The Limb Field and the AER

The Limb Field and the AER

The idea of morphogenetic fields has pervaded embryology so thoroughly that it cannot be shaken off, any more than a religious upbringing.1

Because limb buds are used to illustrate aspects of cell condensation (Chapter 19), chondrogenesis (Chapters 22 and 26), polarity, cell death, morphogenesis and growth (Chapter 38 and 39), I provide a summary of the major morphogenetic and differentiative events of limb-bud development in embryonic chicks up to H.H. 30 (6.5–7 days of incubation) (Table 35.1), in the hope that such a ready reference will be useful. Mapping the presumptive areas of wing buds by implanting carbon particles and following them during development allowed Saunders (1948) to demonstrate that wing-bud mesenchyme is derived from somatic mesoderm of the lateral plate and not from somitic mesoderm (Chapter 16).

THE MESODERMAL LIMB FIELD Demonstrating as they did that limb mesoderm is the site of the limb field the experiments performed by Ross Harrison (1918) on limb development in embryos of Ambystoma are classics in experimental embryology. A field (morphogenetic field) is an area of the early embryo specified to form an organ system. As noted in the epigraph the field concept, although difficult, has persisted. We remain ‘held down still by the gossamer threads of the past, like a giant in a fairy tale, disabled by magic.’2 Evidence for fields arose initially from experimental studies such as those by Harrison, from which it became clear that regions of early embryos were specified to make individual organ systems. Transplant the field

Chapter

35

elsewhere on the embryo and the organ forms ectopically in the new site. Just as, importantly, cells in the original site cannot compensate for the removal of the field – they are not specified to make the particular organ system (Figs 35.1 and 35.2). So we can speak of the heart field, limb field, eye field and so on. Cells outside these fields do not have the ability to make heart, limb or eye. You can think of each of the imaginal discs in Drosophila as a developmental field specified to produce a particular part of the adult. In important recent analyses, the mutation obake (obk) was shown to result in duplication/ multiplication of the antenna morphogenetic field in Drosophila and to be influenced by other mutations affecting antennal imaginal discs and by environmental factors such as larval crowding which suppresses the affect of the mutant.3 Morphogenetic fields are alive and well as evolvable units of morphology. An important property of developmental fields is their ability to regulate, where regulation is the ability of entire early embryos or embryonic fields to compensate for loss in such a way that part of the field can produce the entire structure (see Box 35.1). As an instance, a partial limb field can produce a whole limb but cells outside the field cannot contribute to limb formation. When prospective limb-bud mesoderm is grafted under flank ectoderm away from where limbs would form, the mesoderm grows, flank ectoderm is drawn into limb development and a supernumerary limb grows out from the side of the host (Figs 35.1 and 35.3). No limb develops when mesoderm from outside the prospective limb field is grafted under ectoderm within the limb field. A limb fails to develop if prospective limb mesoderm is removed from neurula-stage embryos but the ectoderm left intact. Specificity of mesoderm as the initial ‘limb inductor’ and evocation of ectoderm in response to limb mesoderm are

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Bones and Cartilage: Developmental and Evolutionary Skeletal Biology Table 35.1 A summary of the major events in the development of the wing bud in the embryonic chick H.H. stagea

Length of H.H. stage (hours)

Developmental event

References

7 10 10–11 12

3 7 10 3

13, 15 3 2 17

15 17

2–3 14

18 20 21 22

6 4 8 6

23 24

12 6

25

6

26

12

27

12

28

6

29 30

12 6

Presumptive limb mesenchyme can chondrify ectopically Earliest point at which flank mesenchyme can become limb A-P axis already fixed; dorsoventral axis fixed at this point Earliest point at which wing mesenchyme will induce formation of an AERb from flank epidermis Earliest age for autonomous differentiation Limb buds present (wing bud adjacent to somites 15–19; leg bud adjacent to somites 26–32); AER present; wing mesenchyme loses ability to induce AER in the flank and flank epithelium loses ability to respond to limb mesenchyme Limb mesenchyme forms cartilage in vitro; humerus specified Vascular pattern initiated; ulna and radius specified Specification of wrist starts (lasts until H.H. 24) S35 uniformly distributed over mesenchyme; sinusoids demark myo- from chondrogenic areas; proximal, central mesenchyme condensing; PNZc irreversibly determined; mitotic index starts to decline, especially centrally Myosin first appears; first innervation of hind limb bud Y-shaped condensation and opaque patch appear; vascular pattern well established; cell packing in prechondrogenic mesenchyme increased 60% over that at H.H. 22; proximo-distal gradient in cell division present; first necrotic cells in PNZ; ability to form cartilage stabilized between H.H. 24 and 25 ECM of cartilage appears; proximal mesenchyme will no longer support an AER (i.e. morphogenetic properties lost), nor will it produce distal structures; innervation reaches stylopod Cell density in chondrogenic mesenchyme increased 10% over that at H.H. 24; ability of cells to segregate appears; dissociated chondrogenic mesenchyme produces condensations; digit III and metacarpals specified (H.H. 25–26); muscle appears Cartilaginous models of limb skeleton present; proximal phalanx specified; innervation reached zeugopod (H.H. 27–29) Opaque patch disappeared; distal phalanx specified; innervation reached autopod AER loses inductive ability Interdigital cell death (H.H. 30 in hind limb, H.H. 31 in wing); definitive nerve pattern

13 10, 16

20, 25 1, 25 25 27

6, 11 8, 23, 26

5, 6, 18

4, 7, 21, 22, 25, 26

5, 6, 25 6, 25, 26 14 16

a

For hours of incubation corresponding to these H.H. stages, see Table 12.3. AER, apical epithelial ridge. c PNZ, posterior necrotic zone. References: (1) Caplan and Koutroupas (1973); (2) Chaube (1959); (3) Dhouailly and Kieny (1972); (4) Ede and Flint (1972); (5) Finch and Zwilling (1971); (6) Fouvet (1970); (7) Hilfer et al. (1973); (8) Hornbruch and Wolpert (1970); (9) Janners and Searls (1970); (10) Kieny (1960); (11) Medoff and Zwilling (1972); (12) Mottet and Hammer (1972); (13) Pinot (1970); (14) Rubin and Saunders (1972); (15) Rudnick (1945); (16) Saunders and Fallon (1967); (17) Saunders and Reuss (1974); (18) Saunders et al. (1959); (19) Searls (1965a); (20) Searls (1968); (21) Searls (1971); (22) Searls (1972); (23) Searls and Janners (1969); (24) Searls et al. (1972); (25) Summerbell (1974b); (26) Thorogood and Hinchliffe (1975); (27) Zwilling (1966). b

both strongly suggested in this pioneering study, which was one of the founding studies of the concept of developmental fields.

