Embryonic timing, axial stem cells, chromatin dynamics, and the Hox clock

Abstract

Collinear regulation of Hox genes in space and time has been an outstanding question ever since the initial work of Ed Lewis in 1978. Here we discuss recent advances in our understanding of this phenomenon in relation to novel concepts associated with large-scale regulation and chromatin structure during the development of both axial and limb patterns. We further discuss how this sequential transcriptional activation marks embryonic stem cell-like axial progenitors in mammals and, consequently, how a temporal genetic system is further translated into spatial coordinates via the fate of these progenitors. In this context, we argue the benefit and necessity of implementing this unique mechanism as well as the difficulty in evolving an alternative strategy to deliver this critical positional information.

Keywords: Hox genes; TAD; chromatin; collinearity; embryos; stem cells; transcription.

Figure 1.

Collinear expression of Hox genes during the development of trunk axial tissues and limbs. (Left) Early: Schematic drawings of posteriorly overlapping transcript domains of HoxD genes in developing trunk axial tissues and early limb buds of an embryonic day 9.5 (E9.5) mouse embryo. Hox-expressing tissues are neural tube (midline); somites (blocks along the neural tube) labeled as cervical (C), thoracic (T) and lumbar (L); forelimb mesoderm (lateral bulges at the level of somites 7–12); and nascent mesoderm and neurectoderm in the tailbud. (t) Time of anterior to posterior development. Colinearity between the positions of the genes in the cluster and the anterior extension of their expression domains along the antero–posterior embryonic axis is illustrated both in the schematic embryo and by the bars under the cluster drawn below. Hox1 is expressed from an anterior limit that is the most rostral of all Hox genes in the embryo (expression not shown in the embryo; no color in the bar), and Hox8 is expressed from an anterior limit that is less rostral than that of Hox1 in the embryo. Posterior to this Hox8 anterior expression boundary, all genes between 1 and 8 are expressed (green color in the schematic embryo and in the bar corresponding to Hox8). Similarly, posterior to the Hox10 expression boundary in the embryo, all genes between Hox1 and Hox10 are expressed (lighter blue in the schematic embryo and in the bar corresponding to Hox10), and similar representations illustrate the expression of Hox11, Hox12, and Hox13. These domains thus tend to overlap posteriorly in the embryo, like Russian dolls. Expression of other Hox genes is not shown. (Right) Late: Hox transcript distribution in the E10.5 developing tailbud and forelimb bud. The two domains in late forelimb buds mark the future proximal (arm and forearm) and distal (digits) parts of the adult limbs, respectively. (t) Time of development of proximal to distal limb structures. Color codes indicate the cumulative amounts of combinations of Hox transcripts. Anterior is to the top in all schemes.

Figure 2.

Temporal collinearity of Hox gene transcription is relayed by axial progenitors into spatially collinear expression domains. Schematics showing the early expression of HoxA genes. The HoxA cluster is represented in reversed genomic orientation in order to appear comparable with HoxD (Fig. 3). Out of the Hox1 to Hox13 genes, only the 3′-most-located Hox1 and 5′-most-located gene expressed at the stages considered (Hox11) are indicated below the cluster (light-gray box). (From top to bottom) Developmental stages and associated genomic events at the Hox locus (left) and transcription domains of Hox genes (embryos at the right) and of their inducers (embryos on the left). (E6.0) Preferential interactions between 3′-located Hox genes and the 3′ topologically associating domain (TAD). No Hox expression was detected, but priming of the 3′ flanking region (open chromatin) is shown in the Wnt3-expressing region. (E7.2) Hox (and Cdx) transcriptional initiation by Wnt3 and Wnt3a via Wnt-dependent enhancers in the proximal 3′ flanking region. (Light green) Expression domain of Hox1 and Cdx. (E7.5) Mid-trunk Hox gene activation (dark-green domain) through the binding of Cdx products in the cluster and further chromatin opening. Mid-trunk Hox domains reach the axial progenitor region (or the posterior growth zone) in the anterior part of the streak. (E8.0; early somite) Gdf11-induced activation of posterior trunk Hox genes (blue). Expansion of trunk Hox gene transcripts (Hox1 [light green] and mid-trunk Hox [dark green]) into the axial progenitor region (end of phase 1). The color code indicates cumulative amounts of Hox transcript combinations. The expression domains overlap posteriorly, as explained in the legend for Figure 1. For all embryos, anterior is to the left. (PS) Primitive streak. (Arrows) transcriptional activation. Genome topology at the top left is from Dixon et al. (2012).

Figure 3.

Early temporal collinearity of Hox gene transcription is relayed by the axial progenitors into spatially collinear expression domains in the emerging differentiated tissues. (E8.25) The transcript domains of Hox1 followed by that of Hox2 to Hox4 (phase 1, temporal collinearity) expand anteriorly and reach the axial progenitor region ([*] MPs and NMPs) before expanding further anteriorly during phase 2. (E9.25) Transcriptional initiation of the next Hox genes has occurred (mid-trunk Hox4 to Hox8), and activation of more posterior Hox genes takes place (as shown with Hox11). The expression domains expand anteriorly toward the axial progenitor area in those tissues generated by the descendants of the MPs and NMPs (phase 2, spatial collinearity). (E10.5) Hox13 is now transcriptionally activated and expressed strongly posteriorly, where it counteracts further axial extension. The Wnt and Fgf pathways are weakened, and the progenitor niche becomes deficient, with fewer and fewer MPs and NMPs (smaller asterisk). The color code identifies the combinations of Hox genes expressed along the axis at the different stages. MPs and NMPs are found around the anterior part of the primitive streak, just posterior to the node (indentation) at E8.25 and in the tailbud at E9.25 and E10.5. For the E8.25 embryo, anterior is to the left; for the E9.25 and E10.5 embryos, anterior is to the top.

Figure 4.

Two temporal sequences in Hoxd gene activation during limb bud development. (A) Limb phase 1. HoxD in the early bud is controlled (green arrow) exclusively by enhancers located in the 3′ TAD. Time sequence of Hoxd activation (t1 to t11) is the same as in the trunk (Fig. 1), and the nested expression domains generated occupy the proximal part of the E12.5 limb bud (right limb bud) and pattern the long bones along with genes from the HoxA cluster. Arrows are transcriptionally active genes, corresponding to open chromatin. (B) Limb phase 2. Between 24 and 48 h after the start of phase 1, enhancers in the 5′ TAD are switched on and activate Hoxd10 to Hoxd13 (purple arrow). HOX13 products then switch off the 3′ TAD enhancers in cells located at the posterior-distal margin. These cells expand and generate the autopods (hands and feet). (C) Combination of limb phases 1 and 2. The 3′ and 5′ TADs operate sequentially, leaving a zone of low Hox expression as the limb bud grows distally, which generates the future mesopods (wrist and ankle). Anterior is to the top, and distal to the right. The color code for Hox gene expression is the same as in Figure 1.