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1.
Curr Biol ; 32(22): 4989-4996.e3, 2022 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-36332617

RESUMO

Early embryogenesis is characterized by rapid and synchronous cleavage divisions, which are often controlled by wave-like patterns of Cdk1 activity. Two mechanisms have been proposed for mitotic waves: sweep and trigger waves.1,2 The two mechanisms give rise to different wave speeds, dependencies on physical and molecular parameters, and spatial profiles of Cdk1 activity: upward sweeping gradients versus traveling wavefronts. Both mechanisms hinge on the transient bistability governing the cell cycle and are differentiated by the speed of the cell-cycle progression: sweep and trigger waves arise for rapid and slow drives, respectively. Here, using quantitative imaging of Cdk1 activity and theory, we illustrate that sweep waves are the dominant mechanism in Drosophila embryos and test two fundamental predictions on the transition from sweep to trigger waves. We demonstrate that sweep waves can be turned into trigger waves if the cell cycle is slowed down genetically or if significant delays in the cell-cycle progression are introduced across the embryo by altering nuclear density. Our genetic experiments demonstrate that Polo kinase is a major rate-limiting regulator of the blastoderm divisions, and genetic perturbations reducing its activity can induce the transition from sweep to trigger waves. Furthermore, we show that changes in temperature cause an essentially uniform slowdown of interphase and mitosis. That results in sweep waves being observed across a wide temperature range despite the cell-cycle durations being significantly different. Collectively, our combination of theory and experiments elucidates the nature of mitotic waves in Drosophila embryogenesis, their control mechanisms, and their mutual transitions.


Assuntos
Proteína Quinase CDC2 , Proteínas de Drosophila , Animais , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Blastoderma/metabolismo , Drosophila/genética , Mitose , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Ciclo Celular/genética
2.
Open Biol ; 12(9): 220147, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-36128719

RESUMO

During early avian development, only a narrow band of cells (the edge cells, also called 'margin of overgrowth') at the rim of the embryo is responsible for blastoderm expansion by crawling over the vitelline membrane (VM) to cover the whole egg yolk in just 4 days (a process called epiboly). Surprisingly, this has not yet been studied in detail. Here we explore the edge cells of the chick embryo using in situ hybridization, immunohistochemistry and live imaging. Morphological and molecular properties reveal that the edge has a distinctive structure, being subdivided into sub-regions, including at least four distinct zones (which we name as leading, trailing, deep and stalk zones). This allows us to study reorganization of the edge region that accompanies reattachment of an explanted blastoderm to the VM. Immunohistochemistry uncovers distinct polarized cellular features resembling the process of collective cell migration described in other systems. Live imaging reveals dynamic lamellipodial and filopodial activity at the leading edge of the outermost cells. Our data provide evidence that edge cells are a distinct tissue. We propose that edge cells may be a useful model system for the study of wound healing and other closure events in epithelial cell sheets.


Assuntos
Blastoderma , Membrana Vitelina , Animais , Movimento Celular , Embrião de Galinha , Células Epiteliais , Cicatrização
3.
Cells Dev ; 171: 203802, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35934285

RESUMO

Segments are repeated anatomical units forming the body of insects. In Drosophila, the specification of the body takes place during the blastoderm through the segmentation cascade. Pair-rule genes such as hairy (h), even-skipped (eve), runt (run), and fushi-tarazu (ftz) are of the intermediate level of the cascade and each pair-rule gene is expressed in seven transversal stripes along the antero-posterior axis of the embryo. Stripes are formed by independent cis-regulatory modules (CRMs) under the regulation of transcription factors of maternal source and of gap proteins of the first level of the cascade. The initial blastoderm of Drosophila is a syncytium and it also coincides with the mid-blastula transition when thousands of zygotic genes are transcribed and their products are able to diffuse in the cytoplasm. Thus, we anticipated a complex regulation of the CRMs of the pair-rule stripes. The CRMs of h 1, eve 1, run 1, ftz 1 are able to be activated by bicoid (bcd) throughout the anterior blastoderm and several lines of evidence indicate that they are repressed by the anterior gap genes slp1 (sloppy-paired 1), tll (tailless) and hkb (huckebein). The modest activity of these repressors led to the premise of a combinatorial mechanism regulating the expression of the CRMs of h 1, eve 1, run 1, ftz 1 in more anterior regions of the embryo. We tested this possibility by progressively removing the repression activities of slp1, tll and hkb. In doing so, we were able to expose a mechanism of additive repression limiting the anterior borders of stripes 1. Stripes 1 respond depending on their distance from the anterior end and repressors operating at different levels.


