 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-29769388

RESUMO

Fate mapping approaches reveal what types of cells, tissues, and organs are derived from specific embryonic cells. Classical fate maps were made by microscopic techniques using embryos comprising small numbers of transparent cells. More complex and opaque embryos require use of a vital or lipophilic dye that labels small groups of cells. Intracellular injection of a lineage tracer that labels the injected cell and all of its descendants can be used to mark a single cell in Xenopus embryos, whose large cells are easy to microinject and usually cleave in regular patterns. Intracellular lineage tracers must be neutral compounds that do not interact with cellular processes that might change the developmental fate of the injected cell, be small enough to diffuse quickly throughout the cytoplasm before the cell divides so that all descendants are labeled, and be large enough to not diffuse to adjacent cells via gap junctions. They should not be diluted by cell division or intracellular degradation, and should be easily detected by histochemical reactions (enzymes) or direct imaging (fluorescent compounds). Several types of lineage tracers have been used, including small, fluorescently tagged dextrans and mRNAs encoding enzymes or fluorescent proteins, described here. Many lineage tracers can be combined with cell type-specific mRNA and protein expression assays, making lineage tracing a powerful tool for testing the function of genes and cell fate commitment.


Assuntos
Diferenciação Celular , Linhagem da Célula , Indicadores e Reagentes , Coloração e Rotulagem/métodos , Xenopus/embriologia , Animais
19.
Cold Spring Harb Protoc ; 2018(12)2018 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-29769401

RESUMO

Microinjecting lineage tracers into a single blastomere in the normal, intact embryo identifies the repertoire of cell types derived from it. In order to reveal the full developmental potential of that blastomere or identify the mechanisms by which its fate is determined, one needs to modify its gene expression under controlled experimental conditions. One method by which this is easily accomplished in Xenopus is by microinjecting synthetic mRNAs or antisense oligonucleotides into an identified blastomere to target altered gene expression specifically to its lineage. Xenopus blastomeres are robust and tolerate pressure-driven microinjection up to a few hundred cells, and they efficiently translate exogenously supplied mRNAs. Targeted microinjections, described here, significantly reduce off-target effects of the mRNAs or oligonucleotides. Many types of constructs can be synthesized to provide specific information about gene function. For example, microinjecting mRNA encoding the wild-type gene in its normal expression domain or in an ectopic site tests whether it promotes or represses target genes or alters the formation of tissues of interest. Mutant forms of a gene transcript can illuminate the function of different domains of the encoded protein or show the developmental consequences of a mutation found in a human disease. mRNAs encoding dominant-negative forms of a protein can elicit a functional knockdown and thereby establish the necessity for that gene in a developmental process. Microinjecting antisense morpholino oligonucleotides (MOs) that are designed to block either endogenous mRNA translation or splicing is an effective method to reduce the levels of endogenous protein.


Assuntos
Blastômeros/metabolismo , Linhagem da Célula , Microinjeções/métodos , Oligonucleotídeos Antissenso/metabolismo , RNA Mensageiro/metabolismo , Coloração e Rotulagem/métodos , Animais , Oligonucleotídeos Antissenso/genética , RNA Mensageiro/genética , Xenopus/embriologia
20.
ACS Chem Neurosci ; 9(8): 2064-2073, 2018 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-29578674

RESUMO

The molecular program by which embryonic ectoderm is induced to form neural tissue is essential to understanding normal and impaired development of the central nervous system. Xenopus has been a powerful vertebrate model in which to elucidate this process. However, abundant vitellogenin (yolk) proteins in cells of the early Xenopus embryo interfere with protein detection by high-resolution mass spectrometry (HRMS), the technology of choice for identifying these gene products. Here, we systematically evaluated strategies of bottom-up proteomics to enhance proteomic detection from the neural ectoderm (NE) of X. laevis using nanoflow high-performance liquid chromatography (nanoLC) HRMS. From whole embryos, high-pH fractionation prior to nanoLC-HRMS yielded 1319 protein groups vs 762 proteins without fractionation (control). Compared to 702 proteins from dorsal halves of embryos (control), 1881 proteins were identified after yolk platelets were depleted via sucrose-gradient centrifugation. We combined these approaches to characterize protein expression in the NE of the early embryo. To guide microdissection of the NE tissues from the gastrula (stage 10), their precursor (midline dorsal-animal, or D111) cells were fate-mapped from the 32-cell embryo using a fluorescent lineage tracer. HRMS of the cell clones identified 2363 proteins, including 147 phosphoproteins (without phosphoprotein enrichment), transcription factors, and members from pathways of cellular signaling. In reference to transcriptomic maps of the developing X. laevis, 76 proteins involved in signaling pathways were gene matched to transcripts with known enrichment in the neural plate. Besides a protocol, this work provides qualitative proteomic data on the early developing NE.


Assuntos
Ectoderma/embriologia , Ectoderma/metabolismo , Espectrometria de Massas , Proteômica/métodos , Processamento Alternativo , Animais , Cromatografia Líquida de Alta Pressão , Ectoderma/citologia , Embrião não Mamífero , Espectrometria de Massas/métodos , Modelos Animais , Neurônios/citologia , Neurônios/metabolismo , Xenopus laevis
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