SPATIO-TEMPORAL DYNAMICS OF EARLY SOMITE SEGMENTATION IN THE CHICKEN EMBRYO

  1. Ana Cristina Maia-Fernandes
  2. Ana Martins-Jesus
  3. Tomás Pais-de-Azevedo
  4. Ramiro Magno
  5. Isabel Duarte
  6. Raquel P. Andrade

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Abstract

During vertebrate embryo development, the body is progressively segmented along the anterior-posterior (A-P) axis early in development. The rate of somite formation is controlled by the somitogenesis embryo clock (EC), which was first described as gene expression oscillations of hairy1 ( hes4 ) in the presomitic mesoderm of chick embryos with 15-20 somites. Here, the EC displays the same periodicity as somite formation, 90 min, whereas the posterior-most somites (44-52) only arise every 150 minutes, matched by a corresponding slower pace of the EC. Evidence suggests that the rostral-most somites are formed faster, however, their periodicity and the EC expression dynamics in these early stages are unknown. In this study, we used time-lapse imaging of chicken embryos from primitive streak to somitogenesis stages with high temporal resolution (3-minute intervals). We measured the length between the anterior-most and the last formed somitic clefts in each captured frame and developed a simple algorithm to automatically infer both the length and time of formation of each somite. We found that the occipital somites (up to somite 5) form at an average rate of 75 minutes, while somites 6 onwards are formed approximately every 90 minutes. We also assessed the expression dynamics of hairy1 using half-embryo explants cultured for different periods of time. This showed that EC hairy1 expression is highly dynamic prior to somitogenesis and assumes a clear oscillatory behaviour as the first somites are formed. Importantly, using ex ovo culture and live-imaging techniques, we showed that the hairy1 expression pattern recapitulates with the formation of each new pair of somites, indicating that somite segmentation is coupled with EC oscillations since the onset of somitogenesis.

Highlights

  • Time of early somite formation can be inferred from sequential length measurements

  • The cranial-most somites are formed faster than trunk somites

  • Oscillations of hairy1 expression are temporally coupled with early somite formation

GRAPHICAL ABSTRACT

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    Reply to the reviewers

    1. General Statements

    The authors greatly appreciate Review Commons’ innovative approach to scientific review and publishing. We thank the reviewers for their kind words regarding the manuscript overall quality and for highlighting the quantitative approach and reproducibility of this work. We further thank the concerns raised and suggestions made that have contributed to improving the manuscript. Below is a point-by-point response to the reviewers, organized into sections that discriminate the alterations already made and plans for further experiments and revisions. We hope that they appropriately address the reviewers' concerns.

    2. Description of the planned revisions

    Reviewer #2: Figure 6 in particular, the number of analyzed embryos is small, given the fact that there is a lot of inter-individual heterogeneity in this process it could well be that the authors got, by chance, two embryos out of three having the same pattern of Hairy1 expression.

    R: The authors appreciate the concern raised by Reviewer#2. This experiment is very time consuming and difficult to execute, which is why the number of samples is limited. Overall, we analyzed 7 embryos and 5 recapitulated the pattern of gene expression. We were particularly interested in the occipital somites and, in this time window, 3 out of 4 showed the same expression pattern. Nevertheless, further experiments will be performed to increase the number of analyzed individuals. We are confident this will contribute to strengthening the conclusions of our work.

    Reviewer #2: I believe that an additional shorter time point (+15 or 30 min) with a different pattern of the oscillatory gene would also add to the characterization of the dynamics (same for Fig 5c). This is particularly true given that the domain of expression of Hairy 1 analyzed in Figure 6 is localized quite rostral which might be interpreted as a phase 1 or a phase 2 as well (as initially described in Palmeirim et al 1997).

    R: We thank Reviewer#2 for this suggestion. We have preliminary data (20-40 min) evidencing different patterns of expression and will perform more experiments to complement these results. Figures will be modified to include samples incubated for shorter time intervals, to evidence different expression patterns obtained in these conditions.

