How do we get five digits?

Hox genes control the number of digits (fingers and toes) in an interesting way.

Once the trail of animal development research was blazed in the fruit fly, mammalian investigators eagerly followed, using similar methods and looking at related genes. Some of the most interesting have been the Hox genes, which control patterning at a very high level- the identities of segments in flies, and the identities and numbers of related body areas in mammals (vertebrae, ribs, limbs, digits, etc.)

Genomic diagram of Hox genes in various organisms. A rough phylogeny is on the left, and diagrams of where some of the genes are expressed and have their effects is on the right. The middle shows the clusters of Hox genes, where the entire clusters has been quadruplicated in the vertebrate lineage, creating A, B, C, and D clusters of genes 1 to 13, though a few are missing here and there due to later loss. Note the general rule of linear expression of Hox genes from front to back in the organism, coordinate with genomic position.

The wiki page on Hox genes supplies this graphic of the Hox gene clusters of various species, related by a rough phylogenetic tree (left; tetrapods are us). Each colored box represents one protein-coding gene, positioned roughly as it appears in the genome (not to scale). Note that vertebrates picked up a quadruplication of the entire Hox cluster, after which a few individual genes were later lost. This expanded the body plan repertoire of this lineage substantially- a significant evolutionary event. The original Hox cluster was incidentally already the result of long-ago duplication of a single gene encoding a transcription regulatory protein. All Hox proteins have very similar structures.

Hox stands for homeobox, which stands for homeotic transforming transcriptional regulator protein containing a diagnostic protein sequence that binds to DNA and was called a "box" of sequence, due to its appearance in sequence alignments. And homeotic? That is not kinky at all, but refers to genetic effects on the body plan, i.e. the transformation of one part of the body into one "like" another via mutation, from the Greek for similar. Hox proteins all have their effects by binding to other genes and controlling their expression, though unfortunately little is known about these details, at least in mice, since this end of things rapidly becomes extremely complex.

Another part of the story of digit control is a different DNA-binding protein, Gli3. When mutated, Gli3 is known to cause polydactyly- the production of typically a sixth digit- as well as many other malformations (see image at bottom, left side). Gli3's activity (details of which are largely unknown) is the effect of a gradient of another protein, called sonic hedgehog (Shh) in the developing limb bud, and which at last is a protein that forms an actual physical gradient in the tissues of a developing limb.

Shh protein forms gradients that help direct development of body patterns during early embryonic times. But it can't do its job alone.

A recent paper showed that these two systems, the Shh/Gli3 system and the Hox system (specifically Hoxa13 & Hoxd11-13, the last genes in the tetrapod clusters above, dark blue) interact to generate the five-finger pattern. The last ingredient believed to be involved is another gradent forming protein, fibroblast growth factor 1 (Fgf1), presumed to be downstream of the various Hox regulators. The researchers speculate that these two gradients, of Fgf1 and Shh, are controlled by different genetic inputs, and interact to create patterns. In this case it is fingers, but in other organisms, similar processes are thought to make zebra stripes, wing patterns, shells, etc..

One molecular gradient can provide some information about where to put things, but probably not anything very consistent or detailed. The interesting part of this story, though the actual biology is unfortunately not yet well developed, is that the combination of two molecular gradients generates far more interesting possibilities. This was, intriguingly enough, pointed out by one of the greatest mathematicians of all time, Alan Turing, who, taking time off from inventing the computer, provided the mathematical foundations of a two-component chemical system which, across a gradient field of both chemicals, which react at different rates as they go forth, can create amazingly stable and interesting patterns, somewhat counter-intuitively.

Abstract model of a Turing wave 2-component system, resolving itself over time spontaneously from a homogenous solution into a complex binary pattern.

Naturally, these researchers made mutations to look at the effects of these genes. The complete deletion of Hoxa13 turns out to be lethal in early embryos. On the other hand, they find that the Hox code is rather complicated, such that deletion of Hox members d11 to d13 causes added effects that mimick or accentuate deletion of a13. In early embryos, they can see the hand region setting up dramatically more fingers (marked by staining for the protein Sox9, a marker of pre-cartilage/bone formation) as they delete either of the gradient-forming or responding genes Gli3 and Hox*.

Early embryonic (day 12.5) limb-buds stained for finger primordia, from mice mutated for various genes, as noted. "+" is wild-type for one or both genes, while "-" is deleted or otherwise inactivated. While deletion of Gli3 alone has some effect on finger number, only with the added deletion of the Hox genes do finger numbers become truly uncontrolled.

There is something amazing going on here. By the time all these genes are deleted (-, or other non-"+" variants) for Gli3, Hoxa13, Hoxd11, Hoxd12, and Hoxd13, there are no individual digits left. The whole zone has turned into a smooth non-digital mess. Lesser amounts of the Hox genes in particular lead to dramatically rising numbers of digits.

Mice mutated for various genes as noted, now at birth, stained for cartilage (blue) and bone (red).

In roughly the same amount of tissue, many different numbers of digits can develop, based on a few genetic alterations.  Clearly the researchers are hot on the trail of how this pattern develops and will be looking for the particular components downstream of the Hox genes that carry out its regulatory directions, especially the protein or other chemical that forms the counter-gradient to Shh. It is a common theme in biology, that most of the action lies in complex layers of regulation (i.e. management) so that the ultimate actors can toe their lines with precision even in a variable genetic and external environment.

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