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Proc Natl Acad Sci U S A. Mar 18, 2008; 105(11): 4185–4190.
Published online Mar 11, 2008. doi:  10.1073/pnas.0707899105
PMCID: PMC2393776
Developmental Biology

Unique SMAD1/5/8 activity at the phalanx-forming region determines digit identity


The zone of polarizing activity is the primary signaling center controlling anterior–posterior patterning of the amniote limb bud. The autopodial interdigits (IDs) are secondary signaling centers proposed to determine digit identity by acting on the cells of the digital ray. Here, we focus on events accompanying digital fate determination and define a region of the digital ray that expresses Sox9 and Bmpr1b and is phosphorylated-SMAD1/5/8 (p-SMAD1/5/8) positive. We name this region the phalanx-forming region (PFR), and show that the PFR cells arise from the distal subridge mesenchyme of digital ray. This phalanx-forming cell lineage is subsequently committed to the cartilage lineage; the fate of these cells is initially labile but becomes fixed as they are incorporated into the condensed cartilage of the digit primordium. Using an in vivo reporter assay, we establish that each digital PFR has a unique p-SMAD1/5/8 activity signature. In addition, we show that changes in this activity correlate with the identity of the digit that forms after experimental manipulation, supporting the idea that threshold signaling levels can lead to different developmental outcomes in a morphogenetic field. Our data define the molecular profile of the PFR, and we propose a model for understanding formation and variation of digits during autopodial development.

Keywords: Bmpr1b, brachydactyly, limb development

Sonic Hedgehog (Shh) is a morphogenetic signal secreted by the cells of the zone of polarizing activity (ZPA) in the early limb bud and is necessary for determining the number and form of amniote digits (1). In the absence of Shh, no digits form in the forelimb and only a biphalangeal digit 1 forms in the hindlimb (2, 3). A variety of observations indicate individual digits require specific levels of SHH for their formation (4). Furthermore, descendants of ZPA cells contribute to the posterior half of digit 3 and all of digits 4 and 5 in the mouse (5). These data support a role for the Shh function and ZPA cells in autopod development but provide little insight into the mechanisms that establish a particular digit's identity. It has been proposed that a direct consequence of ZPA and SHH function is to establish the autopod, which is composed of alternating interdigits (ID) and digital rays. The IDs act as secondary signaling centers that determine digit identity downstream of Shh (6). This hypothesis is based on the observation that manipulations of the ID cause predictable homeotic transformations of the adjacent anterior digit, without effects on other digits. Further, it was proposed that there is a Noggin-sensitive BMP signaling gradient across the autopod, with the highest (posterior) level specifying digit 4, intermediate levels specifying digits 3 and 2, and the lowest (anterior) level specifying digit 1 (6).

In this report, we focus on the cells in the digital ray that give rise to the phalanges and the molecular events that accompany the determination of digital identity, i.e., number, size, and shape of the phalanges. We show that within the digital ray of the autopod, a molecularly distinct cohort of cells, originating from the distal subridge mesenchyme and located immediately distal to the condensed digit primordium, give rise to the phalanges of that digit. We name this region the phalanx-forming region (PFR). Lineage tracing of the PFR cells demonstrates that they form the phalanges. We quantified the levels of SMAD1/5/8 activity for the PFR of each digit and report that there are unique values corresponding to each digit. When digit transformations are experimentally induced, the SMAD1/5/8 activity correlates directly with the digit transformation caused by a particular manipulation. This observation supports the hypothesis that the PFR is the target of interdigital signals and is an example of threshold signaling levels leading to different morphogenetic outcomes.


The Phalanx-Forming Cell Lineage Is Derived from the Distal Mesenchyme Underlying the Apical Ectodermal Ridge.