Assuntos
Blastoderma , Proteínas de Drosophila , Animais , Blastoderma/metabolismo , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Homeodomínio/genética , Fatores de Transcrição/genética
4.
Nat Commun ; 13(1): 3889, 2022 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-35794113

RESUMO

The blastoderm is a broadly conserved stage of early animal development, wherein cells form a layer at the embryo's periphery. The cellular behaviors underlying blastoderm formation are varied and poorly understood. In most insects, the pre-blastoderm embryo is a syncytium: nuclei divide and move throughout the shared cytoplasm, ultimately reaching the cortex. In Drosophila melanogaster, some early nuclear movements result from pulsed cytoplasmic flows that are coupled to synchronous divisions. Here, we show that the cricket Gryllus bimaculatus has a different solution to the problem of creating a blastoderm. We quantified nuclear dynamics during blastoderm formation in G. bimaculatus embryos, finding that: (1) cytoplasmic flows are unimportant for nuclear movement, and (2) division cycles, nuclear speeds, and the directions of nuclear movement are not synchronized, instead being heterogeneous in space and time. Moreover, nuclear divisions and movements co-vary with local nuclear density. We show that several previously proposed models for nuclear movements in D. melanogaster cannot explain the dynamics of G. bimaculatus nuclei. We introduce a geometric model based on asymmetric pulling forces on nuclei, which recapitulates the patterns of nuclear speeds and orientations of both unperturbed G. bimaculatus embryos, and of embryos physically manipulated to have atypical nuclear densities.


Assuntos
Blastoderma , Gryllidae , Animais , Núcleo Celular , Drosophila melanogaster
5.
Development ; 149(12)2022 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-35723262

RESUMO

Classical studies have established that the marginal zone, a ring of extra-embryonic epiblast immediately surrounding the embryonic epiblast (area pellucida) of the chick embryo, is important in setting embryonic polarity by positioning the primitive streak, the site of gastrulation. The more external extra-embryonic region (area opaca) was thought to have only nutritive and support functions. Using experimental embryology approaches, this study reveals three separable functions for this outer region. First, juxtaposition of the area opaca directly onto the area pellucida induces a new marginal zone from the latter; this induced domain is entirely posterior in character. Second, ablation and grafting experiments using an isolated anterior half of the blastoderm and pieces of area opaca suggest that the area opaca can influence the polarity of the adjacent marginal zone. Finally, we show that the loss of the ability of such isolated anterior half-embryos to regulate (re-establish polarity spontaneously) at the early primitive streak stage can be rescued by replacing the area opaca by one from a younger stage. These results uncover new roles of chick extra-embryonic tissues in early development.


Assuntos
Blastoderma , Linha Primitiva , Animais , Embrião de Galinha , Gástrula/fisiologia
6.
PLoS One ; 17(5): e0268524, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35580090

RESUMO

One challenge in avian embryology is establishing a standard developmental timetable, primarily because eggs incubated for identical durations can vary in developmental progress, even within the same species. For remedy, avian development is classified into distinct stages based on the formation of key morphological structures. Developmental stages exist for a few galliform species, but the literature is lacking a description of normal stages for California valley quail (Callipepla californica). Thus, the objective of this study was to stage and document the morphological and structural development of California valley quail. Over two laying seasons, 390 eggs were incubated at 37.8Ö¯ C in 60% RH for ≤23 days. Eggs were opened every ≤6 hours to document embryonic development, including, blastoderm diameter, anterior angle of nostril to beak tip, and lengths of wing, tarsus, third toe, total beak, total foot, and embryo. California valley quail embryos were staged and compared to domestic chicken (Gallus gallus domesticus), the staging standard for galliformes, as well as Japanese quail (Coturnix japonica), blue-breasted quail (Synoicus chinensis) and northern bobwhite quail (Colinus virginianus). This study produced the first description of the 43 normal stages of development for California valley quail. Compared with other galliformes, the California valley quail has a different number of stages and displays developmental heterochrony in stages 1-24, and morphological and developmental differences in stages 25-hatch. The observed differences emphasize the importance of staging individual avian species instead of relying on poultry animal models or close relatives for developmental reference. This is extremely important in species-specific embryological studies that evaluate critical windows of development or evaluate the impacts of environmental change on avian development. This study also suggests that staging frequencies of ≤6 hours and egg transport protocols should be standardized for future staging studies.


