Abstract
The Bicoid (Bcd) protein is a primary determinant of early anterior-posterior (AP) axis specification in Drosophila embryogenesis. This morphogen is spatially distributed in an anterior-high gradient, and affects particular AP cell fates in a concentration-dependent manner. The early distribution and dynamics of the bicoid (bcd) mRNA, the source for the Bcd protein gradient, is not well understood, leaving a number of open questions for how Bcd positional information develops and is regulated. Confocal microscope images of whole early embryos, stained for bcd mRNA or the Staufen (Stau) protein involved in its transport, were processed to extract quantitative AP intensity profiles at two depths (apical-under the embryo surface but above the nuclear layer; and basal-below the nuclei). Each profile was quantified by a two- (or three-) exponential equation. The parameters of these equations were used to analyze the early developmental dynamics of bcd. Analysis of 1D profiles was compared with 2D intensity surfaces from the same images. This approach reveals strong early changes in bcd and Stau, which appear to be coordinated. We can unambiguously discriminate three stages in early development using the exponential parameters: pre-blastoderm (1-9 cleavage cycle, cc), syncytial blastoderm (10-13 cc) and cellularization (from 14A cc). Key features which differ in this period are how fast the first exponential (anterior component) of the apical profile drops with distance and whether it is higher or lower than the basal first exponential. We can further discriminate early and late embryos within the pre-blastoderm stage, depending on how quickly the anterior exponential drops. This relates to the posterior-wards spread of bcd in the first hour of development. Both bcd and Stau show several redistributions in the head cytoplasm, quite probably related to nuclear activity: first shifting inwards towards the core plasm, forming either protrusions (early pre-blastoderm) or round aggregations (early nuclear cleavage cycles, cc, 13 and 14), then moving to the embryo surface and spreading posteriorly. These movements are seen both with the 2D surface study and the 1D profile analysis. The continued spreading of bcd can be tracked from the time of nuclear layer formation (later pre-blastoderm) to the later syncytial blastoderm stages by the progressive loss of steepness of the apical anterior exponential (for both bcd and Stau). Finally, at the beginning of cc14 (cellularization stage) we see a distinctive flip from the basal anterior gradient being higher to the apical gradient being higher (for both bcd and Stau). Quantitative analysis reveals substantial (and correlated) bcd and Stau redistributions during early development, supporting that the distribution and dynamics of bcd mRNA are key factors in the formation and maintenance of the Bcd protein morphogenetic gradient. This analysis reveals the complex and dynamic nature of bcd redistribution, particularly in the head cytoplasm. These resemble observations in oogenesis; their role and significance have yet to be clarified. The observed co-localization during redistribution of bcd and Stau may indicate the involvement of active transport.
Highlights
The concept of morphogenetic gradients in embryo development was introduced in the form of “physiological” gradients of activity many decades ago [1]
The Bcd protein gradient arises from maternally deposited bcd mRNA at the anterior pole of the very early embryo [7]. bcd mRNA is translated at the start of egg deposition, and the protein gradient is established within 3 hours
As discussed in our recent technique paper [60], a two-exponential function (C0ð1Þexp ðað1ÞxÞ þ C0ð2Þexp ðað2ÞxÞ [61]; Fig 2C and 2D) provides a better fit to bcd mRNA profiles than the single-exponential plus uniform background (C0 exp(α x) + B [30], Fig 2A and 2B) more traditionally applied to the Bcd protein gradient, and is especially useful for following the variable bcd mRNA profiles encountered in early developmental stages
Summary
The concept of morphogenetic gradients in embryo development was introduced in the form of “physiological” gradients of activity many decades ago [1]. During the 1960s and early 1970s, this concept was refined in terms of substances (morphogens) which diffused across fields of cells to form spatial concentration gradients [2,3,4]. Simple molecular mechanisms such as localized protein synthesis, diffusion and degradation can produce stationary non-uniform distributions of morphogens that govern embryonic pattern formation. The Bcd protein gradient arises from maternally deposited bcd mRNA at the anterior pole of the very early embryo [7]. The Bcd morphogenetic gradient is subsequently read by ~20–30 target genes, which are expressed in discrete patterns along the AP axis [8,9,10,11,12,13]
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