The chick foot autopod consists of four digital rays (DRs), alternating with mesenchymal IDs. The DRs are composed of the proximal cartilaginous digit primordium, the noncondensed vascular mesenchyme, and the distal-most avascular mesenchyme underlying the proximal-distal axis signaling center, the apical ectodermal ridge (AER) (Fig. 1 A and B). We first sought to determine which cells of the developing digital ray give rise to the phalanges. One current hypothesis proposes that the digit primordium represents all of the digital elements, except the tip or ultimate phalanx, and, through a generalized process of elongation and segmentation by joint formation (7, 8), forms the definitive number, size, and shape of the phalanges. This hypothesis predicts that a marker placed at the apex of the condensed cartilage at any stage should be found just proximal to the distal-most phalanx at day 10 (Fig. 1C). To test this, we placed a tantalum foil barrier at the distal limit of the digit 3 primordium at stage 26, the onset of visible cartilage condensation (Fig. 1D). At day 9, the barrier was imbedded in the joint between the metatarsal and first phalanx (Fig. 1E; 90%, n = 20). We repeated these experiments at progressively later developmental stages and found the barrier at progressively more distal positions in the phalangeal sequence at day 9 [implantation at stage (St.) 28, Fig. 1F: 92%, n = 13; at St. 30, Fig. 1G: 75%, n = 4]. These results are inconsistent with the elongation and segmentation hypothesis (7, 8) for phalanx formation. We propose a hypothesis that the precursor cells of the phalanges arise from the noncondensed mesenchyme distal to the digit primordium and are sequentially determined and incorporated into the elongating digital cartilage.

Fig. 1.
Cells located in the avascular zone give rise to the digital ray. (A) St. 29 chick footplate showing four digital rays (DR1–4) alternating with noncondensed ID tissue. The vascular pattern of the mesenchyme was visualized by injecting India ink ...

To begin to explore this hypothesis further, we carried out lineage analyses of the vascularized, noncondensed mesenchyme and the subridge avascular mesenchyme by using the replication-incompetent retrovirus, RISAP, that expresses the alkaline phosphatase (AP) reporter gene (9). We first infected the vascularized, noncondensed mesenchyme of digital rays 2-3-4 at St. 27 (Fig. 1H). At day 9.5, we found AP activity within the cartilage cells of the proximal phalanges of each digit (Fig. 1I; 100%, n = 10). We repeated these experiments at St. 29/30 and found that AP activity was seen only in the distal phalanges (Fig. 1J; 100%, n = 3). Our data indicate that at a given stage, only a subset of the phalangeal precursor cells are represented in the vascularized, noncondensed mesenchyme of the digital ray. We propose this region is dynamic and is continuously reformed by cells from the subridge avascular mesenchyme as they become vascularized. To test this, we infected the subridge avascular mesenchyme at St. 27 (Fig. 1K). We found AP activity in the cells of the phalanges but, importantly, only in the distal phalanges (Fig. 1L, 76%, n = 13), in a complementary pattern to what is seen in Fig. 1I. These data are supported by grafting experiments of the subridge avascular mesenchyme to a nonlimb site developing phalangeal elements including the ultimate phalanx [supporting information (SI) Fig. 6A]. To determine whether the ID mesenchyme contributes to the digital ray, we performed RISAP infections of the interdigit 2 and 3 and found no significant contribution of these interdigital cells to the phalanges (data not shown). Taken together, these data give strong support for our hypothesis that there is a phalanx-forming cell lineage originating in a dynamic fashion from cells immediately under the AER during autopod stages of development.

The Molecular Profile of the Phalanx-Forming Cell Lineage.

