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Discipline
Biological
Keywords
Embryo Elongation
Morpholgical Landmarks
Chicken Embryo
Observation Type
Standalone
Nature
Standard Data
Submitted
Mar 22nd, 2019
Published
Apr 5th, 2019
  • Abstract

    Early embryo elongation involves coordinated cellular and tissue behaviors that are readily observable in the chick embryo vertebrate model system. Easily identifiable morphological landmarks are crucial to obtain reliable morphometric data, particularly when assessing tissue elongation over time. The posterior end of the primitive streak marks the caudal end of the chick embryonic tissue. However, the identification of its precise location is ambiguous, especially to the untrained eye. Herein, we assessed if the posterior limit of the area pellucida (pPL), which is readily recognizable due to the optical contrast with the area opaca, is a valid proxy for the caudal limit of the primitive streak. Measurements of total embryo length were performed in multiple images of chick embryos over time using both caudal landmarks. We found that the pPL offered greater precision and a higher degree of inter-user reproducibility, when compared to the end of the primitive streak. Importantly, our work uncovers a quantitative proportionality between embryo length measurements using the end of the primitive streak and the pPL as caudal landmarks. We have thus validated the pPL as a reliable morphological proxy for the end of the primitive streak in chick embryo elongation studies.

  • Figure
  • Introduction

    Vertebrate embryo body elongation requires the orchestrated growth of multiple tissues, involving cell proliferation, migration and rearrangements along the anterior-posterior axis. During early development, the embryo body elongates as epiblast cells, migrate through the primitive streak and are specified into multiple tissues. Live-imaging studies have been used with great success to characterize the dynamics of these processes, as well as to evaluate the impact of different culture conditions or drug exposure on embryo elongation. The chick embryo is an ideal model for such experimental approaches, due to the availability of in ovo and ex-ovo culture systems amenable to live imaging approaches, and also because it presents striking morphological similarities to human development in early stages.

    The choice of specific morphological landmarks is a critical step when characterizing embryo elongation. These should be easily and reproducibly identifiable throughout the whole course of the experiment, and also among distinct users. When studying net embryo elongation at the expense of the epiblast tissue, it is useful to measure embryo length until the end of the primitive streak. However, it is not trivial to pinpoint this morphological landmark in a reproducible manner, even when analyzing the same embryo over time. This introduces a degree of inaccuracy to the measurements performed, and strongly hinders automated image recognition and analysis. The posterior limit of the area pellucida (henceforth designated pPL) is located immediately caudal to the end of the primitive streak. The pPL is readily recognizable due to the optical contrast between the area pellucida and the area opaca, and has been used to characterize embryo elongation rates in several studies. However, to what extent the pPL can be used as a reliable proxy for the end of the primitive streak has not yet been assessed.

  • Objective

    The objective of this work was to validate the easily identifiable posterior limit of the area pellucida (pPL) as a reliable proxy for the end of the primitive streak for chick embryo elongation studies.

  • Results & Discussion

    We performed time-lapse imaging of chick embryos cultured in New or EC systems (n=3 each) from early gastrulation to somitogenesis stages (HH4 to HH10) (Fig. 1A). Embryo length was measured from the crown to the end of the primitive streak (C-PS; Fig. 1B, C) and to the pPL (C-pPL; Fig. 1B, D). Measurements were performed in multiple independent frames per developmental stage (n=5 each) for each embryo (total n=6). We found that embryo growth over time displays a similar trend independently of the morphological landmark used (Fig. 1C, D). In fact, both C-PS and C-pPL measurements increase steadily in the embryonic stages analyzed.

    To test the reproducibility of the results when the measurements are performed using each landmark, C-PS and C-pPL were measured in a single embryo over time by 3 operators with different years of experience with the chick embryo model (Fig. 1E, F). Figure 1E evidences significant variability in C-PS length measured by distinct users, which can differ up to 0.84 mm (HH5; 2.81 mm average length). This stems from differences in where each operator positions the end of the primitive streak. Importantly, the variability of the C-pPL measurement performed by the independent users is almost negligible (Fig. 1F). This highlights that the contrast between the posterior border of the area pellucida and the area opaca is an easily recognizable landmark for both experienced and inexperienced experimentalists. The pPL landmark thus allows for high precision measurements for embryo length studies.