    3. Description of the revisions that have already been incorporated in the transferred manuscript

    Reviewer #1 | Major points:

    1. In the RESULTS session, "Occipital somites are formed faster than cervical and trunk somites," the authors argue that the occipital somites form with greater temporal variability than the neck and trunk somites. Judging from Figures 3C and 3D, I feel it is the case. However, the authors should demonstrate it through statistical analysis.

    2. In the RESULTS session, "Anterior-posterior length of rostral somites," the authors concluded that "the large variability in measurements of somites 17-20 most probably results from the rotation of the embryo body in these developmental stages." Probably they mentioned data of the length of #17-#20 somites in Table1. They should demonstrate it through statistical analysis to show the large variability in the specific area. I understand that embryo rotation could be a reason for the variability. The authors should show evidence. Or they should discuss various possibilities from a broad perspective.

    R: The authors thank Reviewer#1 for suggesting a statistical approach to better characterize the data variability obtained. We performed a Brown-Forsythe test and found that, indeed, there is a statistically significant difference between the temporal variability (period) of somites 1-7 and 8-20 (P-value = 0.02319). Application of the Brown-Forsythe test also found a non-equal variability in the length of the somites #17-20 (P-value = 0.005403). A new Supplementary Figure 4 was added, displaying these results.

    The Brown-Forsythe test is a statistical method used to assess the equality of variances in a dataset across different groups, in this case, the period or the length of early and late somites. It is a robust alternative to the traditional Levene's test, particularly useful when the assumption of homogeneity of variances is not met, such as when the data distributions are skewed or contain outliers (which is the case with our data). It calculates the absolute deviations of individual observations from their respective group medians, which makes it less sensitive than the Levene's test to extreme values. By comparing these deviations between groups, the Brown-Forsythe test helps determine whether the variance differs significantly across the groups. We are confident that this result confers robustness to our claims, and hope that it appropriately addresses the reviewer's concerns.

    Regarding the reasons underlying the variability in the length of #17-#20 somites, we believe it is mainly due to technical constraints. In early developmental stages the chicken embryo is flat, and measurements are easily performed along the anterior-posterior (A-P) somite axis. When somites 17-20 are formed, the embryonic axis starts undergoing rotation, meaning that in some cases we may be measuring along a rotated somite axis. In our work somite length is determined as soon as the posterior intersomitic cleft is formed, so an alternative explanation could be that each somite is formed with a variable length, that is soon after consolidated, resulting in the characteristic consistent metameric organization of somites along the embryo body axis. This is highly unlikely because the length of somites 17-20 long after they are formed (Herrmann et al., 1951) is within the same value range we observed.

    The manuscript has been altered to include the above-mentioned information, as follows:

    • Methods section, under Statistical analysis (Line 153): “To assess the homogeneity of variances between early and late somites, we applied the Brown-Forsythe test on both the period and length measurements. This method involves computing the absolute deviations of individual observations from their respective group medians, rendering it less sensitive to extreme values (outliers). Through the comparison of these deviations across groups, the Brown-Forsythe test aids in determining the statistical significance of variance disparities.”
    • Results section, under Anterior-posterior length of rostral somites (Line 201): “A larger variability was obtained for measurements of somites 17-20 (Supplementary Figure 4A), although this most probably results from the rotation of the embryo body in these developmental stages, hindering precise length measurements of the somite A-P axis.”
    • Results section, under Occipital somites are formed faster than cervical and trunk somites (Line 216): “Remarkably, there is substantial variability in the time of formation of the early-most somites (Figure 3C; Supplementary Figures 3), which gradually stabilizes until somite 8 onwards, where both somite formation time and variability is equivalent to that observed for somites 15-20 (Figure 3C, D; Table 1; Supplementary Figure 4B).”
    1. The authors do not describe the expression patterns of hairy1 in the PSM in the manuscript, but they merely judged whether they are different or the same (recapitulate). The description of the expression pattern needs to be revised totally. The authors should describe the expression patterns of hairy1 in the PSM of each sample carefully and in detail. Fortunately, the previous report (Pourquie and Tam, Developmental Cell, 1, 619-620, 2001) categorized the expression patterns of the EC genes into three phases. The authors should at least categorize each sample according to the criterion by Pourque and Tam. If arrows of brackets indicate the area of expression, it is reader-friendly.