Assuntos
Colinus , Galliformes , Animais , Blastoderma , California , Galinhas , Coturnix , Codorniz
7.
BMC Genomics ; 23(1): 262, 2022 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-35379173

RESUMO

BACKGROUND: Cool temperature egg storage prior to incubation is a common practice in the broiler industry; however, prolonged egg storage causes increased embryonic mortality and decreased hatchability and growth in surviving chicks. Exposing eggs to short periods of incubation during egg storage (SPIDES) reduces the adverse consequences of prolonged storage. SPIDES increases blastodermal cell viability by reducing apoptosis, though the counteracting mechanisms are unclear. To define the impact of prolonged storage and SPIDES, transcriptome analysis compared gene expression from blastoderms isolated from eggs exposed to the following treatments: control (CR, stored at 17 °C for 4 days), prolonged storage (NSR, stored at 17 °C for 21 days), SPIDES (SR, stored at 17 °C for 21 days with SPIDES), and incubated control (C2, stored at 17 °C for 4 days followed by incubation to HH (Hamburger-Hamilton) stage 2, used as the ideal standard development) (n = 3/group). Data analysis was performed using the CLC Genomics Workbench platform. Functional annotation was performed using DAVID and QIAGEN Ingenuity Pathway Analysis. RESULTS: In total, 4726 DEGs (differentially expressed genes) were identified across all experimental group comparisons (q < 0.05, FPKM> 20, |fold change| > 1.5). DEGs common across experimental comparisons were involved in cellular homeostasis and cytoskeletal protein binding. The NSR group exhibited activation of ubiquitination, apoptotic, and cell senescence processes. The SR group showed activation of cell viability, division, and metabolic processes. Through comparison analysis, cellular respiration, tRNA charging, cell cycle control, and HMBG1 signaling pathways were significantly impacted by treatment and potential regulatory roles for ribosomal protein L23a (RPL23A) and MYC proto-oncogene, BHLH transcription factor (MYC) were identified. CONCLUSIONS: Prolonged egg storage (NSR) resulted in enriched cell stress and death pathways; while SPIDES (SR) resulted in enriched basic cell and anti-apoptotic pathways. New insights into DNA repair mechanisms, RNA processing, shifts in metabolism, and chromatin dynamics in relation to egg storage treatment were obtained through this study. Although egg storage protocols have been examined through targeted gene expression approaches, this study provided a global view of the extensive molecular networks affected by prolonged storage and SPIDES and helped to identify potential upstream regulators for future experiments to optimize egg storage parameters.


Assuntos
Blastoderma , Galinhas , Animais , Ovos , Perfilação da Expressão Gênica , Fatores de Tempo
8.
In Vitro Cell Dev Biol Anim ; 58(3): 199-209, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-35288810

RESUMO

Chicken blastoderm cells (cBCs) obtained from stage X (EG&K) embryos are easily available materials for the study of cell development. However, cBCs are not widely used because they are hard to maintain in long-term culture in vitro. To solve this problem, ascorbic acid (AA; also known as vitamin C (VC)) and all-trans retinoic acid (ATRA) were added into basic culture medium to promote cell growth. Results suggested that cultured cBCs possessed strongly proliferative activity and maintained their pluripotency on the support of chicken embryonic fibroblast (CEF) feeder. Moreover, when VC or/and ATRA was added, the number and area of cBC colonies increased significantly compared with the control group. The expression of pluripotency genes (Sox2 and Nanog) and cell cycle-regulated genes (CCND1 and CDK6) was upregulated obviously. Furthermore, results showed that 5hmC levels in VC and RA groups increased significantly by DNA dot blot and immunofluorescence staining. These results provide strong evidence that VC and ATRA induced DNA demethylation and enhanced 5hmC level. The level of H3K27me3 was raised, while the level of H3K9me2 was reduced by addition of VC and ATRA. Finally, the expression of Tet1 and Dnmt3b was upregulated remarkably. Therefore, these results indicated that VC and ATRA enhanced DNA demethylation and then promoted cBC survival and proliferation in vitro.