We next examined the gene expression profile of the phalanx-forming cell lineage as they form cartilage. The noncondensed vascularized mesenchymal cells of the digital ray express cartilage markers Sox9 and Col2a as well as Bmpr1b (Fig. 2A). Additionally, immunohistochemistry was performed with an antibody specific for p-SMAD1/5/8. The most extensive staining was seen throughout the subridge avascular region of the entire autopod (black arrow in Fig. 2B). This staining was limited to the distal-most 100 μm of mesenchyme beneath the AER and was clearly down-regulated immediately proximal to this region. This p-SMAD1/5/8 staining in the subridge mesenchyme can be explained by Bmp2/4/7 secreted by the AER and signaling through Bmpr1a, which is highly expressed in the mesenchyme beneath the AER and ubiquitously expressed elsewhere in the autopod (10). We speculate that p-SMAD1/5/8 down-regulation occurs with the elongation of the autopod and as the cells leave AER-BMP influence. Within the digital ray at St. 27, the distal-most 100 μm of subridge mesenchyme is p-SMAD1/5/8-positive, and proximal to that, p-SMAD1/5/8 is down-regulated. Immediately proximal to this down-regulated region (bracket in Fig. 2B), we observe a second focus of p-SMAD1/5/8 staining within the Bmpr1b-expressing cells (red arrows Fig. 2B; high magnification shown in SI Fig. 6B).

Fig. 2.
The molecular profile of the PFR and stages when digit identity is being determined. (A) Section in situ hybridization of the third digital ray of an St. 27 hindlimb showing the expression of Sox9, Bmpr1b, and Col2a. (B) Immunohistochemistry of p-SMAD1/5/8 ...

These data demonstrate that within the phalanx-forming cell lineage, there is a subset of cells expressing Sox9, indicating a commitment to the cartilage lineage. These cells uniquely up-regulate Bmpr1b and show activation of a BMP-receptor (BMPR) pathway as indicated by positive signal for p-SMAD1/5/8 (red arrows in Fig. 2B). We propose these cells are actively being determined to form phalanges; we name this region the PFR.

Quantitation of p-SMAD1/5/8 Activity at Each PFR During Digit-Identity Determination.

Next, we sought to determine whether quantitative levels of SMAD1/5/8 activity could be measured at the individual PFRs of the digital rays. First, we needed to establish at which stages the phalanges are being determined and when each digit's phalangeal complement is fixed and stable (Fig. 2C). Using interdigital removals between stages 25 and 30, we assayed for digit identity transformations (6), indicating when the phalanx cell lineage is responding to ID signals and when the phalangeal elements of each digit are determined, i.e., fixed and stable (red and green arrowheads, respectively, in Fig. 2C). In brief, digit 4 is actively being determined between stages 25 and 27; digit 3 is actively being determined between stages 26 and 29; digit 2 is actively being determined between stages 27 and 30; and digit 1 is actively being determined between stages 28 and 30. Digit identity transformations were not observed after the period of active determination.

Next, we designed an in vivo reporter assay by using the SMAD-responsive element of the Xenopus Vent2 promoter (xVent2). This promoter element responds to p-SMAD1/5/8 signaling through BMP type 1 receptors in vivo but not to TGF-β signaling (11). This element has been used to quantitatively measure SMAD1/5/8 activity in mammalian cell culture (12) and is responsive to areas of BMP signaling in the chick neural tube (see SI Fig. 6 C and D and SI Methods). To use exogenous promoters in the limb bud, we constructed RCANBP retroviruses containing either the xVent2- luciferase or CMV-Renilla luciferase expression cassettes. Transcription of luciferase in the RCANBP-xVent2 cassette is only activated in cells transducing BMPR signaling; whereas the CMV-Renilla cassette is ubiquitously expressed and is an internal control for normalization (Fig. 3A; see Materials and Methods).

Fig. 3.
Unique SMAD1/5/8 activity at each PFR. (A) Diagram of RCAN constructs and infection area at St. 14 and dissection protocol for tissue sources used for the in vivo dual luciferase assay to determine SMAD1/5/8 activity at the PFRs. After injection of retrovirus-expressing ...