    R: A thorough characterization of segmentation clock gene expression (including hairy1) in the PSM of early somitogenesis chick embryos has been previously described (Rodrigues et al, 2006). For this reason, the authors focused mainly on a comparative analysis of the hairy1 expression patterns obtained in explants incubated for different periods of time. The authors acknowledge, however, the need for further description of the expression patterns obtained in early gastrulation stages, which haven’t been previously documented. Overall, the following alterations were made to the manuscript text:

    Line 231: “The regions with greater variability of hairy1 expression included the neural plate, anterior to the node, the epiblast posterior to the node encompassing the precursors of the paraxial mesoderm (Psychoyos & Stern, 1996) and the caudal-most epiblast. hairy2 expression was also very dynamic along the embryo A-P axis (n=20) (Figure 4B), evidencing chevron-like expression domains, that appear at different levels of the primitive streak, as previously described by Jouve and collaborators (Jouve et al., 2002).”

    Line 245: “As somitogenesis takes place, hairy1 and hairy2 expression patterns retain their dynamic properties in the PSM (Figure 5A, B), as previously described (Rodrigues et al., 2006).”

    The authors further thank Reviewer#1’s suggestion to include brackets indicating the areas of hairy1 expression. Figures 4, 5 and 6 have been altered accordingly, which, indeed, makes figure interpretation more reader-friendly. The gene expression phases presented in Palmeirim et al (1997) and then by Pourquié and Tam (2001) mean to summarize a dynamic expression, with continuous intermediate phases, making it difficult to clearly categorize each pattern obtained. Since our purpose was to evaluate if the entire expression pattern was recapitulated (irrespective of the specific phase), we believe that categorizing each sample in phases is not paramount for result interpretation.

    Reviewer #1 | Minor points.

    1. In the RESULTS session, "Anterior-posterior length of rostral somites," the authors described that the average length of somites ranges from 118 - 191 _μ_m. But according to Table-1, the lowest length of somite is 115.92 _μm (∼_116 _μ_m). So, the lower limit should be corrected here.

    R: The authors thank the reviewer for pointing this out. The text has been appropriately modified in Line 199 of the revised manuscript.

    1. In the DISCUSSION section, the authors mentioned that they presented a thorough characterization of the size and time of formation of the first ten somites in the chicken embryo. But based on the tables and figures, it will only be the first nine somites, not the ten.

    R: The authors agree with the reviewer’s comment. The text has been appropriately modified in Lines 128, 176 and 279 of the revised manuscript.

    1. In the GRAPHICAL ABSTRACT, if the color of the oscillation line is the same as the corresponding somites, it is intuitive.

    R: The authors thank the reviewer for this suggestion and have modified the graphical abstract accordingly. We employed multiple shades of the same color (corresponding to different positions along the body axis) to represent different embryos.

    1. It would be helpful if the manuscript contained both page and line numbers.

    R: Page and line numbers were added to the revised manuscript.

    Reviewer #2

    Minor comment: In 5 c similar patterns and newly formed somites should be pointed out by arrows on the figure to help the readers.

    R: We thank Reviewer#2 for this suggestion. Arrows and brackets have been added to the figure to highlight the newly formed somites and gene expression domains, respectively.

    Minor comment: To be more specific the term segmentation clock should be used instead of embryonic clock as I believe there are other embryonic clocks (cell cycle, circadian, etc..)

    R: The authors appreciate the suggestion of Reviewer#2 regarding the term used to identify the molecular oscillator in our work. The term “segmentation clock” or “somitogenesis clock” is commonly used to refer to oscillations in hairy1/2 gene expression because their discovery and subsequent study has mainly focused on the somitogenesis process. Oscillations of hairy1/2 expression (Hes1/7 in mouse), however, have also been described in cells and developmental stages that are not associated with somite formation, and herein we describe dynamic expression in epiblast regions containing precursors that don’t give rise to segmented structures. As discussed in our recent paper (Carraco et al., Front. Cell Dev. Biol, 2022), the broader term Embryo Clock may be used to refer to molecular oscillations in embryonic cells, controlled by negative feedback regulation, that play a role in temporally controlled morphogenetic processes and/or cell fate specification.