ECTODERMAL RESPONSIVENESS The position along the embryonic axis occupied by the limbs is set by the location of the mesodermal limb field and by the ability of flank ectoderm to respond to signals from the limb field. Collagen accumulates beneath the flank ectoderm but not beneath prospective limb ectoderm of embryonic chicks between H.H. 12 and 17 (A. A. Smith et al., 1975).

Deposition of this fibrous collagenous barrier partly explains why flank ectoderm loses its ability to support limb development at and beyond H.H. 17. Limbs fail to form at all in some mutant chick embryos because such a barrier accumulates beneath the apical epithelial ridge (AER), which is the specialized ectodermal thickening that develops a little later in limb development. As discussed in the context of wingless mutants in Chapter 37, the first definitive evidence for a reciprocal interaction between mesoderm and ectoderm came from Zwilling’s experiments on the chick wingless mutant. Experiments similar to Zwilling’s were performed using limb and non-limb sites in wild-type embryos. The site selected was a region of the flank between wing and hind

445

The Limb Field and the AER

1

A

D d

P

P

S5

S3 ot

A

a

S10

D

v

A

P

V

V

2 v a

p

B

d

v

S10

S3

pn

v a

p

S5

a

d

p d

D

4 A

5

eye

P V 3

Figure 35.1 Limb fields, axes and axis determination, illustrated using amphibian embryos. (1) An outline of a neurula (anterior to the right) to show the forelimb bud field (circle) and the four planes: anterior (a), dorsal (d), posterior (p) and ventral (v). Note that the planes and axes (A-P, D-V) of the limb field correspond to those of the embryo (A, D, P, V). The orientation of the posteriorly directed forelimb is shown on the right arising from the limb field. (2) The limb field has been removed, rotated 180 and reimplanted so that limb field and embryonic axes no longer correspond. (3) The A-P but not the D-V axis is reversed after the rotation shown in (2), indicating that the A-P but not the D-V axis had been determined before field rotation, i.e. early in development. (4) The result expected in the experiment in (2) if the D-V but not the A-P axis had already been determined. (5) The result expected in the experiment in (2) if both D-V and A-P axes had been determined before field rotation. Also see Figs 35.2 and 35.3. From Hall (1978a).

limb buds, a region that contains mesenchyme and epithelium but does not produce limbs. Flank ectoderm from embryos of H.H. 10–17 can participate in limb-bud formation if brought into contact with limb-field mesoderm from embryos of H.H. 13–17 (Fig. 35.1). Grafting pre-limbbud mesoderm to the flank elicited an AER from flank epithelium. Formation of a supernumerary wing followed. Timing of this interaction provides a clue to the normal time course of the interaction of limb mesenchyme and epithelium. Wing mesenchyme loses the ability to induce an AER in flank ectoderm at H.H. 17. Reciprocally, flank ectoderm loses the ability to respond at H.H. 17 (see Table 35.1), this being the stage with the first external sign of limb buds. Presumptive limb-area mesoderm from embryos of H.H. 11 (Fig. 16.2) or younger will not elicit a ridge from flank ectoderm. Although mesoderm from embryos of H.H. 12–17 will act, mesoderm from older embryos will not. Localizing the time of interaction

Figure 35.2 Views of the right side of neurula-stage embryos (anterior to the right) of the Mexican axolotl showing the position of the forelimb field – outlined in the square in (A) – and its transplantation to a more posterior region, shown as the square in (B). ot, otocyst; S3, S5, S10, somites three, five and 10; pn, the position of the pronephros (future kidney). Modified from Hamburger (1960).

between H.H. 12 and 17 ensures that once a limb bud forms, additional limbs are unlikely to be produced.4 3 H-thymidine labeling, the ability to identify nuclei in Japanese quail cells, and the type of epidermal differentiation seen in grafts between chick and Japanese quail all indicate that flank ectoderm contributes to the supernumerary limb. Limb-bud mesenchyme from embryos of H.H. 19 and 20 has also been grafted beneath tail-bud epithelium of H.H. 21 and 22 embryos, where it induces tail epithelium to form an AER, followed by limb outgrowth and digit formation. Specificity to initiate limb development resides with mesoderm of the limb field. Mesoderm also specifies the type of limb that will form – forelimb (wing/arm/flipper) or hind limb (leg).5

MESODERM SPECIFIES FORE- VS. HIND LIMB For at least half a century, experimental embryologists have wanted to know how fore- and hind limbs are specified. As discussed above, the limb field is a property of the mesoderm. Whether a limb will be fore or hind also is a mesodermal property, a conclusion based on swapping wing- and hind-limb-bud epithelium and mesenchyme as follows.