Assuntos
Blastoderma , Galinhas , Animais , Ácido Ascórbico/farmacologia , Proliferação de Células , Embrião de Galinha , Desmetilação do DNA , Tretinoína/farmacologia
9.
Development ; 149(2)2022 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-35001104

RESUMO

Biological systems are highly complex, yet notably ordered structures can emerge. During syncytial stage development of the Drosophila melanogaster embryo, nuclei synchronously divide for nine cycles within a single cell, after which most of the nuclei reach the cell cortex. The arrival of nuclei at the cortex occurs with remarkable positional order, which is important for subsequent cellularisation and morphological transformations. Yet, the mechanical principles underlying this lattice-like positional order of nuclei remain untested. Here, using quantification of nuclei position and division orientation together with embryo explants, we show that short-ranged repulsive interactions between microtubule asters ensure the regular distribution and maintenance of nuclear positions in the embryo. Such ordered nuclear positioning still occurs with the loss of actin caps and even the loss of the nuclei themselves; the asters can self-organise with similar distribution to nuclei in the wild-type embryo. The explant assay enabled us to deduce the nature of the mechanical interaction between pairs of nuclei. We used this to predict how the nuclear division axis orientation changes upon nucleus removal from the embryo cortex, which we confirmed in vivo with laser ablation. Overall, we show that short-ranged microtubule-mediated repulsive interactions between asters are important for ordering in the early Drosophila embryo and minimising positional irregularity.


Assuntos
Blastoderma/metabolismo , Divisão do Núcleo Celular , Células Gigantes/metabolismo , Animais , Blastoderma/citologia , Núcleo Celular/metabolismo , Drosophila melanogaster , Células Gigantes/citologia , Microtúbulos/metabolismo , Estresse Mecânico
10.
Sci Rep ; 12(1): 49, 2022 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-34997179

RESUMO

DNA is susceptible to damage by various sources. When the DNA is damaged, the cell repairs the damage through an appropriate DNA repair pathway. When the cell fails to repair DNA damage, apoptosis is initiated. Although several genes are involved in five major DNA repair pathways and two major apoptosis pathways, a comprehensive understanding of those gene expression is not well-understood in chicken tissues. We performed whole-transcriptome sequencing (WTS) analysis in the chicken embryonic fibroblasts (CEFs), stage X blastoderms, and primordial germ cells (PGCs) to uncover this deficiency. Stage X blastoderms mostly consist of undifferentiated progenitor (pluripotent) cells that have the potency to differentiate into all cell types. PGCs are also undifferentiated progenitor cells that later differentiate into male and female germ cells. CEFs are differentiated and abundant somatic cells. Through WTS analysis, we identified that the DNA repair pathway genes were expressed more highly in blastoderms and high in PGCs than CEFs. Besides, the apoptosis pathway genes were expressed low in blastoderms and PGCs than CEFs. We have also examined the WTS-based expression profiling of candidate pluripotency regulating genes due to the conserved properties of blastoderms and PGCs. In the results, a limited number of pluripotency genes, especially the core transcriptional network, were detected higher in both blastoderms and PGCs than CEFs. Next, we treated the CEFs, blastoderm cells, and PGCs with hydrogen peroxide (H2O2) for 1 h to induce DNA damage. Then, the H2O2 treated cells were incubated in fresh media for 3-12 h to observe DNA repair. Subsequent analyses in treated cells found that blastoderm cells and PGCs were more likely to undergo apoptosis along with the loss of pluripotency and less likely to undergo DNA repair, contrasting with CEFs. These properties of blastoderms and PGCs should be necessary to preserve genome stability during the development of early embryos and germ cells, respectively.


