Developmental Biology Evolution Natural History

A head of their time: how invertebrates had it in them all along to form the vertebrate head

David Jandzik, Aaron T. Garnett, Tyler A. Square, Maria V. Cattell, Jr-Kai Yu, Daniel M. Medeiros. Evolution of the new vertebrate head by co-option of an ancient chordate skeletal tissue. Nature. 518, 534-537 (2015). doi: 10.1038/nature14000

Hard-headed vertebrates owe it to their invertebrate ancestors.

“Heads or tails?” might lead on a coin toss, but it’s also a very real question in developmental biology, that is, the study of how cells specialize to form different parts of a complex organism like you or me, a giraffe, a snail, or a primitive fish-like invertebrate known as amphioxus.

We all start more or less in the same place, when a mommy and daddy love each other, and suddenly two cells unite in an event known as fertilization. What happens next is a series of cell divisions to form the basic layers of an embryo that can be thought of as a concentric tube of cells, the ectoderm on the outside, the mesoderm in the middle, and endoderm on the inside. Every cell has identical DNA make-up and hence the potential to be anything it wants to, but what determines what it becomes are the genes that are turned on or off, which is largely decided by variations in the concentrations of chemical signals (gradients) across and along the tube of dividing cells that becomes the embryo.

One reason for studying developmental biology is to understand the evolutionary histories of various taxa (or groups of species). By comparing the developmental patterns of different organisms, scientists can identify places where these organisms diverge, and hence gain an understanding the origin of different morphologies.

One fate for a cell is the production cartilage, the proteinaceous material that provides structural support somewhere between the rigidity of bone and the flexibility of muscle. A defining feature of vertebrate development is the migration of cartilage-producing cells (chondrocytes) originating at the central nervous system into the head of the developing organism to form a rigid skull. By contrast, formation of a cartilaginous skull has not been observed in the development of invertebrates. Hence, the formation of a cartilaginous skull is considered a turning point in the evolution of vertebrates from invertebrates some 500 million years ago. Yet, the idea that an orthogonal mechanism, utterly disconnected from our invertebrate ancestors gave rise to the vertebrate skull, leaves a gaping question—where on earth did we vertebrates get our hard heads?

David Jandzik, Aaron T. Garnett et al. think they’ve made the invertebrate connection in a recent study published in the journal Nature. In examining the development of the oral region of amphioxus, a fish-like invertebrate and most recent living invertebrate ancestor to the vertebrates, they observed a transient form of vertebrate-like cartilage, motivating the hypothesis that vertebrates co-opted an ancient pathway to form the modern skull. Curiously, the oral skeleton of amphioxus consists of a rigid material that does not contain fibrillar collagen, a structural protein component of vertebrate cartilage.

Jandzik and Garnett examined various stages of amphioxus development over time using a chemical cue to synchronize metamorphosis (transition from embryonic to pre-adult larval stage). Using a stain for vertebrate cartilage, they detected formation of structures similar to the precursors of vertebrate cellular cartilage in the oral regions of amphioxus larvae. To support their observation, they compared the structures observed in amphioxus larvae to cartilaginous structures in the larvae of two vertebrates—zebra fish, a well-studied developmental model system, and lampreys, an ancient vertebrate lineage—and showed convincing graphical evidence for transient chondrocytes in the oral region of amphioxus larvae.

Oral tradition. a) box shows oral skeleton region studied in amphioxus, b) oral skeleton of amphioxus stained for vertebrate cartilage, c-e) electron micrographs showing adult morphology of amphioxus oral skeleton, f and i are amphioxus larvae stained for vertebrate cartilage with two different strains compared to vertebrate larvae g-h and j-k, respectively. (Jandzik et al., Science, 2015).

They next asked whether the same signaling pathways from vertebrates were responsible for formation of the oral cavity in amphioxus. Exposing metamorphosing amphioxus larvae to two inhibitors of vertebrate regulators of cartilage differentiation, they observed selective suppression of oral skeletal development; the rest of the organism seemed to develop normally, indicating that they probably had targeted the pathway they intended. Motivated by these results, Jandzik and Garnett probed one step further to see if the same genes that turn on chondrocytes in vertebrates are turned on in the oral region of metamorphosing amphioxus. Designing probes that target RNA, the indicator that a gene is on (or expressing), they were able to detect expression of genes analogous to those responsible for regulation (SoxE) and production (ColA) of cartilage in vertebrates in the oral regions of amphioxus larvae, as well as regulators of SoxE expression (yes, regulators of regulators).

An expressive mouth. Two inhibitors of vertebrate cartilage differentiation result in mouthless amphioxus. a) is a control showing a normally developed oral region, while b) and c) are larvae treated with inhibitors. d)-f) indicate locations of expression of amphioxus analogs to genes involved in vertebrate cartilage regulation and expression.

If the same underlying pathways and genes were conserved between vertebrates and invertebrates, the difference in expression patterns might be due to changes in the regulation of the regulatory genes responsible for cartilage development. Hence, Jandzik and Garnett took the conserved regulatory gene belonging to family known as SoxE out of amphioxus and introduced it along with its neighboring sequences (presumably containing regulatory elements of its expression) into zebra fish. They fused a “reporter” sequence to the SoxE gene that produces a fluorescent protein, thereby allowing visual localization of chondrocytes “turned on” by the regulatory protein. If changes in regulation of SoxE expression are due to changes in regulation of SoxE, then the expression pattern of amphioxus SoxE in zebra fish should mirror that observed in amphioxus. If not, the reporter should be observed in migratory chondrocytes associated with vertebrate head cartilage development. Sure enough they observed the same transient expression pattern in zebra fish as in amphioxus, supporting the view that changes in regulation of SoxE account for differences in the spread and expression of cartilage in zebra fish as compared to amphioxus. RNA-probing for zebra fish SoxE showed normal localization, indicating that amphioxus SoxE and zebra fish SoxE are independently regulated in the same system. Moreover, removal of elements regulating zebra fish SoxE did not disrupt the localization of amphioxus SoxE in the zebra fish host. Hence, it would appear that way back in evolutionary history an invertebrate developed the ability to conduct cells out of central nervous system into the head where, co-opting ancient cartilage signaling pathways, they exuded cartilage to form the cartilaginous pre-adult vertebrate skull. Et voila.

Somebody that I used to know. a) transient expression pattern of SoxE in amphioxus. b) construct containing reporter (GFP)-SoxE and neighborhood c-d) zebrafish embryos exhibiting transient expression of amphioxus SoxE (as shown in blue by reporter) at earlier and later stages, respectfully. e) close-up top-down view of c), d) expression amphioxus GFP-SoxE (blue) and zebra fish SoxE (red) in zebra fish embryo.

Through a series of elegant experiments, Janzik and Garnet et al. demonstrate an example of “how structures in distantly related taxa can arise via conserved developmental mechanisms, revealing unexpected homology.” In other words, they show how vertebrate development unlocked potential already present in invertebrates to give us a good head on our shoulders.

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