    In the beginning of our manuscript (Line 75), we clearly state that we are referring to the embryo clock operating during somitogenesis: “(…) somitogenesis embryo clock (EC), comprising genes with cell-autonomous oscillatory expression in the PSM driven by negative feedback loops (reviewed in Carraco et al., 2022)”, so we believe that the term used will be clearly perceived by the reader. For further clarification, however, the subtitle The Embryo Clock in early somitogenesis in the Discussion section has been modified to (Line 321): “The Embryo Segmentation Clock in early somitogenesis”

    Reviewer #3 | Minor comments:

    The authors did not consider the fact that the first formed somite is the second somite. After the formation of the second somites, the real first somite forms anterior to the second somite. Furthermore, the real first and the third somite seems to be formed simultaneously. It is worthy for the authors to re-examine the data, whether the real first somite and the third somite are formed at the same time. And to check whether the first somite was counted to the segmented region. And this point should be at least discussed.

    R: The authors thank Reviewer#3 for the opportunity to clarify this important issue in our manuscript. It was previously described that the first morphological somite formed is, in fact, the second somite, while the “real” first somite is formed later, anteriorly to this one (Hamburger and Hamilton, 1951). This rostral-most somite-like structure is not anteriorly delimited by a fissure and has thus been termed an “incomplete” or “rudimentary” somite (Hinsch & Hamilton, 1956). Since the methodology used in our work relies on measuring the length between the rostral-most and the posterior-most intersomitic clefts, the “rudimentary” somite is not included in our data, and we considered somite #1 as the first somite delimited both anteriorly and posteriorly by intersomitic clefts. This was stated in the Methods section, under Embryo measurements, and has now also been made explicit under Somite nomenclature (Line 120): “Only structures delimited both anteriorly and posteriorly by intersomitic clefts were counted as somites.”

    We, indeed, observe the formation of the “rudimentary” somite anteriorly to somite #1, when somites 3-4 and formed. This information was included in the Discussion section, under Spatio-temporal properties of the rostral somite segmentation (Line 311): “Note that our analysis did not consider the “rudimentary somite”, as defined by Hinsch and Hamilton (Hinsch & Hamilton, 1956) since it does not possess an anterior somitic cleft. We found that this structure becomes clearly visible, rostrally to somite 1, as somites 3-4 are formed.”

    4. Description of analyses that authors prefer not to carry out

    Reviewer #1 (Significance (Required)):

    General assessment: The results are not conceptually new or surprising. However, their careful quantitative analysis is informative and worthwhile to be published because it has yet to be done in the early somite formation. In this manuscript, the authors focused on the early stage of somitogenesis. It will be more informative if they complete this analysis for the whole somite area including the end of somitogenesis.

    **Reviewer #3 | **Referees cross commenting****

    I find all criticisms are justified. The most advance, as stated by other both reviewers, is the quantitative assay of the somite formation, since this is no yet done previously. As suggested by the first reviewer, it will be more informative if the authors complete this analysis for all regions. For all somites would be too much work, but they can select some representative somites of each region in addition to occipital region, such as 3 somites for one region, including the cervical, thoracic, lumbal, sacral and caudal region. Thus, the dynamic of the temporal somite formation of the whole embryo can be analysed using the same method. This will provide much more impact for this work.