Assuntos
Apoptose/genética , Blastoderma/metabolismo , Galinhas/genética , Reparo do DNA/genética , Instabilidade Genômica/fisiologia , Células Germinativas/metabolismo , Animais , Embrião de Galinha , Dano ao DNA/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Peróxido de Hidrogênio/farmacologia , Células-Tronco Pluripotentes/metabolismo , Transcriptoma
11.
PLoS Genet ; 18(1): e1010002, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34986144

RESUMO

A critical step in animal development is the specification of primordial germ cells (PGCs), the precursors of the germline. Two seemingly mutually exclusive mechanisms are implemented across the animal kingdom: epigenesis and preformation. In epigenesis, PGC specification is non-autonomous and depends on extrinsic signaling pathways. The BMP pathway provides the key PGC specification signals in mammals. Preformation is autonomous and mediated by determinants localized within PGCs. In Drosophila, a classic example of preformation, constituents of the germ plasm localized at the embryonic posterior are thought to be both necessary and sufficient for proper determination of PGCs. Contrary to this longstanding model, here we show that these localized determinants are insufficient by themselves to direct PGC specification in blastoderm stage embryos. Instead, we find that the BMP signaling pathway is required at multiple steps during the specification process and functions in conjunction with components of the germ plasm to orchestrate PGC fate.


Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Células Germinativas/fisiologia , Animais , Blastoderma , Padronização Corporal , Diferenciação Celular , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Epigênese Genética , Feminino , Células Germinativas/metabolismo , Masculino , Transdução de Sinais
12.
Anim Biotechnol ; 33(5): 920-929, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-33970791

RESUMO

The conservation of Taiwan Country chicken (TCC) is important due to concerns for the local breed's adaptability to the area and disease resistance. Furthermore, the genetic resource base of native chickens can be used to improve egg and meat production efficiency in commercial TCC. As the embryonic stem cells (ESCs) hold great potential for regenerative medicine and species conservation, the aims of this study were to isolate and characterize ESCs of TCC. The blastodermal cells (BCs) were isolated from the zona pellucida of stage X chicken embryos and cultured in conditioned medium for the proliferation and maintenance of BCs in vitro. The quantitative real-time polymerase chain reaction (qPCR) results showed that POUV, SOX2 and NANOG were expressed in the putative ESCs. In addition, the expression of pluripotent markers, SSEA-1 and SSEA-4, was detected. The DiI-stained ESCs were injected into the dorsal aorta of the E3.5 recipient fetuses soon after staining and the injected embryos were continuously incubated and checked on day 7 of incubation. It was shown that some DiI-positive cells were found in the 7-d-old chimeric embryos. The results demonstrated that some pluripotent cells existed in the cultured BCs for the production of germline chimeric embryos from TCC.


Assuntos
Blastoderma , Galinhas , Animais , Diferenciação Celular , Embrião de Galinha , Quimera , Meios de Cultivo Condicionados/metabolismo , Células-Tronco Embrionárias/metabolismo , Antígenos CD15/metabolismo , Taiwan
13.
PLoS Comput Biol ; 17(12): e1009614, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34871312

RESUMO

Epithelial tissues form folded structures during embryonic development and organogenesis. Whereas substantial efforts have been devoted to identifying mechanical and biochemical mechanisms that induce folding, whether and how their interplay synergistically shapes epithelial folds remains poorly understood. Here we propose a mechano-biochemical model for dorsal fold formation in the early Drosophila embryo, an epithelial folding event induced by shifts of cell polarity. Based on experimentally observed apical domain homeostasis, we couple cell mechanics to polarity and find that mechanical changes following the initial polarity shifts alter cell geometry, which in turn influences the reaction-diffusion of polarity proteins, thus forming a feedback loop between cell mechanics and polarity. This model can induce spontaneous fold formation in silico, recapitulate polarity and shape changes observed in vivo, and confer robustness to tissue shape change against small fluctuations in mechanics and polarity. These findings reveal emergent properties of a developing epithelium under control of intracellular mechano-polarity coupling.