Using the stages established in Fig. 2C as a framework, we performed dual luciferase assays on the PFRs isolated (see Fig. 3A; also see Materials and Methods for experimental design) from embryos infected with both RCANBP viruses from the onset of digit condensation through digit determination. We found that each digit's PFR has a unique SMAD1/5/8 activity signature during the stages at which each digit identity is being determined (Fig. 3B). Digit 4 (green bars in Fig. 3B) is the first to condense and be determined and shows the first SMAD1/5/8 activity. Digit 3 (orange bars in Fig. 3B) begins to be determined slightly after digit 4 and initially has comparable SMAD1/5/8 activity with digit 4. By the time digit 4 is fixed and stable, the SMAD1/5/8 activity is higher for digit 3. Surprisingly, we found that digit 3 has the highest SMAD1/5/8 activity compared with the other three digits, whereas digit 2 (red bars in Fig. 3B) shows an intermediate SMAD1/5/8 activity compared with digit 1 (blue bars in Fig. 3B), which consistently showed the lowest activity of these three digits. The data demonstrate that, within an individual stage, digit 3 has the highest SMAD1/5/8 activity, digit 1 had the lowest, and digits 2 and 4 have intermediate levels (Fig. 3B). We found that the digit 4 SMAD1/5/8 activity continues to increase beyond the stage when it is fixed and stable, whereas digit 3 SMAD1/5/8 activity declines after it is fixed and stable. The significance of the maintained SMAD1/5/8 activity of digit 4 beyond the stage when its identity is determined remains unclear.

Considering the short time required for digit 4 determination and corresponding SMAD1/5/8 activity observed for these stages, we propose that digit 4 requires the lowest overall SMAD1/5/8 activity. Regardless, the SMAD1/5/8 activity for digit 4 is consistently lower than that of digit 3. Therefore, the proposal (6) of a signaling gradient for digital fate determination with high posterior to low anterior signaling across the autopod is not supported.

SMAD1/5/8 Activity in the PFR Correlates with the Digit That Forms After ID Manipulation.

Using our quantitative assay, we tested SMAD1/5/8 activity after physical manipulations of the interdigits that reproducibly result in digit identity transformations (6). In each of the following experimental conditions, both the manipulated right hindlimb and the control left hindlimb were infected with the RCANBP reporter constructs, and the PFRs were assayed for SMAD1/5/8 activity. In this way, each experimental condition contains its own internal control, provided by comparisons with the luciferase values of the nonmanipulated left limb.

In the first experiment, Noggin-soaked beads were implanted into ID2 at St. 28 to induce anteriorization of digit 2 to a digit 1 (see Fig. 4A; 67%, n = 33). SMAD1/5/8 activity at the digit-2 PFR 24 h after bead placement [hatched red bar corresponding to experimental digit (d)2 in Fig. 4A] was reduced to that observed in the control left and right digit 1-PFR (blue bars, Fig. 4A). Similarly, Noggin-soaked beads implanted into ID3 at St. 27 induce anteriorization of digit 3 to digit 2 (see Fig. 4B; 28%, n = 25). SMAD1/5/8 activity at the digit-3 PFR 24 h after bead placement (hatched orange bar corresponding to experimental d3, Fig. 4B) was reduced to that observed in the control left digit-2 PFR (red bar in Fig. 4B). At the same time, the left control digit 3 showed significantly higher SMAD1/5/8 activity (control orange bar in Fig. 4B). Notably, Noggin beads implanted in either ID2 or ID3 had no effect on SMAD1/5/8 activity of the PFRs of the posterior digit 3 (orange bar, Fig. 4A) or digit 4 (green bar, Fig. 4B), respectively. This demonstrates that Noggin-soaked beads have specific effects limited to the adjacent anterior digital ray, and there is no detectable effect on the adjoining posterior digital ray.

Fig. 4.
Changes in SMAD1/5/8 activity correlate with digit identity. Experimental diagrams are shown on the left and representative skeletal staining is illustrated in the center. The color scheme for the digits is the same as in Fig. 3; hatching indicates luciferase ...