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Box 35.1 Regulation Regulation is the ability of entire early embryos or embryonic fields to compensate for loss in such a way that part of the field can produce the entire structure. Examples include the ability of each of the two blastomeres of a two-cell-stage embryo to produce an entire embryo after being separated, of a bisected limb field to produce an entire limb, or of the left half of the heart field to produce a complete heart. Regulation also occurs on a smaller scale; removing a single somite from a two-day-old chick embryo elicits regulation from the adjacent somite and from intermediate mesoderm, the new somite fitting into the rostro-caudal patterning sequence. Removing a small section of neural crest from zebrafish embryos elicits regulation from the adjacent neural crest.a Regulative ability is lost as soon as regions within fields are specified. Regulation is the embryonic equivalent of regeneration, the difference being – and it is an important difference – that regeneration is the replacement of differentiated cells in structures that have already formed, while regulation is the replacement of cells that have yet to differentiate. Limb fields show the property of regulation. In this case, half a limb field can produce a complete limb bud. Regulative ability also exists during the transition from limb field to limb bud. In this case the limb bud can compensate for a cell population that is lost. Once regulative ability is lost, the cells in question can be considered as specified and their fate set.

A pivotal study and an elegant demonstration of regulation is that published by Searls and Janners in 1969. When prechondrogenic mesenchyme from chick embryos younger than H.H. 24 is grafted to the premyogenic area of a limb bud of a similarly aged embryo, almost all the cells regulate to form myoblasts. If, however, prechondrogenic mesenchyme is taken from limb buds of embryos older than H.H. 25, the majority of the cells produce cartilage, readily identified as nodules of ectopic cartilage within the muscle of the host limb. The ability of central cells to chondrify stabilizes between H.H. 24 and 25 or, to put it another way, the ability to regulate is lost once chondrogenesis has been specified. Several other pieces of evidence, outlined below, lead us to this same conclusion. Zwilling (1966) cultured or CAM-grafted premyogenic, prechondrogenic and what he called intermediate mesenchyme (at the boundary of premyogenic and prechondrogenic regions) limb buds of embryos of H.H. 22–24. Mesenchyme from all three sites chondrified in similar proportions – 67 per cent of the ‘myogenic,’ 64 per cent of the ‘chondrogenic,’ and 71 per cent of ‘intermediate’ mesenchyme. Cells whose progeny would have differentiated into myoblasts differentiated as chondroblasts. Stark and Searls (1974) excised various prospective long-bone areas from wing buds to determine whether adjacent premyogenic mesenchyme would regulate and replace them (Table 35.2). They excised:

Table 35.2 Regulative ability of limb mesenchyme as assessed by

(i) mesenchyme that normally forms the humerus, radius and ulna, representing some 60–70 per cent of limb mesenchyme; (ii) prospective radius and ulna mesenchyme, representing some 20–40 per cent of limb mesenchyme; and (iii) humeral, radial or ulnar mesenchyme independently.

the ability to replace excised prospective humeral, radial or ulna mesenchyme and produce a morphologically normal limba,b Embryonic age at excision (H.H. stage)

Per cent (N) of normal limbs six or seven days post-excision Prospective humerus, radius and ulna all excised

Prospective ulna/ radius or humerus, radius, or ulna excised

19 20 21 22 23 24

67 (8/12) 60 (3/5) 37 (3/8) 33 (4/12) 10 (1/10) 0 (0/2)

100 (9) 100 (18) 77 (27/35) 33 (11/33) 5 (2/40)

a b

Regulative ability decreases with embryonic age and is lost after H.H. 24. Based on data in Stark and Searls (1974).

Procedures (ii) and (iii) gave similar results, and are grouped together in Table 35.3. The greater the amount of mesenchyme removed and the older the embryo from which mesenchyme is removed, the greater the incidence of abnormal limbs and the lower the regulative ability. Regulative ability is lost at H.H. 24, the stage at which prechondrogenic mesenchyme becomes stabilized for chondrogenesis (see above). Finally, regulation occurs irrespective of the position along the proximo-distal axis from which the mesenchyme is excised. In a second experiment Stark and Searls rotated the prospective elbow region through 180 and reimplanted it into the wing bud. Epithelium adjacent to the graft was rotated with the mesenchyme or left in place. Normal development of the joint after rotation would

Table 35.3 Regulative ability of limb mesenchyme as assessed by the ability to compensate for rotation of the prospective elbow region – with or without rotation of adjacent ectoderm – and produce a normal limb buda,b Embryo age at rotation (H.H. stage)

Prospective elbow mesenchyme and ectoderm rotated

Prospective elbow mesenchyme rotated

Dorsal ectoderm rotated

Per cent of specimens with

20 21 22 23 24 a

normal joint

ectopic cartilage

normal joint

ectopic cartilage

normal joint

25 17 5 0 0

50 67 80 100 100

– 100 76 0 0

–

– 60 36 19 –

0 23 100 100

Presence of nodules of ectopic cartilage at the normal site of the joint indicates inability to regulate. Regulative ability is lost after H.H. 22. Results are expressed as per cent of specimens with normal joints or with nodules of ectopic cartilage. Based on data in Stark and Searls (1974) and in Searls (1976).

b

The Limb Field and the AER

447

Box 35.1 (Continued ) signify regulation, while formation of nodules of ectopic cartilage at the joint site would demonstrate failure of regulation. As shown in Table 35.4, provided that the epithelium retains its original orientation, regulation occurs until H.H. 22; rotation of dorsal epithelium of the joint region alone is sufficient to prevent regulation. Regulation at this stage of development begins to appear as if it is not entirely independent of influences from adjacent tissues. Other workers obtained similar results with slightly different stages for onset or offset of regulation: ● ●

●

●

for removal of 90 per cent of the wing mesoderm until H.H. 19; until H.H. 23 after limb mesenchyme from embryos of Japanese quail was grafted into various levels of embryonic chick limb buds; until H.H. 22 after slices along the proximo-distal axis of the chick wing bud were removed; and within the common condensation for the tibia and fibula.b

In summary, regulation is possible before H.H. 22. Between H.H. 22 and 24, cells (regions?) stabilize and are less able to regulate. Regulative ability is lost after H.H. 24 (Table 35.3). Loss of regulative ability in such experiments is interpreted as loss of the ability of mesenchymal cells to change fate. Alternatively, it could be that cells are less capable of filling the larger wounds required to excise

Figure 35.3 Wing-bud mesenchyme can evoke an AER from flank ectoderm, resulting in supernumerary wing formation. (A) An outgrowth, complete with AER formed after wing-bud mesenchyme (H.H. 14) was grafted beneath flank ectoderm of an H.H. 13 host embryo. (B) A supernumerary wing formed after wing-bud mesenchyme from H.H. 13 was grafted beneath flank ectoderm of an H.H. 14 embryo. Also see Fig. 35.1. Modified from Saunders and Reuss (1974).

