The hindbrain is a highly conserved region of the vertebrate embryonic brain that is divided into segments called rhombomeres (r1 to r7) which express a combination of transcription factors (TF) that confer identity and positional information to the cells that make the rhombomeres. These TF notably include the famous group of Hox proteins, Krox20 and others.
A popular example of cell organization into rhombomeric structures following identity information is the grouping of Hoxb1 expressing cells underlying the making of r4 through mutual repression with r3/r5 Krox20-expressing cells. However, this process as well as many of the kind in hindbrain segmentation studies have so far only been postulated based on reporter assays observed at a few time points in fixed embryos. However, considering the highly dynamic nature of the segmentation process, there is a need to monitor the continuity of this series of events at the cellular level in living embryos. Therefore, this PhD project will aim at imaging the events leading to the restriction of Hoxb1 expression within the r4 cells taking advantage of the MS2/MCP system for the very first time in the vertebrate hindbrain allowing one to study transcription at the level of individual cells by tagging nascent mRNA in living embryos. Through this project and using zebrafish as a model organism, I aim at studying some long-term standing segmentation questions at a deeper level of precision, by addressing questions such as cell organization and the underlying transcription process in space in time as well as cell identity at the border between two adjacent rhombomeres, transcription homogeneity within a rhombomere etc. Furthermore, this live-imaging technique could be very useful to study in a deeper level other highly dynamic vertebrate developmental process in the future such as somitogenesis.
Restriction of Hoxb1 expression underlying r4 formation.
(A) Broad expression of Hox PG1 genes from presumptive r3 to presumptive r7/r8 and in the somites (s1-3 are depicted only) in response to Retinoic Acid (RA) signaling gradient in the pre-rhombomeric hindbrain.
(B) Repression of Hoxb1 in r3/r5 by Krox20. Activation of Hoxb1 expression by Hoxa1;
(C) Gradual restriction of Hoxb1 in r4 via mutual repression of Hox PG1 and Krox20. Hoxb1 starts its own auto-regulation.
(D) Hoxb1 expression is sharply restricted to r4 and Hoxb2 and Hoxa2 contribute to cross-regulation of Hoxb1 in r4;
(E) At the onset of neurogenesis, Hoxb1 expression becomes restricted in specific neuron precursors within r4.