In a second set of experiments, we pinned ID3 to the posterior-distal side of the digit 1-PFR at St. 28 to induce a posteriorization of digit 1 to digit 3 (see Fig. 4C; 30%, n = 10). SMAD1/5/8 activity at the digit-1 PFR (hatched blue bar, Fig. 4C) was increased to that of the control left digit-3 PFR (orange bar, Fig. 4C). Similarly, we pinned ID3 to the posterior-distal side of the digit-2 PFR at St. 28 to induce a posteriorization of digit 2 to digit 3 (see Fig. 4D; 68%, n = 19). SMAD1/5/8 activity at the digit-2 PFR (hatched red bar, Fig. 4D) was increased to that of the control left digit-3 PFR (orange bar, Fig. 4D). At the same time, the control left digit 2 (red bar, Fig. 4D) showed significantly lower SMAD1/5/8 activity compared with the manipulated digit 2 (hatched red bar, Fig. 4D).

In a final experiment, ID4 was removed at St. 26 to induce anteriorization of digit 4 to digit 3 (Fig. 4E; 35%, n = 23). SMAD1/5/8 activity at the digit-4 PFR (hatched green bar, Fig. 4E) was increased to that of the control left and right digit-3 PFRs (orange bars, Fig. 4E). These experiments show that manipulations causing homeotic digit transformations result in the PFR of the affected digital ray assuming the SMAD1/5/8 activity of the digit that forms.


A Model for the Origin and Molecular Profile of Phalangeal Precursor Cells (Fig. 5).

Fig. 5.
Model for digital growth and identity determination. The chick limb autopod (A) is composed of digital rays (DR1–4), containing the digit primordia (p1–4), alternating with interdigital mesenchyme (ID1–4). Within each DR (A and ...

In this report, we demonstrate that a specific region of the digital ray, the PFR, is composed of cells that are derived from the subridge mesenchyme. In the early autopod stage, the PFR cells are the vascularized, noncondensed mesenchyme just distal to the condensed cartilage of the metatarsal; they express Sox9 and Bmpr1b, are positive for pSMAD1/5/8, and are the precursor cells of the proximal-most phalanx. As these cells condense to form cartilage, the PFR is replenished with formerly distal subridge cells that will then form the next phalangeal increment. In this dynamic sequence of events, the PFR cells are exposed to signals from the interdigit (blue arrow Fig. 5B) that are responsible for determination of their fate and later morphogenesis (i.e., number, size, and shape of phalanges). The incremental addition of the PFR to the digital cartilage in a proximal-to-distal sequence is a significant mechanism for proximal-distal elongation of the digital ray. Our data on the origin and determination of the phalanx-forming cell lineage demonstrate a complexity not accounted for by the currently held model for digit formation and outgrowth (7, 8). At the same time, we provide no insight into how joint formation producing the individual phalanges occurs, i.e., as discontinuous segments or by segmentation later in development.

To explore the mechanism of phalanx determination required by this model, we describe a powerful tool by using the SMAD1/5/8-responsive xVent2 promoter element to quantitate SMAD1/5/8 activity in vivo. Using this approach, we correlate SMAD1/5/8 activity in the PFR cells with the determination of a particular digit identity, i.e., number, size, and shape of phalanges. In addition, we quantitatively tested the proposal that an interdigital signaling activity gradient is present within the autopod (6). Specifically, we found that the digit-4 PFR has lower SMAD1/5/8 activity than the digit-3 PFR; therefore, the proposal of a BMP signaling gradient across the developing autopod is not supported.

BMP Receptor Signaling and Phalanx Formation.

Our data indicate that there is a phalanx-specific requirement for SMAD1/5/8 activity in the developing chick foot. Activation of SMAD1/5/8 is primarily achieved through type I BMP receptors (11). Conditional inactivation of BMPRIA function in the mouse limb-bud mesoderm results in an almost complete agenesis of the autopod (13), which complicates analysis of BMPRIA function in the formation of the phalanges, whereas genetic removal of BMPR1B function in the mouse results in normal limbs, except for a complete loss of the proximal and middle phalanges of all digits (14) or the formation of a small formless rudimentary element in place of the phalanges (15). In both Bmpr1b mutants (14, 15), the terminal phalanges of all digits form normally, positioned just distal to the relatively normal metacarpal or metatarsal bones. Previous data show that, in the mouse, the proximal and middle phalanges form by endochondral ossification from cartilage models, whereas the distal phalanx forms from membrane bone (16). A further indication for Bmpr1b function in phalanx determination is seen in the rare human homozygous condition where no functional BMPR1B is produced. This study shows severe brachydactyly with hypoplastic proximal phalanges and normal distal-most phalanges of the hands and feet (17).