●

●

●

●

When epithelium from a hind-limb bud is grafted onto wing-bud mesenchyme the distal structures (digits) that develop are wing digits. When epithelium from a wing bud is grafted onto hindlimb-bud mesenchyme leg skeletal elements develop. Conversely, grafting hind-limb-bud mesenchyme in the place of wing-bud mesoderm produces digits (‘toes’) typical of the leg, while wing mesenchyme grafted onto hind-limb bud produces digits typical of wing at the end of the other leg skeletal elements.

From such studies, we see that the distal structures are always determined by the type of limb bud supplying the

mesenchyme in older limb buds, or less able to migrate across the limb bud to fill the gap. This seems unlikely; Barasa (1962, 1964) found that regulation is more complete in larger than in smaller wounds. Mouse embryos can regulate for loss of limb-bud mesenchyme. The experimental approach was to remove forelimb buds from 11.5-day-old embryos and maintain the whole embryos in vitro (well, whole embryos minus the forelimb buds). Within 24 hours, 90 per cent (24/27) of the embryos produced bud-like outgrowths, a third of which formed AERs. In a second approach, a block of mesoderm two to three somites wide was excised from the forelimb bud region of 10-day-old mouse embryos and the embryos were cultured for six to 24 hours. Two thirds of these embryos restored normal morphology and formed AERs as assessed using SEM.c a See Vaglia and Hall (1999) for a discussion of regulation following removal of regions of neural crest, and Liu and Bagnall (1995) for regulation following somite removal. b See Barasa (1962, 1964) and Searls (1976) for regulation after 90 per cent removal, Kieny and Pautou (1976) for the quail–chick chimaeras, Summerbell (1977b, 1981) for removal of P-D slices of wing buds, and Kieny (1967) for regulation within the condensation. c See K. K. H. Lee (1992) and Lee and Chan (1991) for these experimental studies.

mesenchyme. When the exchange is between different species – e.g. between domestic fowl and duck – the results are even more striking, as species-specific characteristics reinforce the evidence of mesenchymal specification of limb type.6 Of course, to say that mesoderm specifies limb type tells us nothing of the molecular basis for that specification, for which see Box 35.2. Once limb epithelium is specified to form an AER, reciprocal interactions between mesenchyme and AER determine the size, outgrowth and differentiation of the limb buds and the resulting limb skeleton. The function of the AER in specification of the proximo-distal sequence of skeletal elements is discussed in Chapter 39. In the present chapter I evaluate evidence for interactions between the AER and underlying mesenchyme under three headings: ● ● ●

the role of the limb-bud epithelium; induction of the AER by limb mesenchyme; and the maintenance of the AER by limb mesenchyme.

ROLES FOR THE ECTODERM ASSOCIATED WITH THE LIMB FIELD By grafting regions of the blastoderm into the body cavities of host embryos and following their fate, Rudnick (1945a) mapped the presumptive wing territory in chick embryos of H.H. 6 and the leg territory in embryos of H.H. 8. Early limb buds of embryonic chicks – indeed the limb buds of any tetrapod – consist of a core of mesenchyme (the mesoblast) underlying a thin cap of cuboidal epithelium. Late in H.H. 17, the epithelial cells at the apex of the limb bud become columnar. By H.H. 19, more rapid development post-axially produces an asymmetrical bud with a nipple-like ridge of ectoderm distally, the apical epithelial ridge

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Bones and Cartilage: Developmental and Evolutionary Skeletal Biology

Box 35.2 Fore or aft

●

●

●

How is the position along the flank where paired appendages will develop specified? What is the molecular basis of specification of limb or fin type: fore or hind? Can differences associated with limb type be detected before the skeletal elements form?

All three are important questions. The first and second are easier in the sense that the genes that specify location and limb type have been identified. That done, we tend to think that we ‘know’ how limb type is specified although, of course, much remains to be uncovered. The third aspect seeks a mechanism or mechanisms: what are the downstream genetic and cellular activities that allow wing or leg to arise from seemingly similar mesenchymea?

Positioning paired appendages Tabin and Laufer (1993), recognizing the serial homology of foreand hind limbs, argued that Hox genes originated in the hind limbs with a homeotic transformation transferring them to the fore limbs. From the elegant study by Burke et al. (1995) we know that a Hox gene code expressed in paraxial and associated mesoderm specifies vertebral type into three broad classes, cervical, thoracic and caudal. Further, we know that both fore- and hind limb buds arise adjacent to the anterior expression boundary of Hoxc-5, even if that boundary is expressed adjacent to different somites along the body axis in different species, which it is; the forelimb boundary is seven somites more posterior in the chick when compared with the mouse. In an important study, Nelson et al. (1996) cloned 23 genes and identified three phases of development, each with different sets of genes in both fore- and hind limb buds. The Hox pattern arises in response to Shh. More recently, Gaunt (2000) reviewed evolutionary shifts in vertebral structures in relation to Hox gene expression, concentrating on the positions of the neck/thorax (cervical/thoracic vertebrae) and forelimbs. Part of such specificity resides in cis-regulatory elements in Hox genes, as demonstrated for Hoxc-8. Hoxc-8 cis-regulation Belting et al. (1998) examined the evolution of cis-regulatory elements of Hoxc-8 in chick and mouse embryos in relation to the divergent axial morphology of birds and mice. Although Hoxc-8 is expressed in the mid-thoracic (brachial) mesoderm and neural tube in both species, activation is delayed in the chick so that expression is more posterior and over a smaller area of mesoderm which, they argue, explains the shorter thorax in chicks as compared with mice.

