Sticklebacks and Hopeless Monsters

What types of genes and mutations underlie the striking anatomical differences found in wild species? New genetic research shows that wild stickleback fish evolve major changes in their defensive armor using the vertebrate homologs of the famous Hox genes of Drosophila. Four-winged fruit flies have been described as "hopeless monsters" that would never survive outside the laboratory. However, sticklebacks with different bony spine patterns are thriving under a full range of fitness constraints in natural environments, providing a striking example of how wild vertebrate species evolve new body structures through repeated regulatory mutations in key developmental control genes.
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When Katie Peichel and I began a new project on stickleback genetics in 1998, we both had backgrounds studying skeletal mutations in mice.  Many different skeletal mutations had been found during simple visual inspection of large breeding colonies of mice in the 20th century. Large morphological changes were controlled by simple Mendelian genes, but their molecular basis was completely unknown (Lyon and Searle, 1989).

For my postdoctoral studies, I had used Drosophila-style chromosome walking approaches to characterize the mouse  “short ear” locus.  My lab’s subsequent studies showed that the short ear gene encoded a secreted signaling molecule called a “bone morphogenetic protein” (BMP) (Kingsley et al., 1992). Soon after, we showed that other classic mouse skeletal mutations were due to mutations in other members of the BMP family, suggesting the overall skeleton was built from the combined activities of multiple BMP family members (Storm et al., 1994). In her own graduate work, Katie had used positional cloning approaches to study the mouse limb mutation “Ulnaless” (Peichel et al., 1996). Her work showed that Ulnaless was likely due to regulatory changes in the mouse HoxD locus, thus linking specific morphological changes in vertebrate limbs to the family of homeo-domain transcription factors originally made famous based on their ability control body part identities in fruit flies (Peichel et al., 1997).

Classic mouse and fly mutations with big morphological effects.   Control and Ulnaless mouse forelimbs (Credit: Katie Peichel).   Four-winged fruit flies with mutations in the Hox gene Ultrabithorax (Credit: Nicolas Gompel).

Although our genetic studies showed it was possible to go from major morphological changes in laboratory mice to underlying genes, how relevant were the results to the many interesting morphological differences that have evolved in wild species?  This was a long-standing question because laboratory mutations with big effects are usually deleterious. short ear mice have a variety of soft tissue defects because BMP molecules play a key role in mesenchymal epithelial interactions as well as cartilage and bone development (King et al., 1994). Ulnaless mice, and many classic homeotic mutations in Drosophila, have reduced fertility and high rates of lethality, because homeodomain transcription factors play a key role in the formation of many different tissues in the body (Peichel et al., 1996). 

Darwin and other classic evolutionary biologists have sometimes referred to large-effect mutations as monsters, and thought that evolution would only proceed by a whole series of smaller, gradual genetic alterations (Darwin, 1859).  This view was reiterated by Ernst Mayr during later debates with Richard Goldschmidt about large-effect “hopeful monster” mutations (Goldschmidt, 1940):  “The occurrence of genetic monstrosities by mutation, for instance the homeotic mutant in Drosophila, is well substantiated, but they are such evident freaks that these monsters can be designated only as 'hopeless’ … It is a general rule, of which every geneticist and breeder can give numerous examples, that the more drastically a mutation affects the phenotype, the more likely it is to reduce fitness. To believe that such a drastic mutation would produce a viable new type, capable of occupying a new adaptive zone, is equivalent to believing in miracles” (Mayr, 1970). 

Some modern critics of evolution have gone even further.  Jonathan Wells describes four-winged fruit flies as  “hopeless cripples”, “sideshow freaks”,  “zombie science”, and a misleading “icon of evolution” (Wells, 2000, 2017). Using impaired laboratory Hox mutants as his favorite example, Wells argues that it would be impossible for natural mutations to ever produce new beneficial body structures or morphological innovations that could survive and thrive outside the laboratory. 

Well, what types of genetic changes do contribute to evolution of new adaptive types in wild species? Following our mouse work, Katie Peichel and I decided to start crossing wild species to track down the molecular basis of real evolutionary traits in nature (Peichel et al., 2001). We chose stickleback fish because they showed dramatic morphological differences that had evolved relatively recently (thousands to millions of years). The fish were small, abundant in nature, and easy to raise in the laboratory. There was a long and rich study of the ecology and significance of variable traits in many populations (Bell and Foster, 1994). And, critically, the reproductive barriers between distinct forms could often be overcome using artificial fertilization in the laboratory.

Fortunately, the awesome power of genetics works as well in fish as it does in mice.  Most of the dramatic differences used to classify sticklebacks turned out to controlled by some genetic loci with large effects.  And by combining both recent meiosis in laboratory crosses (quantitative trait locus mapping), and historical meiosis in natural populations (high resolution association mapping), we found that the major loci controlling evolutionary differences usually corresponded to key developmental control genes encoding essential signalling molecules and transcription factors (Kingsley and Peichel, 2007; Peichel and Marques, 2017).