Taken together, the mouse and human data along with the data we report indicate that there is a phalanx-specific requirement for BMP receptor function in the determination of the cartilage models required for endochondral bone formation of the phalanges of the digits. Although we could not distinguish specific roles for Bmpr1a or Bmpr1b in our experiments, and we acknowledge that both receptors are expressed in the PFR (10), it is relevant that, in the mouse, Bmpr1a expression is not altered in the Bmpr1b mutant (14), indicating that Bmpr1b is required for phalanx formation independent of Bmpr1a.

ID Signals That Mediate BMPR-SMAD1/5/8 Signaling at the PFR Are Unknown.

The experimental and genetic data described above support the hypothesis that BMP receptor signaling is integral to digit identity determination but does not address the ligand(s) involved. Based on the observation that Noggin beads and ID removal caused virtually indistinguishable homeotic digit identity transformations, it was originally proposed that members of the BMP family were candidate molecules mediating these signaling events. The most attractive candidates at the time were BMP2, -4, and -7, all known to be expressed by the ID mesenchyme at the correct time and place. However, BMP4 and BMP7 mouse mutants show no digit identity changes, and BMP2 null is embryonic-lethal (18, 19). A further study (20) reported that the limb-specific conditional BMP2:BMP4 and BMP2:BMP7 compound mouse mutants show no losses or shifts in digit identity. These authors concluded that these ligands are not the interdigital signal(s) affecting digit identity, and the issue of the interdigital ligand remains unresolved. Nevertheless, there is the possibility for other factors playing a role in digital ray outgrowth and identity determination either synergistically or otherwise.

It is pertinent that BMP5 and BMP6 are expressed in the ID mesenchyme throughout the critical stages of digit development and could be signaling candidates (20, 21). Equally likely candidates are the BMP family members GDF5 and GDF6 that are expressed not only in the forming joints later in development, but also in the interdigits (22). Importantly, GDF5 signals through BMPR1B, and its activity is inhibited by Noggin (23, 24). Further, Gdf5−/− mice show a similar phalangeal phenotype to Bmpr1b−/− mice; specifically, the proximal and middle phalanges are fused and reduced to a featureless cartilage nodule (14, 15, 22). Thus, Noggin-sensitive members of the BMP family including GDFs acting singly or in combination are candidates to mediate BMPR-SMAD1/5/8 signaling at the PFR to determine digit identity. Finally, regardless of ligand identity, our data support the hypothesis that the interdigits are signaling centers that determine digit identity by signaling to the PFRs of the digital rays.

Materials and Methods

Embryos and Surgical Manipulations.

Fertile chick eggs were obtained from University of Wisconsin Poultry Science and Charles River Laboratories (SPAFAS strain). Embryos were staged according to ref. 25. ID grafts, tantalum foil (Goodfellow) barrier insertion, and bead implantation were done as described in refs. 6 and 26.

In Situ Hybridization, Immunohistochemistry, and Skeletal Analysis.

In situ hybridization and p-SMAD staining were performed as described in refs. 2628. Briefly, neighboring 20-μm cryosections were processed for tyramide amplification (TSA Indirect Tyramide Signal Amplification Kit; Perkin–Elmer Life Science) and immunoperoxidase labeled (ABC Kit; Santa Cruz Biotechnology). Antigen unmasking was performed in 10 mM sodium citrate (pH 9.0) in an 80°C water bath for 30 min. Skeletal cartilage was visualized as in refs. 6 and 26.

Viral Construction, Infection, and Luciferase Assays.