SO

LPM

Forelimb field Hox code

Until recently, palaeontologists and evolutionary biologists (often one and the same) gave little consideration to the possibility that fore- and hind limbs (or pectoral and pelvic fins) might have evolved independently as anterior and posterior paired appendages. Recently, a non-simultaneous origin appears more likely, primarily because we now know something of how anterior and posterior paired appendages are specified in fishes and tetrapods. As discussed in the text, specification of limb type is a property of limb mesoderm, not epithelium. Traditionally, limb type was assessed by examining the skeletal elements that form after experimental manipulation, e.g. combine wing-bud mesenchyme with leg epithelium and ask whether the skeleton that forms is wing or leg (it is wing). Three further aspects, listed below, are addressed here.

Baleen whales lack four base pairs in element C of Hoxc-8. When expressed in transgenic mice lack of this cis-acting element directs gene expression in the more posterior neural tube but not in the posterior mesoderm (where Hoxc-8 is not normally expressed). Shashikant et al. (1998) correlated his loss with specific traits in whales. Hoxc-8 is expressed in prechondrogenic limb condensations, over-expression leading to the accumulation of dividing chondrocytes. The severity of the over-expression is gene dosage dependent and specific to limb condensations, cranial elements being unaffected. Yueh et al. (1998) argue that Hoxc-8 controls the transition from dividing cells to differentiating cells. Tsumaki et al. (1996) identified separable cis-regulatory elements controlling tissue- and site-specific 2(XI) collagen gene expression in embryonic mouse cartilage. Weatherbee and Carroll (1999) explained regulation of target regions by selector genes in sub-regions of developing limbs through field-specific expression of cis-regulatory elements.

Tbx-5

ECT

FgfR-2 Forelimb bud Fgf-8 outgrowth

Fgf-10

Pitx-1 Hindlimb field Hox code

Fore–hind limb, arm/wing–leg, pectoral fin–pelvic fin

Tbx-4

FgfR-2 Hindlimb bud Fgf-8 outgrowth

Figure 35.4 A model for how the position of the fore- and hindlimbs is specified along the embryonic axis and for how outgrowth of the limb buds is initiated. The right-hand side of an embryos is shown as viewed from the dorsal surface (anterior to the top), with the embryonic axis shown as somites (SO), future limb and flank lateralplate mesoderm (LPM) and the overlying ectoderm (ECT). The axial Hox gene code expressed in LPM activates Tbx-5 or Tbx-4 to specify fore- and hind-limb-bud fields. Outgrowth is controlled via FgfR-2 and Fgf-8. Expression of Tbx-5 and Tbx-4 is maintained by Fgf-10 within the LPM. Additionally Tbx-4 is maintained by Pitx-1. Based on Ruvinsky et al. (2000).

The Limb Field and the AER

449

Box 35.2 (Continued ) Fore or aft? Specification of fore- vs. hind limbs and of pectoral vs. pelvic fins is controlled by members of the T-box gene family. Brachyury, perhaps the best-known and most well-studied family member, is expressed in axial mesoderm, lateral-plate mesoderm and limbbud mesenchyme adjacent to the AER, and may play a role in maintaining the AER (C. Liu et al., 2003). Tbx-5 is expressed in restricted zones along the right and left flanks of teleost and tetrapod embryos before fin or limb buds arise (Fig. 35.4). Tbx-5 is expressed in the wing and flank but not in the hind limb buds of chick embryos, while Tbx-4 is expressed in leg but not wing buds. Implanting Fgf-2 into chick embryo flanks (i.e. in the area between where the wings and the legs arise) up-regulates either Tbx-4 or Tbx-5 (depending on position along the flank) and induces a supernumerary wing or leg, again depending on position along the flank. Limbs that are part wing and part leg do not occur. The response is all or none and mediated by members of the Wnt and Fgf families. Tbx-5 controls wingness; Tbx-4 controls legness.b Pitx-1, which encodes a transcription factor in hind limb but not forelimb buds, is upstream of Tbx-4 and involved in specification of hind-limb identity. Misexpressing Pitx-1 in wing buds elicits more distal expression of Tbx-4, Hoxc-10 and Hoxc-11, resulting in the development of hind-limb characters in wing buds, including hindlimb muscle. Recent analysis demonstrates that Pitx-1 and Pitx-2 are both required for hind-limb development, both being expressed in the mesodermal hind-limb field, Pitx-1 also being expressed throughout hind-limb development.c One paradox is that synthesis of Tbx-5 and Tbx-4 is initiated at H.H. 13, but limb type is specified as early as H.H. 9 (see text, Chapter 35). Saito et al. (2002) speculate that the midline tissue medial to lateralplate mesoderm provides inhibitory signals that regulate Tbx expression, and thus plays an early role in specification of limb type. Signals from midline tissues can transform a potential leg site into wing, the wing field being specified earlier than the leg field. Knocking out Tbx-5 in mice or zebrafish prevents forelimbbud/pectoral fin-bud development but has no effect on hind limbbud/pelvic fin-bud development. The knockout phenotype in mice is essentially that seen in brachyury mutant embryos. The knockout phenotype in zebrafish is essentially that seen in spadetail mutant embryos, spadetail being a mutation of Tbx-5. Both mutations inhibit migration of the lateral-plate mesoderm, which normally migrates to the flank to form fin or limb buds. Recently it has been shown that Holt-Oram syndrome in humans, which is characterized by upper arm and heart defects, results from a haploinsufficiency of TBX-5. In chick and mouse, Tbx-5 is required both for specification of limb buds as fore rather than hind early in development and for limb growth later in development (Rallis et al., 2003). Tbx-6 (mice) and Tbx-16 (zebrafish) are expressed in restricted zones along the right and left flanks before limb or fin buds arise. Knocking out Tbx-6 in mice or Tbx-16 in zebrafish results in lack of hind limb-bud/pelvic fin-bud development but has no effect on forelimb-bud or pectoral fin-bud development.d