How about spine number itself? Prominent bony spines decorate the back of various sticklebacks, which are commonly referred to as threespine-, fourspine-,  fivespine-, or ninespine- sticklebacks for the main species groups found in North America (Gasterosteus aculeatus, Apeltes quadracus, Culaea inconstans, and Pungititus pungitius; respectively).  Interestingly spine patterns also vary within each of these groups, providing the basis for new genetic and molecular studies reported in (Wucherpfennig et al., 2022).

In a genetic cross between wild threespine-stickleback (Gasterosteus aculeatus) populations that had either three or two dorsal spines, graduate student Tim Howes found a surprising result.  Some of the sticklebacks produced in the cross had four spines, a count that exceeded the number in either original population.  Tim’s own Ph.D. work focused on other traits, but he mapped a significant locus for increased spine number to the distal end of stickleback linkage group VI.   At the time, nothing obvious appeared in that region of the genome.  But a new graduate student, Julia Wucherpfennig recognized that the Lunapark and Mtx2 genes also mapped in the key spine control region.  Julia realized that those genes normally flank vertebrate orthologs of Hox clusters, and soon confirmed that a previously unannotated Hox cluster mapped in the region.  Julia also quickly developed efficient genome-editing methods in sticklebacks, making it possible to test whether a particular gene contributed to a particular trait (Wucherpfennig et al., 2019).   Julia’s very first CRISPR tests of the chromosome VI locus showed a significant effect on dorsal spine patterns, confirming that we were likely on the right track.

Spines in the lab:  The large projecting structures on the back of a stickleback are some of the most prominent structures seen in stickleback X-rays.   By measuring the length and number of spines in both lab crosses and CRISPR fish, Julia Wucherpfennig found that spine patterns were controlled by variants at the stickleback HOXDB locus. (Credit:  David Kingsley). 

Because four-spined fish are not found in the particular wild Gasterosteus populations from which we started our cross, we also looked for other sticklebacks that showed abundant spine number changes in nature. In the 1980s, Max Blouw and Don Hagen had published a beautiful series of papers on the adaptive significance of very common spine number changes in Apeltes quadracus, the “fourspine” stickleback of Nova Scotia ( Hagen and Blouw, 1983; Blouw and Hagen, 1984a, 1984b, 1984c, 1984d, 1987). Their work showed that Apeltesquadrucus” had become “quinticus” or even “sextacus” in some wild populations, and that fish and bird predators preyed selectively on sticklebacks with different spine numbers.  Julia Wucherpfennig and Amy Herbert traveled to Nova Scotia to revisit some of these classic populations.  When we began association mapping of dramatic morphological differences in these thriving sticklebacks, we were thrilled to see that genotypes at the HOXDB locus could predict the spine number of wild-caught fish!   Julia then carried out a whole series of elegant experiments using expression analysis, high-resolution genetic mapping, and enhancer testing with even more transgenic and CRISPR sticklebacks.  Her results identified a specific enhancer within the HOXDB locus that recapitulates axial expression patterns during development, and has undergone independent sequence changes linked to spine identity changes in wild Gasterosteus and Apeltes populations.

Fish in the wild:  Although our genetic hunt began with laboratory crosses, we also visited dozens of lakes and streams across North America to collect wild sticklebacks and study their spines and Hox alleles.  Many thanks to the diverse stickleback experts who collaborated with us on interesting Gasterosteus and Apeltes populations from Haida Gwaii, British Columbia (Thomas Reimchen), Cook Inlet, Alaska (Michael Bell), and Nova Scotia, Canada (Anne Dalziel).  Credit:  Michael Bell. 

I think the experiments in this paper are a beautiful demonstration of applying the powerful emerging methods of molecular genetics to classic problems in evolutionary biology.   The combined results from both Gasterosteus and Apeltes are clear.  Striking axial skeletal changes evolving in wild fish are controlled by regulatory changes that either increase or decrease expression of Hox genes.  Fittingly, the particular locus involved in wild spine variation is the stickleback ortholog of the Ulnaless locus studied by Katie Peichel in laboratory mice so many years ago.  And while the Ulnaless or Ultrabithorax mutations may be deleterious, the stickleback Hox variants are changing body morphology and adding new structures under a full range of fitness constraints in the wild.  Our results add to several other example of natural variants in Hox genes that have previously been linked to natural trichome and pigmentation variation in insects  (Stern, 1998; Tian et al., 2019), or to adaptive tail length changes in deer mice (Kingsley et al., 2021).  Four-winged Drosophila may only survive in laboratories. But sticklebacks and other wild species show that obvious morphology mutants do not have to be hopeless monsters, and that nature is using repeated changes in powerful developmental control genes to generate endless forms most beautiful in natural populations. 

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