Assuntos
Fenômenos Biomecânicos/fisiologia , Blastoderma , Polaridade Celular/fisiologia , Desenvolvimento Embrionário/fisiologia , Epitélio/fisiologia , Animais , Blastoderma/citologia , Blastoderma/fisiologia , Drosophila/embriologia , Células Epiteliais/citologia , Células Epiteliais/fisiologia , Modelos Biológicos
14.
Development ; 148(16)2021 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-34351412

RESUMO

The neuroblast timer genes hunchback, Krüppel, nubbin and castor are expressed in temporal sequence in neural stem cells, and in corresponding spatial sequence along the Drosophila blastoderm. As canonical gap genes, hunchback and Krüppel play a crucial role in insect segmentation, but the roles of nubbin and castor in this process remain ambiguous. We have investigated the expression and functions of nubbin and castor during segmentation in the beetle Tribolium. We show that Tc-hunchback, Tc-Krüppel, Tc-nubbin and Tc-castor are expressed sequentially in the segment addition zone, and that Tc-nubbin regulates segment identity redundantly with two previously described gap/gap-like genes, Tc-giant and Tc-knirps. Simultaneous knockdown of Tc-nubbin, Tc-giant and Tc-knirps results in the formation of ectopic legs on abdominal segments. This homeotic transformation is caused by loss of abdominal Hox gene expression, likely due to expanded Tc-Krüppel expression. Our findings support the theory that the neuroblast timer series was co-opted for use in insect segment patterning, and contribute to our growing understanding of the evolution and function of the gap gene network outside of Drosophila.


Assuntos
Padronização Corporal/genética , Genes Homeobox , Proteínas de Homeodomínio/genética , Proteínas de Insetos/genética , Células-Tronco Neurais/metabolismo , Fatores do Domínio POU/genética , Tribolium/embriologia , Tribolium/genética , Animais , Blastoderma/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Drosophila/genética , Drosophila/crescimento & desenvolvimento , Desenvolvimento Embrionário/genética , Feminino , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Proteínas de Homeodomínio/metabolismo , Proteínas de Insetos/metabolismo , Masculino , Fatores do Domínio POU/metabolismo , Interferência de RNA , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo
15.
J Vis Exp ; (173)2021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34398141

RESUMO

Due to their optical clarity and rapid development, zebrafish embryos are an excellent system for examining cell behaviors and developmental processes. However, because of the complexity and redundancy of embryonic signals, it can be challenging to discern the complete role of any single signal during early embryogenesis. By explanting the animal region of the zebrafish blastoderm, relatively naïve clusters of embryonic cells are generated that can be easily cultured and manipulated ex vivo. By introducing a gene of interest by RNA injection before explantation, one can assess the effect of this molecule on gene expression, cell behaviors, and other developmental processes in relative isolation. Furthermore, cells from embryos of different genotypes or conditions can be combined in a single chimeric explant to examine cell/tissue interactions and tissue-specific gene functions. This article provides instructions for generating zebrafish blastoderm explants and demonstrates that a single signaling molecule - a Nodal ligand - is sufficient to induce germ layer formation and extension morphogenesis in otherwise naïve embryonic tissues. Due to their ability to recapitulate embryonic cell behaviors, morphogen gradients, and gene expression patterns in a simplified ex vivo system, these explants are anticipated to be of great utility to many zebrafish researchers.


Assuntos
Blastoderma , Peixe-Zebra , Animais , Blastoderma/metabolismo , Padronização Corporal , Regulação da Expressão Gênica no Desenvolvimento , Morfogênese , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismo
16.
Poult Sci ; 100(8): 101227, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34175796

