Divide and conquer: Segmentation and patterning of the anteroposterior axis

Abstract

The formation of the anteroposterior (AP) axis is one of the key events that occur during embryogenesis. Here we investigate the dual processes of patterning and segmentation of the AP axis. To study the role of Hox genes in AP patterning, we decided to analyse the function of the PG1 (paralogous group 1) genes (Hoxa1, -b1, and -d1), the first Hox genes to be expressed in the embryo (Chapter II). PG1 genes are expressed in a widely overlapping domain in the future hindbrain and its associated neural crest. To overcome the functional redundancy of the Hox genes we knocked down the complete paralogous group using morpholinos (MOs) against all three PG1 genes. In the triple PG1 knockdown embryos, the hindbrain lacks segments and the posterior expansion of the rhombomere 1 (r1) marker, Gbx-2, suggests that in the absence of Hox PG1 expression the hindbrain acquires an r1-like fate. This effect could be due to the reduction of other Hox genes, as we show that the ‘Hox code’ is severely perturbed in the PG1 knockdown. Furthermore, PG1 function is also necessary for the migration of the neural crest cells and the formation of the gill cartilages.

In Chapter III we study the expression and function of X-Delta-2, one of the genes shown previously to be involved in the somitogenesis of Xenopus laevis. We show that X-Delta-2 pre-patterns the presomitic mesoderm (PSM) from the end of gastrulation on, much earlier than the formation of the first pair of somites. The expression analysis suggests that X-Delta-2 is oscillating in the posterior part of the PSM, leading to the formation of a stripe of expression marking the presumptive somites. Complementary to the expression in the paraxial mesoderm, X-Delta-2 is also expressed in the central nervous system (CNS) and in the cranial placodes. Loss of function studies showed that X-Delta-2 is also involved in hindbrain segmentation and in regulating gene expression throughout the brain. Furthermore, X-Delta-2 also seems to be involved in the determination of eye size and in the neurogenesis and migration of the cranial placode cells.

In vertebrates, somites are formed by segmentation of the Hox expressing paraxial mesoderm. Hox genes confer specific identity to the somites, and their anterior boundaries are maintained at the same somite number. This suggests that somitogenesis and AP patterning are coordinated at some level. In Chapter IV we study the interaction between these two events, and more precisely between X-Delta-2 and PG1 genes. We show that besides its role in segmentation, X-Delta-2 is also vital for Hox gene expression. X-Delta-2 function is necessary for both ectodermal and mesodermal expression, not only during somitogenesis, as previously shown, but also during gastrulation when the 'Hox code' starts to be set. We also show evidence that the regulation of Hox genes is via the intracellular domain of X-Delta-2, and not via the Notch receptor, as is the case in the somitogenesis process. For the first time evidence is shown for a clear involvement of the Hox genes in the somitogenesis process. The loss of PG1 gene function not only prevents segmentation in the hindbrain, as shown in Chapter II, but also stops the formation of somites in the paraxial mesoderm. This effect is likely to be via X-Delta-2, as we show that its expression in the PSM is downregulated.

Our results show that the same molecules, X-Delta-2 and Hox genes, are involved in the tightly linked processes of segmentation and patterning throughout the AP axis, as discussed in Chapter V.