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The fickle finger of fate
Luis de la Fuente and Jill A. Helms
Department of Surgery, Stanford University, Stanford, California, USA.
Address correspondence to: Jill A. Helms, Department of Surgery, Stanford University, 257 Campus Drive, GK 207, Stanford, California 94305, USA. Phone: (650) 736-0919; Fax: (650) 736-4374; E-mail: jhelms@stanford.edu.
Find articles by de la Fuente, L. in: JCI | PubMed | Google Scholar
Department of Surgery, Stanford University, Stanford, California, USA.
Address correspondence to: Jill A. Helms, Department of Surgery, Stanford University, 257 Campus Drive, GK 207, Stanford, California 94305, USA. Phone: (650) 736-0919; Fax: (650) 736-4374; E-mail: jhelms@stanford.edu.
Find articles by Helms, J. in: JCI | PubMed | Google Scholar
Published April 1, 2005 - More info
J Clin Invest. 2005;115(4):833–836. https://doi.org/10.1172/JCI24840.
© 2005 The American Society for Clinical Investigation
Ancient physicians viewed the skeleton as “the foundation of the rest of the parts of the body and all the members rest upon them and are supported, as proceeding from a primary base” (1). Defects in this structural foundation also serve as portholes through which the process of fetal skeletogenesis can be analyzed. For example, studies of genetic perturbations that result in distal limb truncations have shown that the morphogen Sonic hedgehog (Shh) establishes precisely where skeletogenic condensations will form in the tips of the hand- and footplates (2, 3). This spatial patterning information is further refined by bone morphogenetic protein (Bmp) signaling, as shown by the fact that disruptions in the Bmp signaling pathway lead to fusions, or syndactyly, of the digits (4). Once the spatial pattern of the skeletogenic condensations is achieved, a closely related cousin, Indian hedgehog (Ihh), takes over and plays an instrumental role in segregating inner chondrogenic cells from the flattened, elongated perichondrial cells at the periphery (5). Ihh secreted from chondrocytes stimulates the differentiation of perichondrial cells into osteoblasts (5–7), mesodermal cells that give rise to bone.
Another critical feature of limb skeletogenesis is the creation of the articulations, or joint spaces, between the skeletal elements. In the fingers, joint spaces are created when a single, larger skeletogenic condensation cleaves into 2 or 3 smaller segments, each of which will give rise to a phalange (8). Wnt14 is critical in determining where a joint space will form (9), but precisely how the cleavage event is controlled remains uncertain. One thing is clear, however: when a separation fails to take place, the phenotypic consequence is brachydactyly (shortened digits). Thus, while we have a fairly complete picture of the range of skeletal malformations that can occur, how these disruptions are related to one another and to the basic program of skeletogenesis remains unknown.
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Reading the bones
In this issue of the JCI, Niedermaier et al. provide new insights into the process of skeletal morphogenesis, describing the phenotypic consequences of a chromosomal inversion that disrupts both temporal and spatial regulation of Shh (10). Chromosomal inversions are genetic mishaps that take place when a region of the chromosome breaks and rejoins in a new orientation (Figure 1). This structural aberration can serve as a window into embryogenesis, especially when genes within the inverted region are subjected to new regulation as a result of enhancers or promoters located on the other side of the breakpoint.
Figure 1A chromosomal inversion causes Shh relocation. Schematic representation of the inversion event leading to development of Dsh mutant mice; the Shh gene, normally influenced by distal enhancer sequences (blue circles), is moved nearer to other regulatory sequences (pink triangles).
In the current study Niedermaier and colleagues examined heterozygous mice in which an inversion caused foreshortening of the distal limb skeletal elements, hence their nom de plume, Short digits (Dsh) (10). In the homozygous state, the Dsh phenotype is far more severe; in fact, Dsh/Dsh embryos bear a striking resemblance to Shh–/– embryos in that both exhibit cyclopia and incomplete cleavage of the embryonic forebrain. But the emphasis in this study was on the skeletal anomalies of the limb; Dsh/Dsh embryos showed an absence of all distal limb skeletal elements. Because Dsh/+;Shh+/– mice had a phenotype nearly identical to that of Shh–/– and Dsh/Dsh mice, the authors concluded that Dsh and Shh were allelic.
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It’s about time; it’s about space
Perhaps the most unexpected finding came from studying the Dsh/+ phenotype: here, the authors found that the chromosomal inversion functionally removed Shh activity from a number of its endogenous sites of activity, but also directed ectopic Shh expression to — of all places — the region where Ihh is normally expressed, the limb skeletogenic condensations (Figure 2) (10). Ectopic expression of Shh occurred simultaneously with the loss of Ihh in the digit condensations. While the 2 events — induction of Shh and loss of Ihh — might be controlled by separate mechanisms, a more plausible explanation is that the loss of normal Ihh expression is a secondary consequence of ectopic Shh expression, since previous investigators have shown that a negative feedback loop controls Hedgehog signaling in the skeleton (11).
Figure 2The chromosomal inversion causes Shh to be expressed ectopically in the phalanges. (A and B) In WT embryos (A), Ihh is expressed in mesenchymal aggregates of cells called cartilage condensations, up to the cartilage-perichondrium boundary (dotted lines); whereas in Dsh/+ embryos (B), Ihh is lost from the phalangeal condensations. (C and D) Whereas in WT embryos (C), Shh is never expressed in skeletal condensations, in Dsh/+ embryos (D), Shh expression replaces Ihh, albeit in a larger domain that extends past the cartilage-perichondrium boundary. u, ulna; r, radius.
