Dynamics of growth zone patterning in the milkweed bug Oncopeltus fasciatus

Abstract

We describe the dynamic process of abdominal segment generation in the milkweed bug Oncopeltus fasciatus We present detailed morphological measurements of the growing germband throughout segmentation. Our data are complemented by cell division profiles and expression patterns of key genes, including invected and even-skipped as markers for different stages of segment formation. We describe morphological and mechanistic changes in the growth zone and in nascent segments during the generation of individual segments and throughout segmentation, and examine the relative contribution of newly formed versus existing tissue to segment formation. Although abdominal segment addition is primarily generated through the rearrangement of a pool of undifferentiated cells, there is nonetheless proliferation in the posterior. By correlating proliferation with gene expression in the growth zone, we propose a model for growth zone dynamics during segmentation in which the growth zone is functionally subdivided into two distinct regions: a posterior region devoted to a slow rate of growth among undifferentiated cells, and an anterior region in which segmental differentiation is initiated and proliferation inhibited.

Keywords: Arthropod; Cell division; Growth zone; Segmentation; Terminal addition.

Fig. 1.

Measurements made on the growth zone and segments. (A) Illustration of an Oncopeltus fasciatus embryo after segmentation is complete. Abdominal segments are color-coded, with the posterior inv stripe of each segment in a lighter shade. (B) The growth zone and newly formed segments, indicating the measurements made. (C-E) Violin plots representing the distribution of measurements on the growth zone by developmental stage (number of inv stripes). Pairwise one-way ANOVAs were performed to assess statistically significant changes in dimensions from one stage to the next. *P<0.05, **P<0.01, ***P<0.001. (F-H) Violin plots representing the distribution of measurements on the two most recently formed segments, and the three most recently formed inv stripes, by developmental stage (number of inv stripes). The colors correspond to the segment colors in A. Pairwise one-way ANOVAs were performed to assess statistically significant changes in dimensions of the same segment from one stage to the next. *P<0.05, **P<0.01, ***P<0.001 (colors correspond to the segment in question). In F, asterisks above the plot concern the first pair of measurements of a segment (e.g. stage A1 versus A2 for segment 1).

Fig. 2.

Growth and segmentation. (A) (a) The areas used for the calculation of growth. (b) Growth at stage n was measured by dividing the combined area of the growth zone and the most recently formed segment by the average area of the growth zone in stage n−1. (c) Owing to the way that growth is calculated (i.e. using the averages of all individuals in the same stage), the developmental time for which growth is calculated is offset with respect to the segmental stage. The schematic shows how stages used on the y-axis for C and the x-axis for B relate to each other. (B) Growth calculated as shown in A, using measurement averages. Error bars indicate the (propagated) s.e.m. (C) Staged embryos collected in 60-min windows (n=123), from 44-45 to 55-56 hAEL, as well as the linear model fitting these data (red line).

Fig. 3.

Changes in the growth zone throughout the segmentation process. (A) Embryos of increasing age (stages A3-A9) stained for expression of inv. Note the gradual decrease in growth zone size as segmentation proceeds. (B) Early embryos in stage A0-A1 (during formation of the first abdominal segment). Embryos from a single clutch fixed in 30-min intervals demonstrate changes in growth zone dimensions during the formation of a single segment. The growth zone begins round, gradually elongating and assuming a teardrop shape as the new segment slowly buds from the anterior growth zone. hl, head lobe; md, mandibular segment; mx, maxillary segment; lb, labial segment; T3, third thoracic segment; A1, first abdominal segment.

Fig. 4.

Developmental gene expression patterns during germband segmentation. Expression pattern of cad (A), eve (B) and Dl (C) mRNA at different developmental stages, from the earliest germband (∼40 hAEL) to the final stages of abdominal segmentation (∼55 hAEL). (A) cad is stably expressed in the posterior growth zone throughout germband segmentation. (B) eve displays a more complex expression pattern, in which the entire posterior growth zone expresses eve, yet in the anterior growth zone eve is expressed in a dynamic striped pattern. (C) Dl is expressed in the anterior growth zone in a varying number of stripes, with no expression in the posterior growth zone. More anteriorly, Dl is expressed in the nervous system, seen as two mediolateral lines of punctate expression extending posterior from the head lobes to (but not including) the growth zone. GZ, growth zone; hl, head lobe; md, mandibular segment; mx, maxillary segment; lb, labial segment.

Fig. 5.

Cell division and gene expression in the growth zone. Double stainings of anti-PH3 (green), as an indicator of cell division, with in situ hybridization for segmentation genes (red pseudocolor, detected using brightfield). DAPI is used as nuclear counterstain (blue). The precise age of the embryos is not known, but they are all towards the end of posterior segmentation (∼50 hAEL). Brackets labeled with an asterisk mark a gap in cell proliferation in the anterior growth zone. (A) Embryo stained with anti-PH3 and DAPI without in situ staining. A gap in cell proliferation (asterisk) is noticeable in the anterior growth zone. (B) cad staining correlates with an area with increased PH3⁺ cells. Note that the anterior red staining is an artifact of the image merge process and is not seen in single-stained embryos (compare with Fig. 4A). (C) eve in the posterior overlaps with cad expression. The striped expression pattern of eve corresponds to an area with decreased PH3 staining. (D) inv marks the anterior border of the growth zone and the boundary between high PH3 staining (anteriorly) and low PH3 staining (posteriorly).

Fig. 6.

Cell proliferation in different areas of the germband. (A) Heat map of cell proliferation in the germband. Merged image of 35 PH3-stained germbands in various stages, aged from 46-54 hAEL, aligned at the widest part of the growth zone, translated to a look-up table (legend beneath the panel). The dotted lines delineate the zone of low proliferation. (B) Illustration of eve expression, indicating the different areas for which cell proliferation was calculated in C. (C) Proportion of cells in mitosis in each area (see B), calculated from the number of PH3⁺ cells among total cells (DAPI staining). Error bars indicate s.e.m. *P<0.05, ***P<0.001 (ANOVA).

Fig. 7.

Model of the Oncopeltus growth zone. The segmentation process takes place over three distinct domains. The posterior growth zone is characterized by the expression of cad and the stable expression of eve and probably other pair-rule gene orthologs (jointly indicated in yellow). It is also characterized by a relatively high level of cell division (densely packed large dots). The anterior growth zone is characterized by the dynamic expression of pair-rule gene orthologs; in some other arthropods Notch pathway ligands have a similar expression domain (jointly indicated by blue stripes). Cell division levels are significantly lower (sparsely distributed small dots) than in the domains anterior and posterior to it. Cell movements in this domain lead to the constriction of the growth zone and to the extension of the posterior growth zone posteriorly. The dynamic cyclical expression of genes in this domain leads to the sequential sequestering of the anteriormost tissue into the segmented germband. The posterior of the germband is defined by the expression of inv (red stripe). Cell division levels in this area are higher (densely packed large dots) than in the anterior growth zone.