Transducing positional information to the Hox genes: critical interaction of cdx gene products with position-sensitive regulatory elements

Abstract

Studies of pattern formation in the vertebrate central nervous system indicate that anteroposterior positional information is generated in the embryo by signalling gradients of an as yet unknown nature. We searched for transcription factors that transduce this information to the Hox genes. Based on the assumption that the activity levels of such factors might vary with position along the anteroposterior axis, we devised an in vivo assay to detect responsiveness of cis-acting sequences to such differentially active factors. We used this assay to analyze a Hoxb8 regulatory element, and detected the most pronounced response in a short stretch of DNA containing a cluster of potential CDX binding sites. We show that differentially expressed DNA binding proteins are present in gastrulating embryos that bind to these sites in vitro, that cdx gene products are among these, and that binding site mutations that abolish binding of these proteins completely destroy the ability of the regulatory element to drive regionally restricted expression in the embryo. Finally, we show that ectopic expression of cdx gene products anteriorizes expression of reporter transgenes driven by this regulatory element, as well as that of the endogenous Hoxb8 gene, in a manner that is consistent with them being essential transducers of positional information. These data suggest that, in contrast to Drosophila Caudal, vertebrate cdx gene products transduce positional information directly to the Hox genes, acting through CDX binding sites in their enhancers. This may represent the ancestral mode of action of caudal homologues, which are involved in anteroposterior patterning in organisms with widely divergent body plans and modes of development.

Early anteroposterior regionalisation of human neural crest is shaped by a pro-mesodermal factor

“…Our proposed model synthesises previous findings showing that WNT-driven posterior axial identity acquisition in neural derivatives of NMPs takes place prior to neural induction ( Lippmann et al, 2015 ; Metzis et al, 2018 ; Neijts et al, 2017 ; Neijts et al, 2016 ; Takemoto et al, 2006 ) with data illustrating that temporally discrete modes of trunk axial patterning in CNS spinal cord/NC rely on both FGF and WNT activities ( Bel-Vialar et al, 2002 ; Delfino-Machín et al, 2005 ; Dunty et al, 2014 ; Hackland et al, 2019 ; Liu et al, 2001 ; Mazzoni et al, 2013 ; Mouilleau et al, 2021 ; Muhr et al, 1999 ; Nordström et al, 2006 ; Sanchez-Ferras et al, 2016 ; Sanchez-Ferras et al, 2014 ; Sanchez-Ferras et al, 2012 ; van Rooijen et al, 2012 ; Zhao et al, 2014 ). Our work also reflects results showing the existence of distinct phases of Hox gene expression program implementation in vivo : (i) an early ‘plastic’ phase linked to A-P patterning of multipotent axial progenitors/NMPs within their posterior niche and (ii) a later phase, which marks the instalment and fixing of lineage-specific Hox gene expression patterns and the sharpening of their final boundaries in the neural and mesodermal derivatives of axial progenitors as and after they exit the posterior growth zone ( Ahn et al, 2014 ; Brend et al, 2003 ; Charité et al, 1998 ; Deschamps and Wijgerde, 1993 ; Forlani et al, 2003 ; Hayward et al, 2015 ; McGrew et al, 2008 ; Wymeersch et al, 2019 ).…”

Cdx1 Interacts Physically with a Subset of Hox Proteins

“…To determine if Hox and Cdx members interacted on common target promoters, we used a ChIP−re-ChIP approach to assess the occupancy of the Hoxb8 regulatory element, which is Cdx target. 18,28 To this end, P19 cells were transfected with Cdx1 in combination with Hoxd4 or Hoxd4 R,R,R→G,G,G and the response element. As expected, Cdx1, Hoxd4, and Hoxd4 R,R,R→G,G,G were all enriched at this locus (data not shown), and mutation of Hoxd4 did not abrogate its ability to bind to DNA (Figure 5B, right panel).…”

“…To determine if the interaction between Cdx1 and Hox proteins has potential biological impact, we assessed the effect of coexpression of Cdx1 and Hoxd4 on transcription directed by the Hoxb8 promoter region, which contains four consensus Cdx binding sites that can be driven by Cdx1. 18 Transfection analyses in P19 embryocarcinoma cells revealed that Cdx1 and Hoxd4, but not Cdx1 and Hoxd4 R,R,R→G,G,G , activated this reporter (Figure 5C). These data are consistent with the physical interaction between Cdx and Hox having an impact on transcription of at least some common target genes.…”

“…14−17 This is likely a direct relationship, as Cdx binding sites have been identified in numerous Hox promoters, some of which are capable of conveying Hox-like spatiotemporal expression patterns in transgenic models. 18,19 As with many other transcription factors, Cdx1 and Cdx2 have been shown to physically interact with other effectors of transcription. For example, sequences within the Cdx2 homeodomain are involved in interactions with Brn-4 on the proglucagon promoter 20 as well as with HNF1α on the lactase promoter.…”

“…A number of signaling pathways and transcription factors are known to impact Hox expression, among which are the vertebrate Cdx (caudal) family of homeodomain transcription factors, Cdx1, Cdx2, and Cdx4. − A critical role for Cdx upstream of Hox expression was initially suggested by the finding of vertebral homeoses and posterior shifts in the expression of a number of Hox genes in Cdx1 null mutants and Cdx2 heterozygotes. − This is likely a direct relationship, as Cdx binding sites have been identified in numerous Hox promoters, some of which are capable of conveying Hox- like spatiotemporal expression patterns in transgenic models. , …”