Identification of In Vivo Hox13-Binding Sites Reveals an Essential Locus Controlling Zebrafish Brachyury Expression
This week, we profile a recent publication in Development from Zhi Ye (pictured, left) in the lab of David Kimelman (right) at UW.
Can you provide a brief overview of your lab’s current research focus?
My lab studies how the early vertebrate embryo forms using zebrafish as a model system. While it has been known for a very long time that the vertebrate embryo forms progressively from the head to the tail, one of the key recent discoveries in this area has been the finding from several labs, including ours, that most of the vertebrate body, particularly the musculature and spinal cord, comes from a unique bipotent progenitor that can produce either neurons or muscle cells, depending on the signals that the progenitor cell receives. This finding has overturned decades of dogma about how cell fate is determined in an early vertebrate embryo.
What is the significance of the findings in this publication?
In a recent publication, we showed that a set of transcription factors from the Hox13 family act to help the bipotent progenitor population maintain its ability to stay bipotent during the early embryonic stages. This alone was a surprising finding since a number of previous papers had proposed the opposite, that the Hox13 factors act to limit how this progenitor population contributes to the growing embryo.
What was missing from our story, however, was an idea of the direct targets of the Hox13 factors. While the many Hox factors are widely known to be very important in embryonic development, finding their direct transcriptional targets has been a major challenge. ChIP-seq is a widely used technique, but it is essentially hopeless in an embryo where we are studying small domains within the embryo that contain tiny numbers of cells compared to cell culture studies.
Using an amazing recent technology developed by Steven Henikoff’s lab at the Fred Hutchinson Cancer Center, together with specially engineered transgenic zebrafish we produced, we were able to identify in vivo Hox13 target genes. Excitingly, two of the Hox13 binding sites were in enhancer elements located approximately 30 kb upstream of one of the key genes (tbxta) that regulates the fate of the bipotent progenitors. These enhancer elements have been highly conserved through hundreds of millions of years of evolution, demonstrating their importance.
We were able to show that these enhancer elements, when coupled to the proximal promoter for tbxta, specifically drove expression in the bipotent progenitors, and removal of just one of these elements produced fish embryos with shorter tails. All of this now gives us a clear model for how the Hox13 factors act to regulate the bipotent progenitors through the direct action on one of the major essential genes.
What are the next steps for this research?
Depending on the criteria used, there are somewhere between 600 and 1400 Hox13 target genes we identified, and there is a lot to learn about which of these genes are also important in regulating embryonic development. In addition, the tbxta enhancer elements that bind Hox13 also have multiple other conserved sites. We identified the proteins that bind two of these sites, but there are several other sites where we don’t know the binding factors, nor do we know how all of these transcription factors interact on the enhancers to precisely regulate the expression of this key gene, tbxta, within the bipotent progenitors.
This work was funded by:
This work was funded by the NIH, with grants to the Kimelman lab (R01GM079203) and a grant to our bioinformatics colleague Andrea Wills (R01NS099124).