Hox complexity

Here's a prediction for you: the image below is going to appear in a lot of textbooks in the near future.

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Confocal image of septuple in situ hybridization exhibiting the spatial expression of Hox gene transcripts in a developing Drosophila embryo. Stage 11 germband extended embryo (anterior to the left) is stained for labial (lab), Deformed (Dfd), Sex combs reduced (Scr), Antennapedia (Antp), Ultrabithorax (Ubx), abdominal-A (abd-A), Abdominal-B (Abd-B). Their orthologous relationships to vertebrate Hox homology groups are indicated below each gene.

That's a technical tour-de-force: it's a confocal image of a Drosophila embryo, stained with 7 fluorescent probes against different Hox genes. You can clearly see how they are laid out in order from the head end (at the left) to the tail end (which extends to the right, and then jackknifes over the top). Canonically, that order of expression along the body axis corresponds to the order of the genes in a cluster on the DNA, a property called colinearity. I've recently described work that shows that, in some organisms, colinearity breaks down. That colinearity seems to be a consequence of a primitive pattern of regulation that coupled the timing of development to the spatial arrangements of the tissues, and many organisms have evolved more sophisticated control of these patterning genes, making the old regulators obsolete…and allowing the clusters to break up without extreme consequences to the animal. A new review in Science by Lemons and McGinnis that surveys Hox gene clusters in different lineages shows that the control of the Hox genes is much, much more complicated than previously thought.

Here, for instance, is a diagram of Hox arrangements in various animals.

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Cladogram depicting Hox gene chromosomal organization for representative animals. At the base is shown a cnidarian (Nematostella vectensis), which has a dispersed genomic organization of Hox genes and lacks posterior Hox paralogs. The left branch displays fragmented Hox clusters for the lophotrochozoan flatworm Schistosoma mansoni and the ecdysozoan fruit fly (Drosophila melanogaster) and nematode (Caenorhabditis elegans). The right (deuterostome) branch portrays the rearranged but coherent Hox cluster of the sea urchin Strongylocentrotus purpuratus, the "prototypical" Hox cluster of Branchiostoma floridae (a cephalochordate), the dispersed genomic organization of the Hox genes of a urochordate (Oikopleura dioica), and the quadruplicated Hox clusters of a mammal (Mus musculus), which remain coherent but have experienced losses of multiple paralogs. Similar to the mammals but not shown diagrammatically, the ray-finned fish have multiple duplicate Hox clusters that are mostly coherent and have experienced gene loss, as exemplified by the zebrafish (Danio rerio), pufferfish (Takifugu rubripes), and medaka (Oryzias latipes). At the base of the cladogram is the likely Hox cluster organization of the last common ancestor of bilaterians. Genes are typically assigned to the Hox class if they encode homeodomain sequences that group with the founder HOX protein sequences from Drosophila and vertebrate clusters, and then into Hox homology groups arbitrarily designated 1 through 14. Even having a Hox-like homeobox sequence and mapping in a cluster of Hox genes is not an invariably useful standard for Hox axial patterning function in some animals, because one of the Drosophila Hox clusters contains the ftz (marked 6* in fly Hox cluster) gene, derived from Hox ancestors, but with novel developmental functions.

Look first at the cephalochordate (on the right, about in the middle). It has the purest, simplest, cleanest arrangement: there are 14 Hox genes, all in one string, all in order, and all with the same orientation. This is also a perfect example of colinearity, with those genes expressed in a tidy front-to-back order in the animal's body. As you can see looking around the diagram, though, it's also an exception.

Take the fly, at the top left, for example. It's Hox cluster is broken in two pieces. Or look at the sea urchin—what a mess. It looks like part of the cluster has been swapped around. Vertebrates, represented by the mammals here, have kept everything in order, but they've duplicated whole clusters, and individual clusters have gaps. Most dramatically, look at the urochordate (this is Oikopleura, which I wrote about before: the cluster is completely broken up, the individual genes scattered throughout the genome with no apparent relationship to one another anymore.

Gene arrangements clearly tell us about the evolutionary history of these organisms, but if we want to understand how gene expression is translated into form, it's going to be a red herring in most cases. There has to be more going on.

One of those things going on, and also one of the current big buzzwords in molecular and developmental biology, is non-coding RNA. Scattered around and between the genes are sequences that are transcribed into RNA, but are not translated into protein—the RNA itself modulates the activity of other genes. In particular, there are microRNAs (miRNAs) interlaced throughout the Hox cluster, that in some cases have been shown to bind to and modify the expression of adjacent Hox genes. Our cartoons of ranks of colored arrows are going to have to be expanded to include squiggles of non-coding RNA, and more arrows showing which genes these RNAs modulate.

/>Diagram of the D. melanogaster BX-C showing Hox genes (arrows) Ultrabithorax (Ubx), abdominal-A (abd-A), and Abdominal-B (Abd-B), as well as a sampling of noncoding RNAs that derive from the intergenic regions. The intergenic regions bxd, iab-4, and iab-7 are transcribed and are known to be involved in proper segment-specific expression of the BX-C Hox genes (interactions with supporting evidence are shown as solid lines). The ability of these regions to affect activation states of the Hox genes is dependent on specific DNA binding sites within these regions and on ASH1 binding to noncoding transcripts such as RNAs from the bxd region, as well as on regulators possibly binding to other BX-C transcripts (dotted lines). (B) Depictions of the mouse Hoxa and Hoxb clusters. Indicated as hairpins are miRNAs miR-10a, miR-196a-1, and miR-196b along with verified (solid lines) and predicted (dotted lines) interactions with Hox target genes. Also indicated is the position of Hoxa11 antisense transcripts (a11-AS).

The summary of this article speaks for itself.

Genomic analyses have revealed surprising diversity in Hox gene number, organization, and expression patterns in different animals. There are still many animal groups about which little genomic sequence is known, and it remains to be seen how much more variation in Hox gene organization and function will emerge, including the numbers and functions of non-protein-coding RNAs. The property of HOX proteins working as a loosely coordinated system, often with overlapping patterns of expression and function, has apparently fostered their abilities to contribute to morphological change during the evolution of animals. Their colinear arrangement and coordinated regulation in many animals may assist in the maintenance of their overlapping expression patterns. This may have allowed some members of the clusters to subtly and slowly alter their expression patterns and functions to drive groups of cells toward novel structures. But Hox genes still can work as an axial patterning system even when partially dispersed in the genome, and dispersal may foster their rate of functional evolution.

I think the key concept is that Hox genes form a loose network, with multiple factors — the temporal sequencing of colinearity, transcription factors, and miRNAs — that all work together to generate form from genes, and that while this may look like a daunting mess, the complexity of regulation actually facilitates evolutionary change.

Lemons D, McGinnis W (2006) Genomic evolution of Hox gene clusters. Science 313:1918-1922.