New tools for self-organised pattern formation

K. Bernhardt, N.S. Chand, J. Haseloff, J.M. Goncalves, X. Zhu, J.W. Ajioka, Y. Xu, D. Rowe, G. Micklem, J. Lee, E.J. Carter
2007 IET Synthetic Biology  
A fundamental question in developmental biology is how the early embryo breaks initial symmetry to establish the spatial coordinate system later important for the organisation of the embryonic body plan. In zebrafish, this is thought to depend on the inheritance of maternal mRNAs [1] [2] [3] , cortical rotation to generate a dorsal pole of beta-catenin activity [4] [5] [6] [7] [8] and the release of Nodal signals from the yolk syncytial layer (YSL) [9] [10] [11] [12] . Recent work aggregating
more » ... work aggregating mouse embryonic stem cells has shown that symmetry breaking can occur in the absence of extra-embryonic tissue [19, 20] . To test whether this is also true in zebrafish, we separated embryonic cells from the yolk and allowed them to develop as aggregates. These aggregates break symmetry autonomously to form elongated structures with an anterior-posterior pattern. Extensive cell mixing shows that any pre-existing asymmetry is lost prior to the breaking morphological symmetry, revealing that the maternal pre-pattern is not strictly required for early embryo patterning. Following early signalling events after isolation of embryonic cells reveals that a pole of Nodal activity precedes and is required for elongation. The blocking of PCP-dependent convergence and extension movements disrupts the establishment of opposing poles of BMP and Wnt/TCF activity and the patterning of anterior-posterior neural tissue. These results lead us to suggest that convergence and extension plays a causal role in the establishment of morphogen gradients and pattern formation during zebrafish gastrulation. Our current understanding of pattern formation during early development relies heavily on the notion of opposing signalling gradients that set-up rudimentary body plans [17] . These gradients establish cell fates in space that in turn lead to the population specific cell behaviours that dictate the complex cell and tissue rearrangement of gastrulation and axial elongation. In zebrafish, opposing Nodal and BMP signalling gradients are thought to be necessary and su cient for the establishment of the body plan as shown by experiments in which deployment of such gradients in animal caps leads to the formation of a complete AP axis [13] . In addition to controlling cell fate assignments, a recent study has demonstrated that Nodal signalling is a key driver of convergence and extension movements and is su cient to generate these behaviours when expressed within zebrafish animal caps [14] . Furthermore, BMP levels have been shown to be important for controlling cell movements during both gastrulation [21] and posterior body elongation [22] . These observations raise the possibility that opposing BMP and nodal signalling gradients are upstream of both morphogenesis and patterning. However, the causal relationships of these processes are di cult to dissociate in situations where continuous external signalling sources are present, either from overexpression experiments or from the extra-embryonic signals present during early development. To follow how cells can develop and pattern in the absence of external signals, we used primary culture of cells from zebrafish embryos at the 256 cell stage i.e. before the midblastula transition ( Figure 1A ). This stage also precedes the formation of the zebrafish extra-embryonic yolk syncytial YSL which has been suggested to shape the embryonic axes through mesendodermal induction [9-12] and regulation of epiboly [15] . Explants from embryos at di↵erent stages between the 64 cell and 512 cells, all exhibited a similar behaviour (Figure S1A-B): they self-organised to form polarised aggregates with a protrusion emerging from one pole. We focused our studies on 256 cells stage embryos that exhibit this behaviour in more than 60% of explants from each experiment (n=20, 80-100 explants per experiment; Video S1). Quantification of aspect ratio of the longest vs. shortest axis of each aggregate over time revealed a coordinated onset of elongation at 7 hours post culture (hpc; Figure S1A ), demonstrating a degree of synchrony in the symmetry breaking event. Explants from Tbx16:GFP reporter embryos [16] revealed mesoderm specification within the elongating end of the aggregate ( Figure 1B ; Video S2), accompanied by polarised expression of tbxta ( Figure 1C ). These results showed how symmetry breaking and mesoderm patterning can occur spontaneously in embryonic cells when separated from the yolk. Fixing aggregates at the onset of elongation revealed a pole of BMP activity ( Figure 1D ) opposite the elongating end that was characterized by ndr1 and ndr2 expression and signalling as revealed by a phospho-Smad2
doi:10.1049/iet-stb:20070020 fatcat:mmso6z2jk5ed5dujycy34cgv6m