To produce RCANBPBP(A)-CMV-Renilla luciferase and RCANBPBP(B)-xVent2-Luciferase constructs, the CMV-Renilla region of pRL-CMV (Promega) was moved into pGL3 Basic by using BglII-XbaI. To produce the xVent2-Luciferase construct, the xVent2 promoter (11) was cloned upstream of luciferase gene in pGL3 Basic by using BglII-HindIII. Both constructs were then subcloned into the RCANBP shuttle vector, Cla12, by using SmaI-XbaI. ClaI fragments from each of the Cla12 vectors were inserted into RCANBPBP(A) and BP(B). Retroviral vectors were cotransfected into DF-1 cells by using Lipofectamine.

The prospective hindlimb regions of SPAFAS embryos were injected with DF-1 cells expressing both viruses at St. 14. At the desired stage, the noncondensed region of each visible digit was surgically isolated, and the tissue was used for dual luciferase analysis according to the manufacturer's instructions (dual-luciferase reporter assay system; Promega). Luciferase assays were performed in triplicate by using pooled tissue of 3–5 PFRs for each specific digit. For Noggin bead implantation or ID removal, virus-expressing cells were injected into the right and left hindlimbs at St. 14. The manipulations were performed on the right hindlimb 24 h before collecting the PFR tissues of both the experimental right and control left limbs. Luciferase value was measured by using an EG&G Berthold Lumat LB9507 luminometer. Data were analyzed for significance by using an unpaired t test.

Supplementary Material

Supporting Information:


We thank Michael Sheets for helpful discussions that changed the direction of this work; M. Harris, M. Sheets, X. Sun, and members of the J.F.F. laboratory for helpful suggestions on the manuscript; and K. W. Y, Cho (University of California, Irvine, CA), K. Miyazono (University of Tokyo, Tokyo), C. Tabin (Harvard Medical School, Boston), A. Lough (Medical College of Wisconsin, Milwaukee), Y. Kawakami (The Salk Institute, La Jolla, CA), P. Beachy (The Johns Hopkins University, Baltimore), S. Hughes and A. Ferris (National Cancer Institute, Frederick, MD), and C. Cepko (Harvard Medical School) for providing plasmids. T.S. thanks M. Martowicz and S. Smith for technical assistance. This work was funded by National Institutes of Health Grant HD032551 (to J.F.F.) and a Japan Society for the Promotion of Science Fellowship (to T.S.).


The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/cgi/content/full/0707899105/DC1.