    For pPL to be a valid proxy for the end of the primitive streak, the distance between the two landmarks (PS-pPL) should be approximately constant over time; i.e., any variation in C-pPL length should be exclusively due to a respective variation in C-PS. We found that this is the case, since the natural variations observed in PS-pPL distances over time (Fig. 1G) were neglectable when compared to the progressive increase in C-pPL length (Fig. 1D). These findings evidence that C-pPL variation over time reliably represents C-PS dynamics, thus validating the use of the pPL as a precise and reliable morphological landmark for chick embryo elongation studies.

    Finally, when C-PS values were plotted against their respective C-pPL, for all embryos and developmental stages analyzed, a direct proportionality was found represented by the equation [C-PS] = -0.378 + 0.947[C-pPL] with R2 = 0.949 (mean error of estimated C-PS = 0.206±0.132). This means that the absolute value of C-PS length can be directly calculated from C-pPL measurement.

  • Conclusions

    Altogether, our results evidence that the posterior limit of the area pellucida (pPL) is a reliable morphological proxy for the end of the primitive streak. The pPL landmark provides higher precision measurements and minimizes inter-user variability. Importantly, we describe a novel quantitative relationship between C-PS and C-pPL, which represents a powerful tool to directly infer C-PS from experimentally-measured C-pPL.

  • Limitations

    This study was performed in HH4-HH10 embryonic stages, thus the pPL landmark was only validated for this developmental time window. Although the findings are consistent in both New and EC culture systems, the sample size is limited and may not reflect the variability that could be found with a larger population set.

  • Conjectures

    With the recent technological developments, there is an increased usage of live-imaging approaches to study embryo elongation and elucidate the mechanisms underlying this highly coordinated event. This may involve experimentally challenging the system by altering gene expression, signaling pathway modulation, microsurgical approaches, among others. As such, a thorough and precise characterization of chick embryo elongation in control conditions is more relevant than ever. The pPL herein validated is a powerful landmark for such studies, as it provides greater precision and inter-user reproducibility in embryo length measurements. Finally, the clear optical contrast between the area pellucida and area opaca may allow the precise identification of the pPL landmark by automated image recognition and analysis tools.

  • Methods

    Chicken embryos and culture

    Fertilized chicken (Gallus gallus) eggs were provided by commercial sources and incubated in a humidified atmosphere and controlled temperature (38ºC). HH4-5 embryos were cultured in either Newor EC culture systems for up to 24 h at 38ºC in a humidified atmosphere.

    Image acquisition and analysis

    Time-lapse movies were performed using a Zeiss SteREOLumarV12 Stereomicroscope coupled with a Zeiss Axiocam MRc camera. Measurements were performed in 5 independent frames per developmental stage for each embryo analyzed (150 images in total), using Axiovision Se 64 Rel 4.9.1 (Carl Zeiss) or FIJI software. Data analysis was performed using Excel and SPSS software.

  • Funding statement

    This work was supported by FCT, Portugal (grant PTDC/BEX-BID/5410/2014) and Research Center Grant UID/BIM/04773/2013 CBMR 1334. The Light Microscopy Unit was partially funded by PPBI-POCI-01-0145-FEDER-022122. TPA and ACMF were supported by FCT fellowships SFRH/BD/84825/2012 and PTDC/BEX-BID/5410/2014, respectively.

  • Acknowledgements

    We thank Gil Carraco for help in time-lapse imaging and Ana P. Martins-Jesus for performing measurements as an independent user. We thank Gil Carraco, Ana P. Martins-Jesus, Isabel Duarte, Ramiro Magno and Paulo Martel for helpful discussions. We acknowledge the staff of the Light Microscopy Unit of CBMR-UAlg for technical support. ACMF thanks José and Ana Paula Fernandes for their constant support in her growth.

  • Ethics statement

    Not Applicable.