Early differences Differences between wing- and leg-bud mesenchyme have been identified as early as the condensation stage. One difference is

(AER, but see Box 35.3 for terminology). As development proceeds, this asymmetry becomes more pronounced as the ridge directs limb bud growth posteriorly. It has been known for 125 years that a portion of the ectodermal covering of the developing limb bud thickens

morphogenetic. Leg and wing mesenchyme from H.H. 24 embryos – which means that the leg mesenchyme is a little younger – both chondrify when placed in culture, but leg mesenchyme forms nodules of cartilage while wing mesenchyme forms sheets (Downie and Newman, 1994). The differences are apparent as early as condensation and relate in part to fibronectin; prechondrogenic mesenchyme from hind limb buds has higher levels of fibronectin mRNA and fibronectin than does mesenchyme from wing buds. Wing condensations are broad and flat with much diffusely organized fibronectin; leg condensations are compact and spherical and connected by fibronectin-rich fibres. Leg- and wing-bud mesenchyme also respond differentially to foetal bovine serum, Tgf-1 and retinoic acid. Wing-bud condensations are more sensitive to Tgf, which up-regulates fibronectin levels (see Chapter 20), and in turn increases condensation size. Treating cultured wing-bud mesenchyme with an antibody against the amino-terminal heparin-binding domain of fibronectin inhibits condensation formation in wing- but not in leg-bud mesenchyme. Comparative analyses of wing- and leg-bud condensations would be a profitable way to analyze how differences between fore- and hind-limb mesenchyme are established.e Misexpressing Hoxa-13 leads to reduction of the zeugopod and arrest of cartilage growth in chick limb buds. A homeotic transformation of the long bones of the zeugopod into distal carpals/tarsals then occurs. Yokouchi et al. (1995) concluded that Hoxa-13 plays an essential role in switching long bones to short bones by altering cell-adhesion properties. Newman (1996) related this fascinating study to his own extensive studies on the differential role of cell adhesion molecules such as N-CAM in wing- and leg-bud chondrogenesis. Tgf-2 also plays a role in sorting out prechondrogenic cells and in governing their differentiation. Exogenous Tgf-2 enhances chondrogenesis from mouse limb buds and promotes the production of Tgf-2 mRNA in a positive feedback loop. Beads soaked in Tgf-2 suppress chondrogenesis, suggesting lateral inhibition within aggregations. Tgf-2 is chemotactic for proximal and distal limb mesenchyme and promotes the expression of N-cadherin, which plays a central role in condensation.f a The same issue arises when we ask how proximal and distal skeletal elements are specified from apparently similar chondrocytes, proximal chondrocytes making a humerus, distal ones a digit. b See Isaac et al. (1998), Rodriguez-Esteban et al. (1999), Takeuchi et al. (2003) and Yang (2003). c See Logan and Tabin (1999) for Pitx-1, Niswander (1999) and Kawakami et al. (2003) for overviews, and Marcil et al. (2003) for Pitx-1 and Pitx-2 and hind-limb development. d See Ruvinsky et al. (1998). Tbx-16 from zebrafish is an orthologue of chicken Tbx-6. Zebrafish and mouse Tbx-6 are not orthologues but distantly related paralogues (Ruvinsky et al., 1998). e See Leonard et al. (1991) for Tgf as a stimulator of fibronectin gene expression and initiation of condensation, and Downie and Newman (1995) for differential responses of wing and leg mesenchyme. See Newman and Cooper (1990), Newman (1992), Newman and Tomasek (1996) for morphogenetic mechanisms operating at condensation, and Newman (1996) for the importance of cell interactions and adhesivity in condensation and for differences between fore- and hind limbs. f See Oberlender and Tuan (1994) and Miura and Shiota (2000) for these studies with TGF-2.

into a ridge. It was not until 1948, however, that experimental evidence indicated that this ectodermal ridge directs developmental events in the underlying mesoderm.7 In that year, the talented American experimental embryologist John Saunders surgically removed the

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ridge – which he named the apical ectodermal ridge (see Box 35.3) – from wing buds of embryonic chicks. Subsequent outgrowth of wing mesenchyme ceased. The partial wing that developed lacked some or all of such distal skeletal elements as radius, ulna and/or digits. Proximal limb elements – humerus, pectoral girdle – were normal in morphology and size; overall shortening of the limb resulted from absence of the distal skeletal elements, not from a global decrease in limb length. Limb-bud growth The initiation of growth within a limb bud is a complex series of processes that includes initiating cell division, altering rates of division over time, establishing gradients in cell division across or along limb buds, and, as limbs develop, altering such cell-surface-related properties as attachment and cell movement. Cell proliferation In Chapters 19 and 20, I discussed rates of proliferation of progenitor cells and the accumulation of precursor cells into condensations. Here, I concentrate on initiation of

Box 35.3 Mesoderm is not synonymous with mesenchyme, nor ectoderm with epithelium It is most unfortunate that Saunders named the AER an ectodermal and not an epithelial ridge. The stage in development when the AER appears is way beyond any stage when ectoderm is still present, ectoderm being the name of a germ layer, epithelium (epithelia) being a term for the type of cellular organization in which cells exist as a layer(s) of connected cells on a basement membrane. Similarly, limb mesenchyme is often, perhaps usually, referred to as limb mesoderm. However, the embryonic stages when limb buds arise are well beyond the germ-layer stage, and mesoderm is a germ layer. I will try to speak of limb-bud mesenchyme and limb-bud epithelium rather than mesoderm and ectoderm, and encourage you to begin to do the same. Mesoderm, ectoderm and endoderm are three germ layers; epithelia and mesenchyme are two types of cellular organization. Mesoderm
mesenchyme, nor does ectoderm
epithelium. Most embryologists of the 1940s probably thought that epithelia only arose from ectoderm or endoderm, and so the terms ectoderm and epithelium were often used interchangeably. However, epithelia can arise from mesoderm; mesodermal somites are initially epithelial. Indeed, all cells of metazoans are organized either as an epithelium or as mesenchyme, for these are the only two types of cellular organization. This is true even when the mesenchymal matrix may be liquid (as in blood) or when it is solid (as in bone) or whether the epithelium is a single layer or multiple layers of cells. I doubt that many will begin to refer to the apical ectoderm ridge as the apical epithelial ridge, but I do hope that we begin to subconsciously replace epithelial for ectodermal when we speak of the AER, which is an ectodermally derived epithelial ridge. Similarly, limb-bud mesoderm or limb mesoderm is mesenchyme derived from mesoderm.