Another phenotypic peculiarity was that the molecular substitution of Shh for Ihh only took place in the digits, while more proximal skeletal elements (i.e., humerus, radius, and ulna) were spared. Here, the answer may lie in the timing: the limb skeleton develops in a proximal-to-distal direction, and the inversion disrupted Shh expression relatively late in gestation, perhaps after the proximal skeletal elements were specified. But what remains a puzzle — and a very exciting one at that — is just how this inversion resulted in ectopic Shh expression in the digits. In other words, where did the instructions driving Shh expression in the digits originate? The chromosomal rearrangement appears to have created a new, chimeric regulatory domain for the Shh gene, composed partially of resident enhancer sequences and partially of enhancer sequences that were translocated along with the Shh gene.
Another perplexing question arises: how was the information in this chimeric regulatory domain derived? One possibility is that the inversion positioned the Shh coding sequence so that it was adjacent to a new enhancer that drives the expression of one or more genes in the digits. This possibility seems unlikely, however, as none of the genes near the insert point were expressed in the digits (10). A more plausible and attention-grabbing possibility is that the instructions for driving spatiotemporal expression of Shh are contained within the normal Shh regulatory region but are somehow masked. If this is the case, then the inversion event may have resulted in the loss of a repressor activity that normally restricts Shh from being expressed in skeletogenic condensations.
The implications of this finding are far reaching: if a repressor sequence was disrupted by chromosomal rearrangement, then the ectopic Shh represents part of a more ancient pattern of expression. Furthermore, these data suggest that the normal Shh regulatory sequence may have buried within it instructions for targeting Shh expression to skeletogenic condensations. That Shh and Ihh might have shared expression patterns sometime in the distant past is not a large stretch, since gene duplication is thought to be one of the chief mechanisms responsible for the diversification of gene function.
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A molecular switcheroo
Niedermaier et al.’s molecular dissection of the Dsh/+ phenotype has provided additional insights into the molecular underpinning of brachydactyly. Specifically, the investigators show that Shh does not simply replace Ihh in Dsh/+ chondrocytes; rather, Shh is more widely distributed in the cartilage and perichondrium of the digits, and it is active in this larger domain, as illustrated by the expansion of Hedgehog target genes Gli1 and Ptch (Figure 3) (10).
Figure 3Disrupted expression boundaries in Dsh/+ animals lead to perichondrial and joint defects (1). A transverse schematic of the phalangeal skeletal condensation, showing expression domains corresponding to a specific Hedgehog gene (Ihh in WT, Shh in Dsh/+), the Hedgehog target Ptch, and the Hedgehog repressor Gli3. Dotted lines indicate the normal cartilage-perichondrium boundary. In WT phalanges, Ihh (purple) is initially expressed (E12.5) in a small domain that expands over time to encompass the skeletal condensation. At E12.5 in Dsh/+ phalanges, Shh expression (blue) encompasses the normal Ihh domain and over time extends beyond what would normally be the cartilage-perichondrium boundary. Ptch (orange), an indicator of Hedgehog responsiveness, is first expressed in WT phalanges coincident with Ihh; eventually, Ptch is downregulated in cells expressing Ihh and upregulated in cells adjacent to the Ihh domain, where it functions to limit the spread of Hedgehog expression. Note the absence of color within the element, which shows the relative lack of Ptch in this domain (10). In Dsh/+ phalanges, Ptch is expressed coincident with the broader domain of Shh; however, in contrast to what occurs in WT tissues, Ptch does not become downregulated in chondrocytes. Gli3 (green), a repressor of Hedgehog signaling, is initially expressed in WT phalanges in a domain overlapping with Ihh; over time, this domain is restricted to the perichondrium, where Gli3 limits the activity of the Hedgehog protein. In Dsh/+ phalanges the Gli3 expression domain is initially broader, and only after an extended period of time does it become restricted to the perichondrium.
The consequences of ectopic Hedgehog expression are delayed chondrocyte differentiation, retardation of the maturation of perichondrium into periosteum, and loss of distal joint spaces. Given that all 3 defects occur simultaneously, one cannot help but wonder if they are interrelated features, all initially dependent upon a single precipitating event. We speculate that this event is the establishment of a boundary that segregates chondrocytes and osteoblasts into adjacent but immiscible cell populations. This boundary is, to some extent, a consequence of local Hedgehog signaling.
Around the same time that the cartilage-perichondrium boundary is being specified, some cells within the Ihh-positive skeletal condensation are faced with another decision: whether to progress along a chondrogenic pathway and undergo hypertrophy or to differentiate into a specialized type of perichondrial cell that will line the joint space (16). This cell fate decision, along with formation of a proper perichondrium, is disrupted in the digits of Dsh/+ mice as a consequence of ectopic Hedgehog activity (10). Just how does this imbalance between Hedgehog responsiveness and repression cause cells to alter their fate? Organisms use boundaries, the juxtaposition of like and unlike cells, in a myriad of ways: to define compartments, convey positional information, and even establish new tissues and signaling centers that can only form at the interface between 2 populations. In a wide variety of other developmental contexts, Hedgehog proteins play a critical role in specifying boundaries and thus defining cell and tissue compartments (17–19); the same may be true during skeletogenesis, but data to support this hypothesis are still sparse. What the Niedermaier et al. study reveals, however, is that removal or expansion of one of the factors that contributes to the establishment of a boundary can cause a multitude of processes, including those that shape and control development of the skeleton, to go awry.
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