  • References
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    Matters13/20

    The posterior limit of the area pellucida (pPL) as a reliable proxy for the end of the primitive streak in chick elongation studies

    Affiliation listing not available.
    Abstractlink

    Early embryo elongation involves coordinated cellular and tissue behaviors that are readily observable in the chick embryo vertebrate model system. Easily identifiable morphological landmarks are crucial to obtain reliable morphometric data, particularly when assessing tissue elongation over time. The posterior end of the primitive streak marks the caudal end of the chick embryonic tissue. However, the identification of its precise location is ambiguous, especially to the untrained eye. Herein, we assessed if the posterior limit of the area pellucida (pPL), which is readily recognizable due to the optical contrast with the area opaca, is a valid proxy for the caudal limit of the primitive streak. Measurements of total embryo length were performed in multiple images of chick embryos over time using both caudal landmarks. We found that the pPL offered greater precision and a higher degree of inter-user reproducibility, when compared to the end of the primitive streak. Importantly, our work uncovers a quantitative proportionality between embryo length measurements using the end of the primitive streak and the pPL as caudal landmarks. We have thus validated the pPL as a reliable morphological proxy for the end of the primitive streak in chick embryo elongation studies.

    Figurelink

    Figure 1. The posterior limit of the area pellucida (pPL) is a valid and reproducible landmark for chick embryo elongation studies.

    (A) Representative images of the developmental stages analyzed, obtained from a single embryo over time. Scale bar: 0.5 mm.

    (B) Illustration of the measurements performed. C-PS: distance from the crown to the caudal end of the primitive streak (red line); C-pPL: distance from the crown to the pPL (blue line); PS-pPL: distance from the caudal end of the primitive streak to the pPL (green line).

    (C) Measured C-PS length per developmental stage. Colored symbols represent different embryos (emb; n=6).

    (D) Measured C-pPL length per developmental stage. Colored symbols represent different embryos (emb; n=6).

    (E) C-PS length of multiple images of a single embryo per developmental stage (n=5), independently measured by 3 different users.

    (F) C-pPL length of multiple images of a single embryo per developmental stage (n=5), independently measured by 3 different users.

    (G) Measured distance between the end of the primitive streak and the pPL (PS-pPL) per developmental stage. Colored symbols represent different embryos (emb; n=6).

    (H) C-PS measures plotted against respective C-pPL lengths. Colored symbols represent different embryos (emb; n=6).

    Introductionlink

    Vertebrate embryo body elongation requires the orchestrated growth of multiple tissues, involving cell proliferation, migration and rearrangements along the anterior-posterior axis[1]. During early development, the embryo body elongates as epiblast cells, migrate through the primitive streak and are specified into multiple tissues. Live-imaging studies have been used with great success to characterize the dynamics of these processes, as well as to evaluate the impact of different culture conditions or drug exposure on embryo elongation[1]. The chick embryo is an ideal model for such experimental approaches, due to the availability of in ovo and ex-ovo culture systems amenable to live imaging approaches, and also because it presents striking morphological similarities to human development in early stages[2].

    The choice of specific morphological landmarks is a critical step when characterizing embryo elongation. These should be easily and reproducibly identifiable throughout the whole course of the experiment, and also among distinct users. When studying net embryo elongation at the expense of the epiblast tissue, it is useful to measure embryo length until the end of the primitive streak. However, it is not trivial to pinpoint this morphological landmark in a reproducible manner, even when analyzing the same embryo over time. This introduces a degree of inaccuracy to the measurements performed, and strongly hinders automated image recognition and analysis. The posterior limit of the area pellucida (henceforth designated pPL) is located immediately caudal to the end of the primitive streak. The pPL is readily recognizable due to the optical contrast between the area pellucida and the area opaca, and has been used to characterize embryo elongation rates in several studies[3][4][5][6]. However, to what extent the pPL can be used as a reliable proxy for the end of the primitive streak has not yet been assessed.

    Objectivelink

    The objective of this work was to validate the easily identifiable posterior limit of the area pellucida (pPL) as a reliable proxy for the end of the primitive streak for chick embryo elongation studies.

