Vertebrate limb development: moving from classical morphogen gradients to an integrated 4-dimensional patterning system

Abstract

A wealth of classical embryological manipulation experiments taking mainly advantage of the chicken limb buds identified the apical ectodermal ridge (AER) and the zone of polarizing activity (ZPA) as the respective ectodermal and mesenchymal key signaling centers coordinating proximodistal (PD) and anteroposterior (AP) limb axis development. These experiments inspired Wolpert's French flag model, which is a classic among morphogen gradient models. Subsequent molecular and genetic analysis in the mouse identified retinoic acid as proximal signal, and fibroblast growth factors (FGFs) and sonic hedgehog (SHH) as the essential instructive signals produced by AER and ZPA, respectively. Recent studies provide good evidence that progenitors are specified early with respect to their PD and AP fates and that morpho-regulatory signaling is also required for subsequent proliferative expansion of the specified progenitor pools. The determination of particular fates seems to occur rather late and depends on additional signals such as bone morphogenetic proteins (BMPs), which indicates that cells integrate signaling inputs over time and space. The coordinate regulation of PD and AP axis patterning is controlled by an epithelial-mesenchymal feedback signaling system, in which transcriptional regulation of the BMP antagonist Gremlin1 integrates inputs from the BMP, SHH, and FGF pathways. Vertebrate limb-bud development is controlled by a 4-dimensional (4D) patterning system integrating positive and negative regulatory feedback loops, rather than thresholds set by morphogen gradients.

Figure 1.

Two morpho-regulatory signaling centers control vertebrate limb-bud development. (A) Skeletal preparation of a mouse forelimb at birth. (B) Skeletal preparation of a fetal chicken wing at day 15 of embryonic development. Red and blue histological stains mark ossified bone and cartilage, respectively. Despite morphological differences, the basic bauplan along both axes is conserved. (Prox-dist) Proximodistal axis, (ant-post) anteroposterior axis, (Sc) scapula, (Cl) clavicle, (Hu) humerus, (Ra) radius, (Ul) ulna. Digit identities are indicated by numbers. (C) Visualization of the AER by in situ detection of Fgf8 transcripts in a mouse limb bud. (D) The ZPA expresses the Shh morphogen. (E) Wolpert's French flag model: A concentration gradient forms by diffusion of a morphogen from a source and positional information is determined in groups of cells by inducing distinct responses to specific concentration thresholds (indicated by blue, white, and red).

Figure 2.

Models and mechanisms of PD limb axis morphogenesis. (A) The original progress zone model. PD positional information values depend on the time cells have spent in the progress zone under the influence of the AER. Stylopod identity is acquired early, whereas zeugopod and autopod identities are specified at progressively later time points. The sequence of skeletal elements is specified from proximal to distal. (B) Early specification/expansion model. PD positional information is specified very early during initiation of limb-bud development and the specified territories expand sequentially during distal progression of limb-bud outgrowth. (C) Two signal gradient model. Cells are specified by a proximal to distal RA gradient emanating from the embryonic flank/proximal limb bud and by a distal to proximal gradient of AER-FGF signaling. Integration of these two signals over space and time provides the cells with their positional values. The Meis1/2, Hoxa11, and Hoxa13 expression domains mark the three PD territories. (D) The differentiation front model. AER-FGF signaling keeps the distal mesenchyme in an undifferentiated state. Sprouty4 (Spry4) and AP2 are molecular markers of this undifferentiated zone, while Sox9 marks differentiating chondrocytes. The differentiation front separates these two domains and is displaced distally during progression of limb-bud outgrowth.

Figure 3.

Models and mechanisms for SHH-mediated AP limb axis patterning (A) The early limb bud is already prepatterned by an antagonistic interaction between HAND2 (orange) and the repressor form of GLI3 (GLI3R, dark blue) transcription factors. Nested expression of 5′Hoxd genes and HAND2 participate in activation of Shh expression. (B) Skeletal preparation of a Shh deficient mouse limb at birth. (C) Spatial gradient model. Diffusion of the SHH peptide secreted by the ZPA generates a GLI3R gradient across the limb bud (graded blue) by inhibiting processing of full-length GLI3. The red line indicates the threshold values predicted by Wolpert's French flag model (Fig. 1E). (D) Temporal gradient model. Descendants of Shh expressing ZPA cells contribute to the progenitor domains of digit 3 to 5. Cells having expressed Shh for a short time contribute to digit 3, whereas the progenitor domains of digits 4 and 5 contain cells having expressed Shh for progressively longer times. Progenitors forming digit 2 and parts of digit 3 are specified by long-range SHH signaling. (E) Genetic analysis of the temporal requirement of SHH in the mouse shows digit identities are specified early. Subsequently, SHH is required for proliferative expansion of progenitor pools and determination of specified identities. Determination of digit identities in the mouse occurs in the following sequence: digit 4 (first), 2, 5, and 3 (last).

Figure 4.

The role of BMP signaling from the interdigital mesenchyme in determination of digit identities. Graded BMP signaling from the interdigital (ID) mesenchyme (blue) in the chicken foot primordia is involved in determining the identities of digits 1 to 4 at late developmental stages. The distal phalanx of individual digits form from the sub-AER mesenchyme, which is therefore called phalanx forming region (PFR). The activity of phosphorylated SMAD (pSMAD) proteins, which are the intracellular mediators of BMP signal transduction, is graded within the PFR (green), such that each digit has its characteristic pSMAD activity signature. Note that the pSMAD activity in the PFR of the posterior-most digit 4 is lower than the one of digit 3. AER is indicated in red.

Figure 5.

Interlinked signaling feedback loops control initiation, propagation, and termination of E–M feedback signaling. The SHH/GREM1/FGF E–M feedback loop is required for maintaining and propagating SHH signaling by the ZPA and up-regulation of FGF signaling in the AER. In mouse limb buds lacking Grem1, establishment of this E–M feedback signaling loop, distal progression of limb-bud development, and specification of digit identities is disrupted. The BMP antagonist GREM1 defines a regulatory node in this at least in parts self-regulatory limb signaling system as its transcription is positively regulated by BMPs (predominant during initiation) and SHH (predominant during progression), and inhibited by high FGF levels (predominant during termination of E–M feedback signaling).