RESUMO

At the time of oviposition, the chicken embryo is in its blastodermal stage. The blastoderm displays the unique ability to undergo developmental arrest at low temperatures in a process called "embryonic diapause." In the wild, diapause occurs in freshly laid eggs until the last egg of the clutch has been laid, providing an evolutionary advantage to hens that can synchronously hatch their eggs. The poultry industry utilizes the diapause phenomenon to store eggs before incubation, thereby mitigating their logistic problems. The embryos can only be stored at particular embryonic stages-termed "diapause developmental window" (DW)-if they are to continue to develop normally thereafter. Both cellular and molecular mechanisms define the limits of this DW which broadly comply with onset of blastulation to early gastrulation. Storage conditions affect the cellular and molecular characteristics of the embryo during this window and their ability to successfully resume development (SRD). At storage temperatures of ~12°C to 18°C, embryos can undergo diapause for a short period (up to 7 days (d)) without affecting SRD. However, following longer period of diapause (up to 28 d), embryo stored at ~12°C, but not at ~18°C, can resume development normally. Moreover, eggs can be heated before or during the storage period which will lead to their commencing in development; however, unlike the non-heated embryos, the storage temperature for heated embryos, which are more advance in developing, is not clear. Thus, based on SRD, this review brings evidence supporting the notion that a lower storage temperature is beneficial for early-stage blastoderms whereas a higher storage temperature is favorable for later-stage/gastrulating embryos. Our understanding of the molecular mechanisms underlying the relationship between storage temperature and development stage within the DW is rather limited. However, it is expected to become relevant in light of the effect of selective breeding of modern avian birds on the advancement of embryonic development stage. Thus, this review discusses parameters that are regulated during the DW and affect SRD, and presents the need to adopt new storage techniques. The pre-managerial decision of required duration of storage with manipulation of storage temperature in the currently used storage techniques may improve SRD characteristics.


Assuntos
Galinhas , Diapausa , Animais , Blastoderma , Embrião de Galinha , Temperatura Baixa , Feminino , Óvulo
17.
Dev Biol ; 476: 249-258, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33905721

RESUMO

Embryogenesis proceeds by a highly regulated series of events. In animals, maternal factors that accumulate in the egg cytoplasm control cell cycle progression at the initial stage of cleavage. However, cell cycle regulation is switched to a system governed by the activated nuclear genome at a specific stage of development, referred to as maternal-to-zygotic transition (MZT). Detailed molecular analyses have been performed on maternal factors and activated zygotic genes in MZT in mammals, fishes and chicken; however, the underlying mechanisms remain unclear in quail. In the present study, we demonstrated that MZT occurred at blastoderm stage V in the Japanese quail using novel gene targeting technology in which the CRISPR/Cas9 and intracytoplasmic sperm injection (ICSI) systems were combined. At blastoderm stage V, we found that maternal retinoblastoma 1 (RB1) protein expression was down-regulated, whereas the gene expression of cyclin D1 (CCND1) was initiated. When a microinjection of sgRNA containing CCND1-targeted sequencing and Cas9 mRNA was administered at the pronuclear stage, blastoderm development stopped at stage V and the down-regulation of RB1 did not occur. This result indicates the most notable difference from mammals in which CCND-knockout embryos are capable of developing beyond MZT. We also showed that CCND1 induced the phosphorylation of the serine/threonine residues of the RB1 protein, which resulted in the degradation of this protein. These results suggest that CCND1 is one of the key factors for RB1 protein degradation at MZT, and the elimination of RB1 may contribute to cell cycle progression after MZT during blastoderm development in the Japanese quail. Our novel technology, which combined the CRISPR/Cas9 system and ICSI, has the potential to become a powerful tool for avian-targeted mutagenesis.


Assuntos
Coturnix/embriologia , Coturnix/genética , Ciclina D1/genética , Animais , Blastoderma/embriologia , Blastoderma/metabolismo , Ciclo Celular/genética , Pontos de Checagem do Ciclo Celular/genética , Ciclina D1/metabolismo , Desenvolvimento Embrionário/genética , Expressão Gênica/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Genoma/genética , RNA Mensageiro/genética , Ativação Transcricional/genética , Zigoto/metabolismo
18.
Cell ; 184(7): 1914-1928.e19, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33730596

RESUMO

Embryo morphogenesis is impacted by dynamic changes in tissue material properties, which have been proposed to occur via processes akin to phase transitions (PTs). Here, we show that rigidity percolation provides a simple and robust theoretical framework to predict material/structural PTs of embryonic tissues from local cell connectivity. By using percolation theory, combined with directly monitoring dynamic changes in tissue rheology and cell contact mechanics, we demonstrate that the zebrafish blastoderm undergoes a genuine rigidity PT, brought about by a small reduction in adhesion-dependent cell connectivity below a critical value. We quantitatively predict and experimentally verify hallmarks of PTs, including power-law exponents and associated discontinuities of macroscopic observables. Finally, we show that this uniform PT depends on blastoderm cells undergoing meta-synchronous divisions causing random and, consequently, uniform changes in cell connectivity. Collectively, our theoretical and experimental findings reveal the structural basis of material PTs in an organismal context.