    Results & Discussionlink

    We performed time-lapse imaging of chick embryos cultured in New[7] or EC[8] systems (n=3 each) from early gastrulation to somitogenesis stages (HH4 to HH10[9]) (Fig. 1A). Embryo length was measured from the crown to the end of the primitive streak (C-PS; Fig. 1B, C) and to the pPL (C-pPL; Fig. 1B, D). Measurements were performed in multiple independent frames per developmental stage (n=5 each) for each embryo (total n=6). We found that embryo growth over time displays a similar trend independently of the morphological landmark used (Fig. 1C, D). In fact, both C-PS and C-pPL measurements increase steadily in the embryonic stages analyzed.

    To test the reproducibility of the results when the measurements are performed using each landmark, C-PS and C-pPL were measured in a single embryo over time by 3 operators with different years of experience with the chick embryo model (Fig. 1E, F). Figure 1E evidences significant variability in C-PS length measured by distinct users, which can differ up to 0.84 mm (HH5; 2.81 mm average length). This stems from differences in where each operator positions the end of the primitive streak. Importantly, the variability of the C-pPL measurement performed by the independent users is almost negligible (Fig. 1F). This highlights that the contrast between the posterior border of the area pellucida and the area opaca is an easily recognizable landmark for both experienced and inexperienced experimentalists. The pPL landmark thus allows for high precision measurements for embryo length studies.

    For pPL to be a valid proxy for the end of the primitive streak, the distance between the two landmarks (PS-pPL) should be approximately constant over time; i.e., any variation in C-pPL length should be exclusively due to a respective variation in C-PS. We found that this is the case, since the natural variations observed in PS-pPL distances over time (Fig. 1G) were neglectable when compared to the progressive increase in C-pPL length (Fig. 1D). These findings evidence that C-pPL variation over time reliably represents C-PS dynamics, thus validating the use of the pPL as a precise and reliable morphological landmark for chick embryo elongation studies.

    Finally, when C-PS values were plotted against their respective C-pPL, for all embryos and developmental stages analyzed, a direct proportionality was found represented by the equation [C-PS] = -0.378 + 0.947[C-pPL] with R2 = 0.949 (mean error of estimated C-PS = 0.206±0.132). This means that the absolute value of C-PS length can be directly calculated from C-pPL measurement.

    Conclusionslink

    Altogether, our results evidence that the posterior limit of the area pellucida (pPL) is a reliable morphological proxy for the end of the primitive streak. The pPL landmark provides higher precision measurements and minimizes inter-user variability. Importantly, we describe a novel quantitative relationship between C-PS and C-pPL, which represents a powerful tool to directly infer C-PS from experimentally-measured C-pPL.

    Limitationslink

    This study was performed in HH4-HH10[9] embryonic stages, thus the pPL landmark was only validated for this developmental time window. Although the findings are consistent in both New and EC culture systems, the sample size is limited and may not reflect the variability that could be found with a larger population set.

    Conjectureslink

    With the recent technological developments, there is an increased usage of live-imaging approaches to study embryo elongation and elucidate the mechanisms underlying this highly coordinated event. This may involve experimentally challenging the system by altering gene expression, signaling pathway modulation, microsurgical approaches, among others. As such, a thorough and precise characterization of chick embryo elongation in control conditions is more relevant than ever. The pPL herein validated is a powerful landmark for such studies, as it provides greater precision and inter-user reproducibility in embryo length measurements. Finally, the clear optical contrast between the area pellucida and area opaca may allow the precise identification of the pPL landmark by automated image recognition and analysis tools.

    Methodslink

    Chicken embryos and culture

    Fertilized chicken (Gallus gallus) eggs were provided by commercial sources and incubated in a humidified atmosphere and controlled temperature (38ºC). HH4-5[9] embryos were cultured in either New[7]or EC[8] culture systems for up to 24 h at 38ºC in a humidified atmosphere.

    Image acquisition and analysis

    Time-lapse movies were performed using a Zeiss SteREOLumarV12 Stereomicroscope coupled with a Zeiss Axiocam MRc camera. Measurements were performed in 5 independent frames per developmental stage for each embryo analyzed (150 images in total), using Axiovision Se 64 Rel 4.9.1 (Carl Zeiss) or FIJI[10] software. Data analysis was performed using Excel and SPSS software.