    R: The authors thank the Reviewers #1 and #3 for the kind words and for highlighting the quantitative approach taken. Regarding completing the analysis for the whole somite area including the end of somitogenesis, the authors agree that this would be interesting for the community. The focus of this work, however, was a detailed understanding of early somite segmentation, where measurements of somites 14-20 were performed for validation purposes alone of the technical approach developed, since their time of formation has been previously well established. Characterization of somite formation dynamics along the entire embryonic axis, while informative, would entail significant technical challenges, which are beyond the focus of this work. Briefly, we performed live imaging using the EC culture system (Chapman et al, 2001). This appropriately reproduces in ovo development of early embryos but imposes significant constraints on embryo development in older developmental stages, including the ones corresponding to the formation of the last somites. A possible alternative to perform these measurements would be to apply the tissue explant culture system developed by Palmeirim et al., 1997 to different portions of the embryo body, and optimize it for real-time imaging. However, we believe that the time and effort required are beyond the scope of this work and would not significantly contribute to elucidating the main questions addressed in this manuscript.

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    Referee #3

    Evidence, reproducibility and clarity

    Somites form consecutively along the anterior to posterior (AP) axis. The time of the formation of a somite is controlled by the segmentation clock, oscillation of cyclic genes in the presomitic mesoderm. The length of an oscillation cycle differs between species and should also differ between the axial levels. In chicken embryos, one cycle for a trunk somite requires 90 minutes, while it is much slower (150 minutes) for a posterior-most somite. Is this quicker or slower for an anterior-most somite? Andrade' group addressed this question and measured the time of the formation of each occipital somites (somite 1-5). They found that the formation of an occipital somite requires only 75 minutes, while somites from somite 6 onwards takes as long as the trunk somites (about 90 minutes). The faster formation of occipital somites is correlated with the time of the cyclic expression of hairy1 and hairy2.

    Major comments:

    The conclusion is well supported by the data. The measurement of the length increments of the segmented region and then assay using algorithm are well established. Thus, the data are well reproducible.

    Minor comments:

    The authors did not consider the fact that the first formed somite is the second somite. After the formation of the second somites, the real first somite forms anterior to the second somite. Furthermore, the real first and the third somite seems to be formed simultaneously. It is worthy for the authors to re-examine the data, whether the real first somite and the third somite are formed at the same time. And to check whether the first somite was counted to the segmented region. And this point should be at least discussed.

    Referees cross commenting

    I find all criticisms are justified. The most advance, as stated by other both reviewers, is the quantitative assay of the somite formation, since this is no yet done previously. As suggested by the first reviewer, it will be more informative if the authors complete this analysis for all regions. For all somites would be too much work, but they can select some representative somites of each region in addition to occipital region, such as 3 somites for one region, including the cervical, thoracic, lumbal, sacral and caudal region. Thus, the dynamic of the temporal somite formation of the whole embryo can be analysed using the same method. This will provide much more impact for this work.

    Significance

    Significance: The measurement of the length increments of the segmented region and then assay using algorithm are the novelty and strengths of this study. So, the data are reproducible and objective.

    The results of this study extend our understanding about the dynamic process of the somitogenesis. Especially, the most interesting point is that based on this result, we can see that the segmentation clock runs faster in the head region, and then slow down gradually along the AP axis.

    Audience: specialized, basic research
    The developmental biologist will be interested in this topic.

    My expertise is the somite development, somite differentiation, mesoderm development.

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    Referee #2

    Evidence, reproducibility and clarity

    In this study, Maia-Fernandez et al used time-lapse imaging of chicken embryos to analyze the formation of the first formed somite, by doing in situ hybridization, they checked the dynamic nature of well-known segmentation clock genes during this process. They found that the segmentation clock period is faster for the formation of the first five somites (most anterior) and that this process is underlain by dynamic/cyclic expression of Hairy 1 and Hairy 2 as it has been described for more posterior somites.

    • I believe that there few issues that should be addressed to strengthen the conclusions of the manuscript:
      Figure 6 in particular, the number of analyzed embryos is small, given the fact that there is a lot of inter-individual heterogeneity in this process it could well be that the authors got, by chance, two embryos out of three having the same pattern of Hairy1 expression.
    • I believe that an additional shorter time point (+15 or 30 min) with a different pattern of the oscillatory gene would also add to the characterization of the dynamics (same for Fig 5c). This is particularly true given that the domain of expression of Hairy 1 analyzed in Figure 6 is localized quite rostral which might be interpreted as a phase 1 or a phase 2 as well (as initially described in Palmeirim et al 1997).