1. McGlinn E, Tabin CJ. Mechanistic insight into how Shh patterns the vertebrate limb. Curr Opin Genet Dev. 2006;16:426–432. [PubMed]
2. Chiang C, et al. Manifestation of the limb prepattern: Limb development in the absence of sonic hedgehog function. Dev Biol. 2001;236:421–435. [PubMed]
3. Ros MA, et al. The chick oligozeugodactyly (ozd) mutant lacks sonic hedgehog function in the limb. Development. 2003;130:527–537. [PubMed]
4. Yang Y, et al. Relationship between dose, distance and time in sonic hedgehog-mediated regulation of anteroposterior polarity in the chick limb. Development. 1997;124:4393–4404. [PubMed]
5. Harfe BD, Scherz PJ, Tian H, McMahon AP, Tabin CJ. Evidence for an expansion-based temporal Shh gradient in specifying vertebrate digit identities. Cell. 2004;118:517–528. [PubMed]
6. Dahn RD, Fallon JF. Interdigital regulation of digit identity and homeotic transformation by modulated BMP signaling. Science. 2000;289:438–441. [PubMed]
7. Casanova JC, Sanz-Ezquerro JJ. Digit morphogenesis: Is the tip different? Dev Growth Differ. 2007;49:479–491. [PubMed]
8. Sanz-Ezquerro JJ, Tickle C. Fgf signaling controls the number of phalanges and tip formation in developing digits. Curr Biol. 2003;13:1830–1836. [PubMed]
9. Chen CM, et al. Production and design of more effective avian replication-incompetent retroviral vectors. Dev Biol. 1999;214:370–384. [PubMed]
10. Zou H, Wieser R, Massague J, Niswander L. Distinct roles of type 1 BMP receptors in the formation and differentiation of cartilage. Genes Dev. 1997;11:2191–2203. [PMC free article] [PubMed]
11. Candia AF, et al. Cellular interpretation of multiple TGF-beta signals: Intracellular antagonism between activin/BVg1 and BMP-2/4 signaling mediated by smads. Development. 1997;124:4467–4480. [PubMed]
12. Kida Y, Maeda Y, Shiraishi T, Suzuki T, Ogura T. Chick Dach1 interacts with the Smad complex and Sin3a to control AER formation and limb development along the proximodistal axis. Development. 2004;131:4179–4187. [PubMed]
13. Ovchinnikov DA, et al. BMP receptor type IA in limb bud mesenchyme regulates distal outgrowth and patterning. Dev Biol. 2006;295:103–115. [PubMed]
14. Baur ST, Mai JJ, Dymecki SM. Combinatorial signaling through BMP receptor IB and GDF5: Shaping of the distal mouse limb and the genetics of distal limb diversity. Development. 2000;127:605–619. [PubMed]
15. Yi SE, Daluiski A, Pederson R, Rosen V, Lyons KM. The type I BMP receptor BMPRIB is required for chondrogenesis in the mouse limb. Development. 2000;127:621–630. [PubMed]
16. Gruneberg H, Lee AJ. The anatomy and development of brachypodism in the mouse. J Embryol Exp Morphol. 1973;30:119–141. [PubMed]
17. Demirhan O, et al. A homozygous BMPR1B mutation causes a new subtype of acromesomelic chondrodysplasia with genital anomalies. J Med Genet. 2005;42:314–317. [PMC free article] [PubMed]
18. Katagiri T, Boorla S, Frendo J-L, Hogan BL, Karsenty G. Skeletal abnormalities in doubly heterozygous Bmp4 and Bmp7 mice. Dev Genet. 1998;22:340–348. [PubMed]
19. Selever J, Liu W, Lu M-F, Behringer RR, Martin JF. Bmp4 in limb bud mesoderm regulates digit pattern by controlling AER development. Dev Biol. 2004;276:268–279. [PubMed]
20. Bandyopadhyay A, et al. Genetic analysis of the roles of BMP2, BMP4, and BMP7 in limb patterning and skeletogenesis. PLoS Genet. 2006;2:2116–2130. [PMC free article] [PubMed]
21. Zuzarte-Luís V, et al. A new role for bmp5 during limb development acting through the synergic activation of Smad and MAPK pathways. Dev Biol. 2004;272:39–52. [PubMed]
22. Settle SH, et al. Multiple joint and skeletal patterning defects caused by single and double mutations in the mouse Gdf6 and Gdf5 genes. Dev Biol. 2003;254:116–130. [PubMed]
23. Seemann P, et al. Activating and deactivating mutations in the receptor interaction site of GDF5 cause symphalangism or brachydactyly type A2. J Clin Invest. 2005;115:2373–2381. [PMC free article] [PubMed]
24. Merino R, et al. Expression and function of GDF-5 during digit skeletogenesis in the embryonic chick leg bud. Dev Biol. 1999;206:2161–2170. [PubMed]
25. Hamburger V, Hamilton HL. A series of normal stages in the development of the chick embryo. J Morphol. 1951;88:49–92. [PubMed]
26. Suzuki T, Takeuchi J, Koshiba-Takeuchi K. Tbx genes specify posterior digit identity through Shh and BMP signaling. Dev Cell. 2004;6:43–53. [PubMed]
27. Ros MA, Simandl BK, Clark AW, Fallon JF. In: Development Biology Protocols. Tuan RS, Lo CW, editors. Totowa, NJ: Humana; 2000. pp. 245–266.
28. Ahn K, Mishina Y, Hanks MC, Behringer RR, Crenshaw EB. Bmpr-IA signaling is required for the formation of the apical ectodermal ridge and dorsal-ventral patterning of the limb. Development. 2001;128:4449–4461. [PubMed]

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