    Funding Statementlink

    This work was supported by FCT, Portugal (grant PTDC/BEX-BID/5410/2014) and Research Center Grant UID/BIM/04773/2013 CBMR 1334. The Light Microscopy Unit was partially funded by PPBI-POCI-01-0145-FEDER-022122. TPA and ACMF were supported by FCT fellowships SFRH/BD/84825/2012 and PTDC/BEX-BID/5410/2014, respectively.

    Acknowledgementslink

    We thank Gil Carraco for help in time-lapse imaging and Ana P. Martins-Jesus for performing measurements as an independent user. We thank Gil Carraco, Ana P. Martins-Jesus, Isabel Duarte, Ramiro Magno and Paulo Martel for helpful discussions. We acknowledge the staff of the Light Microscopy Unit of CBMR-UAlg for technical support. ACMF thanks José and Ana Paula Fernandes for their constant support in her growth.

    Conflict of interestlink

    The authors declare no conflicts of interest.

    Ethics Statementlink

    Not Applicable.

    No fraudulence is committed in performing these experiments or during processing of the data. We understand that in the case of fraudulence, the study can be retracted by ScienceMatters.

    Referenceslink
    1. Bertrand Bénazéraf
      Dynamics and mechanisms of posterior axis elongation in the vertebrate embryo
      Cellular and Molecular Life Sciences, 76/2019, pages 89-98 DOI: 10.1007/s00018-018-2927-4chrome_reader_mode
    2. Claudio Stern
      The chick model system: a distinguished past and a great future
      The International Journal of Developmental Biology, 62/2018, pages 1-4 DOI: 10.1387/ijdb.170270cschrome_reader_mode
    3. Ruth Bellairs, D. R. Bromham, C. C. Wylie
      The influence of the area opaca on the development of the young chick embryo
      Journal of Embryology and Experimental Morphology, 17/1967, pages 195-212 chrome_reader_mode
    4. Claudio D. Stern, Ruth Bellairs
      The roles of node regression and elongation of the area pellucida in the formation of somites in avian embryos
      Journal of Experimental Morphology, 81/1984, pages 75-92 chrome_reader_mode
    5. Kaichiro Sawada, Hirohiko Aoyama
      Fate maps of the primitive streak in chick and quail embryo: ingression timing of progenitor cells of each rostro-caudal axial level of somites.
      The International Journal of Developmental Biology, 43/1999, pages 809-815 chrome_reader_mode
    6. Chompunut Lumsangkul, Yang-Kwang Fan, Shen-Chang Chang, Yh-Cherng Ju, Hsin-I. Chiang
      Characterizing early embryonic development of Brown Tsaiya Ducks (Anas platyrhynchos) in comparison with Taiwan Country Chicken (Gallus gallus domestics)
    7. D. A. T. New
      A New Technique for the Cultivation of the Chick Embryo in vitro
      Journal of Embryology and Experimental Morphology, 3/1955, page 320–331 chrome_reader_mode
    8. Susan C. Chapman, Jérôme Collignon, Gary C. Schoenwolf, Andrew Lumsden
      Improved method for chick whole‐embryo culture using a filter paper carrier
    9. Viktor Hamburger, Howard L. Hamilton
      A series of normal stages in the development of the chick embryo
      Journal of Morphology, 88/1951, pages 49-92 DOI: 10.1002/jmor.1050880104chrome_reader_mode
    10. Johannes Schindelin, Ignacio Arganda-Carreras, Erwin Frise, Verena Kaynig, Mark Longair, Tobias Pietzsch, Stephan Preibisch, Curtis Rueden, Stephan Saalfeld, Benjamin Schmid, Jean-Yves Tinevez, Daniel James White, Volker Hartenstein, Kevin Eliceiri, Pavel Tomancak, Albert Cardona
      Fiji: an open-source platform for biological-image analysis
      Nature Methods, 9/2012, pages 676-682 DOI: 10.1038/nmeth.2019chrome_reader_mode
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