cell proliferation as a factor in growth. The outgrowth of avian embryonic limb buds received the most attention and provides perhaps the best-understood system for the analysis of gradients of cell proliferation and their role in outgrowth of structures such as limb buds.8 Since the carbon-marking experiments by Saunders (1948), we have known that limb buds grow from their tips, i.e. growth is apical. Continued bud outgrowth depends on the presence of the AER, but removing the AER does not affect mitotic activity in flank mesoderm (Janners and Searls, 1971). Limb-bud growth consists of more than the production of more cells. Outgrowth involves the number of cells, their proliferation, position, movement, size, shape, packing density and constraint from the limb epithelium. In a brave attempt to discriminate between these factors and to determine the least number of components necessary to produce a limb bud, Ede and Law (1969) formulated a computer simulation of limb-bud outgrowth. A combination of cell proliferation in a proximo-distal gradient with distal movement of the cells produced form and growth patterns remarkably similar to those seen in normal limb buds. The computer simulation required a gradient of cell division within limb buds; Amprino (1965) had produced the first evidence of such a gradient in ovo. Mitotic activity is relatively constant in wing buds between H.H. 16 and early stage 22, when mitotic activity declines to a lower but again relatively constant rate. The greatest decline is in proximal chondrogenic mesenchyme, where the labeling index drops by 75 per cent between H.H. 19 and 24. Stark and Searls (1973) concluded that cell proliferation without cell migration was sufficient to account for limb-bud outgrowth.9 Suppressing the flank Since limb buds protrude from the flank, we assume that the growth mechanisms lie completely within limb-bud cells, i.e. limb buds grow out and, indeed, limb-bud cells do divide at a higher rate than flank cells (see below). However, initial limb-bud outgrowth results from suppressing mitotic activity in the flank between H.H. 16 – when the limb bud appears – and H.H. 20. During this period the mitotic rate in flank mesenchyme declines by 25 per cent while the mitotic rate of limb-bud mesenchyme remains constant (Searls and Janners, 1971). What appears to be outgrowth of limb mesenchyme is really regression of flank mesenchyme. So, initial ‘outgrowth’ of the limb bud is more apparent than real; the flank recedes away from the limb buds because mitotic activity in flank mesenchyme declines. This also is true in other regions, such as the facial processes from which the jaws and face arise. Differential rates of decline in cell division and/or the appearance of more slowly cycling subpopulations are responsible for the growth and morphogenesis of the frontonasal and maxillary processes in embryonic chicks. The pattern is

The Limb Field and the AER one of proliferation declining proximally within each facial process but remaining high distally or at boundaries. Mitotic rate in limb mesenchyme Hornbruch and Wolpert (1970) counted mitotic activity in wing buds from embryos between H.H. 18 and 30. Throughout all stages, mitotic activity was constant within the epithelium at around two per cent. Mitotic activity in the limb mesenchyme declined with developmental stage – from 12 per cent at H.H. 18 to 2 per cent at H.H. 30. No proximo-distal gradient was observed until H.H. 24, by which stage the commitment of mesenchymal cells as chondrogenic had been stabilized, and at which stage the mitotic index in distal mesenchyme exceeds that in proximal mesenchyme. Although they found no statistically significant gradient before H.H. 24, there was a difference in mitotic activity at H.H. 20 of 6.5 per cent distally to 9.5 per cent proximally. This became apparent when Lewis (1975) reworked their data, which I present in another form in Figure 32 in Hall (1978a), using as a common base the length of the limb bud, with the distal tip as zero and the proximal base of the limb bud at that H.H. stage as 10; the per cent mitotic index is then plotted as a per cent of the rate at the distal tip at that H.H. (rate at distal tip as 100 per cent). These plots show no gradient at H.H. 18, a slight decline in distal-proximal rate during H.H. 19/20, with a rise at H.H. 21/22 midway along the limb bud, steepening considerably between H.H. 21 and 23, the latter stage corresponding to condensation of proximal skeletal elements. A sharp decline in mitotic activity at H.H. 24 and establishment of a distal-proximal gradient at stage 25 complete the pattern. A proximo-distal decline in agglutination of limb mesenchymal cells to the plant lectin concanavalin between H.H. 19 and 26 parallels establishment of the mitotic gradient, stabilization of cells for chondrogenesis and a decline in morphogenetic potential. The possibility that cell-surface changes and cell-to-cell adhesion play a role in establishing the mitotic gradient should be considered.10 More detailed information shows a gradient in mitotic rate within the chondrogenic mesenchyme during H.H. 24–27: highest distally, a sharp drop in the distal one-third of the limb bud, and a slower decline proximally. A much more gradual gradient is found in myogenic mesenchyme, suggesting that mitotic activity may be differentially controlled in chondro- and myogenic mesenchymal cells. Blocking mitosis at 6.5 days does block mitosis in myogenic but not chondrogenic mesenchyme. Similar studies on earlier embryos might: provide valuable information on the establishment of regional differences in mitotic activity across limb buds; show that the differences reflect central (chondrogenic) mesenchyme as more homogeneous than peripheral (myogenic, fibroblastic) mesenchyme; or else show that myogenic and chondrogenic