Assuntos
Embrião não Mamífero/fisiologia , Desenvolvimento Embrionário , Animais , Blastoderma/citologia , Blastoderma/fisiologia , Caderinas/antagonistas & inibidores , Caderinas/genética , Caderinas/metabolismo , Adesão Celular , Embrião não Mamífero/citologia , Morfolinos/metabolismo , Reologia , Viscosidade , Peixe-Zebra/crescimento & desenvolvimento
19.
J Cell Physiol ; 236(2): 1391-1400, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-32749682

RESUMO

The development of primordial germ cells (PGCs) undergoes epigenetic modifications. The study of histone methylation in regulating PGCs is beneficial to understand the development and differentiation mechanism of germ stem cells. Notably, it provides a theoretical basis for directed induction and mass acquisition in vitro. However, little is known about the regulation of PGC formation by histone methylation. Here, we found the high enrichment of H3K4me2 in the blastoderm, genital ridges, and testis. Chromatin immunoprecipitation sequencing was performed and the results revealed that genomic H3K4me2 is dynamic in embryonic stem cells, PGCs, and spermatogonial stem cells. This trend was consistent with the H3K4me2 enrichment in the gene promoter region. Additionally, narrow region triggered PGC-related genes (Bmp4, Wnt5a, and Tcf7l2) and signaling pathways (Wnt and transforming growth factor-ß). After knocking down histone methylase Mll2 in vitro and vivo, the level of H3K4me2 decreased, inhibiting Cvh and Blimp1 expression, then repressing the formation of PGCs. Taken together, our study revealed the whole genome map of H3K4me2 in the formation of PGCs, contributing to improve the epigenetic study in PGC formation and providing materials for bird gene editing and rescue of endangered birds.


Assuntos
Proteína Morfogenética Óssea 4/genética , Epigênese Genética/genética , Histona Metiltransferases/genética , Testículo/crescimento & desenvolvimento , Células-Tronco Germinativas Adultas/citologia , Células-Tronco Germinativas Adultas/metabolismo , Animais , Blastoderma/crescimento & desenvolvimento , Diferenciação Celular/genética , Galinhas/genética , Galinhas/crescimento & desenvolvimento , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Genitália/crescimento & desenvolvimento , Células Germinativas/crescimento & desenvolvimento , Masculino , Transdução de Sinais/genética , Testículo/metabolismo , Proteína 2 Semelhante ao Fator 7 de Transcrição/genética , Fator de Crescimento Transformador beta/genética , Proteína Wnt-5a/genética
20.
J Exp Zool B Mol Dev Evol ; 336(8): 606-619, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-32649025

RESUMO

Land colonization was a major event in the history of life. Among animals, insects exerted a staggering terrestrialization success, due to traits usually associated with postembryonic life stages, while the egg stage has been largely overlooked in comparative studies. In many insects, after blastoderm differentiation, the extraembryonic serosal tissue wraps the embryo and synthesizes the serosal cuticle, an extracellular matrix that lies beneath the eggshell and protects the egg against water loss. In contrast, in noninsect hexapods such as springtails (Collembola) the early blastodermal cells synthesize a blastodermal cuticle. Here, we investigate the relationship between blastodermal cuticle formation and egg resistance to desiccation in the springtails Orchesella cincta and Folsomia candida, two species with different oviposition environments and developmental rates. The blastodermal cuticle becomes externally visible in O. cincta and F. candida at 22% and 29% of embryogenesis, respectively. To contextualize, we describe the stages of springtail embryogenesis, exemplified by F. candida. Our physiological assays then showed that blastodermal cuticle formation coincides with an increase in egg viability in a dry environment, significantly contributing to hatching success. However, protection differs between species: while O. cincta eggs survive at least 2 hr outside a humid environment, the survival period recorded for F. candida eggs is only 15 min, which correlates with this species' requirement for humid microhabitats. We suggest that the formation of this cuticle protects the eggs, constituting an ancestral trait among hexapods that predated and facilitated the process of terrestrialization that occurred during insect evolution.


Assuntos
Artrópodes , Blastoderma , Óvulo , Animais , Dessecação , Feminino , Oviposição , Óvulo/fisiologia
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