    Minor comments:

    In 5 c similar patterns and newly formed somites should be pointed out by arrows on the figure to help the readers.
    To be more specific the term segmentation clock should be used instead of embryonic clock as I believe there are other embryonic clocks (cell cycle, circadian, etc..)

    Significance

    In this study, the authors address the question of the formation of the first-formed somites using bird a model system; this is a conceptual advance in the sense that our knowledge of the dynamics of these critical morphological events is minimal. The technical advances (time-lapse, image analysis, dissection) made by the authors are quite remarkable and allow for filling the gap of knowledge the community has in this particular domain. The article is well written, data are well presented and it is interesting for a large community of developmental biologists.

    My expertise is in cell and tissue morphogenesis of amniote embryos

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    Referee #1

    Evidence, reproducibility and clarity

    Maia-Fernandes et al. investigated somite formation dynamics in the chick embryo's early stage in this manuscript. They found that the cranial most somites (1-5) form faster than the trunk. They also show that the oscillatory expression pattern of hairy1, regarded as the somitogenesis embryo clock (EC), is coupled to the somite segmentation in the occipital somites. The results are not conceptually new or surprising; they merely show what has been widely believed. However, their careful quantitative analysis is informative and worthwhile to be published because it has yet to be done in the early somite formation. To improve the manuscript, I have several concerns to be addressed.

    Major points.

    1. In the RESULTS session, "Occipital somites are formed faster than cervical and trunk somites," the authors argue that the occipital somites form with greater temporal variability than the neck and trunk somites. Judging from Figures 3C and 3D, I feel it is the case. However, the authors should demonstrate it through statistical analysis.
    2. The authors do not describe the expression patterns of hairy1 in the PSM in the manuscript, but they merely judged whether they are different or the same (recapitulate). The description of the expression pattern needs to be revised totally. The authors should describe the expression patterns of hairy1 in the PSM of each sample carefully and in detail. Fortunately, the previous report (Pourquie and Tam, Developmental Cell, 1, 619-620, 2001) categorized the expression patterns of the EC genes into three phases. The authors should at least categorize each sample according to the criterion by Pourque and Tam. If arrows of brackets indicate the area of expression, it is reader-friendly.
    3. In the RESULTS session, "Anterior-posterior length of rostral somites," the authors concluded that "the large variability in measurements of somites 17-20 most probably results from the rotation of the embryo body in these developmental stages." Probably they mentioned data of the length of #17-#20 somites in Table1. They should demonstrate it through statistical analysis to show the large variability in the specific area. I understand that embryo rotation could be a reason for the variability. The authors should show evidence. Or they should discuss various possibilities from a broad perspective.

    Minor points.

    1. In the RESULTS session, "Anterior-posterior length of rostral somites," the authors described that the average length of somites ranges from 118 - 191 μm. But according to Table-1, the lowest length of somite is 115.92 μm (∼116 μm). So, the lower limit should be corrected here.
    2. In the DISCUSSION section, the authors mentioned that they presented a thorough characterization of the size and time of formation of the first ten somites in the chicken embryo. But based on the tables and figures, it will only be the first nine somites, not the ten.
    3. In the GRAPHICAL ABSTRACT, if the color of the oscillation line is the same as the corresponding somites, it is intuitive.
    4. It would be helpful if the manuscript contained both page and line numbers.

    Significance

    General assessment: The results are not conceptually new or surprising. However, their careful quantitative analysis is informative and worthwhile to be published because it has yet to be done in the early somite formation. In this manuscript, the authors focused on the early stage of somitogenesis. It will be more informative if they complete this analysis for the whole somite area including the end of somitogenesis.

    Advance: Previously no one provided quantitative data for somite formation. In this viewpoint, this manuscript has an advantage.

    Audience: Their data could be helpful in the generation of mathematical models.

    My field: Developmental Biology

  9. Version published to 10.1101/2023.04.05.535703v1 on bioRxiv