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

Abstract

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.

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 into the circulation. (B) High magnification of boxed area in A. The digital ray shows a constant proximal to distal tissue arrangement consisting of three morphologically distinguishable proximal to distal regions. First is the condensed avascular cartilage of the digit primordium (p). Second is ≈100 μm of noncondensed, vascularized mesenchyme (arrowhead). Third is 100 μm of noncondensed, avascular mesenchyme subjacent to the AER (arrow). Hereafter, we refer to this proximal-distal arrangement as the digital ray, and we refer to the digit primordia as the condensed, avascular cartilage of the digital ray. (C) Cartilage stain of a day-10 chick foot illustrating the number, size, and shape of the phalanges of the digits. (D, H, and K) Diagrams of experimental manipulations performed. In E–G, only the manipulated digit 3 is shown; the distal edge of the metatarsal is indicated (MT). (D) A tantalum foil barrier placed at the distal extent of p3 at St. 26 was found in the joint of the metatarsal and first phalanx at day 9.0 (black arrowhead in E). Barriers placed at the distal extent of p3 at St. 28 and St. 30 were found in the middle phalanges or distal phalanx (black arrowheads in F and G), respectively. AP staining was seen in the proximal phalanges after RISAP injections at the PFRs of St. 27 DRs (I) and in the distal phalanges after injection at the PFRs of St. 29/30 DRs (J). RISAP injection of the subridge avascular cells of the DRs at St. 27 resulted in AP staining only in the distal phalanges at day 9.5 (L).

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 and neighboring in situ hybridization of Bmpr1b in the autopod. Note strong p-SMAD1/5/8 staining and Bmpr1b expression distal to the digit primordium at the PFR (red arrows). Also note the down-regulation of p-SMAD1/5/8 staining (bracket) between the signal in the subridge mesenchyme (black arrow) and in the Bmpr1b domain (red arrow). (C) Time course of ID removals to determine the stages when the phalanges become fixed and stable. The stage at which the digit primordium (condensed cartilage) is first visible is indicated by the red arrowheads. The green arrowheads indicate the stage at which digit identity is fixed and stable, i.e., when digit identity transformations were no longer observed after ID removal. Phalanx determination occurs in a proximal-to-distal sequence. The digit that forms depends on the stage at which the ID was removed. ID removals (n > 10 for each stage) at the onset of digit primordia condensation produced full digit (d) identity transformations (d2→d1, d3→d2, and d4→d3). Subsequent removals produced chimeric digits (data not shown). We define chimeric digits as a combination of normal proximal phalanges or phalanx of a digit in tandem with transformed distal phalanges or phalanx.

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 cells, individual PFRs were harvested at different stages and used for the dual luciferase assay. (B) Each PFR reports unique signaling activity throughout all stages. Black arrows show when each digit identity is fixed and stable (see Fig. 2). Data were analyzed by unpaired t test. Statistical comparisons between the PFRs at St. 28 were as follows: digit 1 (d1):d4, d3:d4, and d2:d3 were P < 0.01, and d2:d4 was P < 0.05.

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 activity after manipulation of the ID. (A) Noggin bead implantation into ID2 (red circle) is accompanied by decreased SMAD1/5/8 activity at the digit-2 PFR similar to the left and right control digit-1 PFRs, corresponding to anteriorization of digit 2 to digit 1. (B) Noggin bead implantation into ID3 (red circle) is accompanied by decreased SMAD1/5/8 activity at the digit-3 PFR, similar to the left and right control digit-2 PFRs, corresponding to anteriorization of digit 3 to digit 2. Note that there is no change in SMAD1/5/8 activity in the PFRs posterior to the beads in both A and B. (C) Pinning ID3 to the posterior-distal position of the digit-1 PFR is accompanied by an increase in SMAD1/5/8 activity at the digit-1 PFR to levels similar to that of the control right and left digit-3 PFRs, corresponding to a posterization of digit 1 to a digit 3. (D) Pinning ID3 to the posterior-distal position of the digit-2 PFR induces up-regulation of SMAD1/5/8 activity at the digit-2 PFR to levels seen in the left control digit-3 PFR, corresponding to the posteriorization of digit 2 to a digit 3. (E) ID4 removal up-regulates SMAD1/5/8 activity of the digit-4 PFR to that of the left and right digit-3 PFRs, corresponding to the anteriorization of digit 4 to a digit 3. Data were analyzed by unpaired t test; significance, P < 0.05.

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 enlargement of distal DR represented in B), we propose that proximal-distal elongation occurs in a dynamic fashion driven by the distal subridge cells proliferating. A result of this is that a subset of cells is removed from AER influence. These DR cells up-regulate Sox9 expression (red dashed line) committing them to the cartilage differentiation pathway. These cells take on the character of the PFR (red crescent) by up-regulating Bmpr1b and becoming immunohistochemically positive for p-SMAD1/5/8. Under the influence of unidentified signals arising from the posterior ID (blue arrow), unique levels of p-SMAD1/5/8 can be measured for each PFR, and this activity signature correlates with digit identity, supporting the notion that threshold signaling levels (graded purple color of ID1–3, red for ID4) in A lead to different developmental outcomes in a morphogenetic field. Because the PFR cells are determined to form a particular digital element, they are incorporated into the cartilage of the digit primordium (black dashed line indicates path of incorporation).