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cells are at different stages of cell proliferation.11 The AER does not maintain the high mitotic activity in distal mesenchyme, although influences from the epithelium as a resistant layer have not been investigated adequately. Since time spent within the distal tip of the limb bud (the progress zone) determines positional morphology (Chapter 38), any change in mitotic rate should have pronounced effects on the length of particular segments of the skeleton. Between four and 10 days of incubation, each mesenchymal cell divides three times. Given the number of starting cells, rate of proliferation, and rate of accretion of extracellular matrix (ECM), growth rates and final size should be predictable with considerable accuracy, especially as specification of the length of the humerus, ulna and digits between H.H. 22 and 36 (4–10 days of incubation) is accurate to  4–5 per cent. Such are the bounds within which wild-type development must remain.12 Proximo-distal patterning of the limb skeleton Saunders noted that the older the embryo from which the AER was removed the less marked were the deficiencies in wing development. In particular, Saunders observed that increasingly distal cartilages were present when the ridge was removed from progressively older embryos, which he interpreted as the AER controlling skeletal development by specifying a proximo-distal sequence of limb elements and limb-bud outgrowth. Early in development, mesenchyme adjacent to the AER would be specified for proximal skeletal elements such as the humerus. Progressively later in development, mesenchyme near the ridge (distal mesenchyme) would be specified for increasingly distal elements. Limb cartilages are patterned according to a proximodistal sequence. Rowe and Fallon (1982) demonstrated proximo-distal specification of hind-limb skeletal elements, the most proximal element – the humerus – being specified earliest and the most distal elements – the phalanges of the digits – being specified last. Furthermore, and surprisingly, it takes much longer to specify a short complex region with multiple skeletal elements than to specify a longer region with a single element. Specification of the humerus takes some 12 hours; elements of wrist or ankle – the carpal and tarsal regions – take 24 hours. The ridge is active from H.H. 17 to 19 (see Table 35.1). It follows that the form of the mature limb skeleton can be mapped out in the limb bud as a fate map. Indeed, as far back as the early 1920s Murray and Huxley had used CAMgrafting to test whether the limb bud is a mosaic, i.e. whether all the parts are prefigured in the earliest limb buds (Box 12.3). Is the maintenance of an AER an intrinsic property of limb-bud epithelium or does it depend on influences from the adjacent flank epithelium or from limb mesenchyme?

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MESENCHYMAL FACTORS MAINTAIN THE AER As will be discussed in the context of limbless mutants (Chapter 37), much of the evidence for a mesenchymal factor that maintains the AER – an apical epidermal maintenance factor (AEMF ) – comes from studies on wingless and polydactylous mutants in domestic fowl. A succinct summary of wingless is that the AER in wingless embryos regresses because AEMF is lacking; wingless mesenchyme grafted to wild-type limb epithelium is followed by regression of the AER and a wingless phenotype.13

30 µm

AEMF AEMF is distributed within the postaxial region of each limb bud close to an area with high levels of cell death known as the posterior necrotic zone (PNZ; see below). The initial evidence for this conclusion was experimental, showing that you don’t have to isolate a factor to know that one must exist. Placing a filter across the long (proximo-distal) axis of a limb bud divides it into pre- and post-axial regions and is followed by regression of the AER (Saunders and Gasseling, 1963). If AEMF is only found post-axially, then 180 rotation of the distal half of the limb bud would place AEMF into the anterior (preaxial) face of the limb bud, where it should maintain a second AER, resulting in limb duplication. Saunders and Gasseling (1968) showed that this is just what happens. Polarity of the AER is maintained by a gradient of AEMF. MacCabe and Parker (1975) found that the AER flattens – is not maintained as a specialized ridge – if preaxial (anterior) wing mesenchyme is cultured alone, with flank mesenchyme or with anterior limb mesenchyme. However, the AER is maintained with no indication of cell death when anterior and posterior (preaxial and postaxial) mesenchymes are co-cultured. When cultured with mesenchyme from the mid-region of the limb bud – i.e. mesenchyme in a lower part of the proposed gradient of AEMF – some loss of AER and some cell death ensues, interpreted as indicating lower levels of AEMF in that mesenchyme. Consistent with the presence of a diffusible molecular factor, a cell-free extract of post-axial – but not preaxial – limb buds has AEMF activity, shown to result from two diffusible components of high (300 000-kDa) molecular weight. Conversely, mitotic inhibitors administered in ovo diminish the rate of accumulation of mesenchymal cells, a diminution that in turn leads to premature loss of the AER and loss of skeletal elements.14 Mesenchymal cells derived from limb buds produce AEMF when maintained in monolayer culture. If covered with a sheet of limb epithelium containing an AER, the monolayered mesenchymal cells grow out and pile up to produce a limb-like bud with a normal AER that is maintained morphologically and functionally (Fig. 35.5).

60 µm

30 µm Figure 35.5 Embryonic chick-limb development mimicked in vitro. (A) A monolayered culture of mesenchyme cells from limb buds of embryos of H.H. 19/20 differentiates cartilage nodules (C) after 48 hours’ culture. F, filter substrate. (B, C) Substantial mesenchyme accumulates when monolayered limb mesenchyme (LM) is cultured in contact with an AER. Modified from Globus and Vethamany-Globus (1976).

Limb-bud epithelium lacking the AER (i.e. non-AER epithelium) does not support outgrowth under the same culture conditions (Globus and Vethamany-Globus, 1976). Whether non-limb mesenchymal cells would have maintained the AER was not tested, but would not be expected. The chick mutant diplopodia4 is polydactylous with extra digits preaxially (Figs 35.6 and 35.7). Additional AEMF is produced in anterior or preaxial limb mesenchyme in diplopodia4 mutants, resulting in preaxial thickening of the ectoderm and preaxial polydactyly. The same argument is used for the mechanism underlying the polydactylous mutants talpid 2 (Box 20.1), and for similar mutants in murine embryos (Fig. 35.8).15 The PNZ The posterior necrotic zone (PNZ ) was described in wing buds in 1968.

The Limb Field and the AER

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Figure 35.6 Hind-limb development in diplopodia4, a polydactylous mutant of domestic fowl. (A) A diplopodia4 embryo at 11 days of incubation to show the extra digits. (B, C) The hind limbs of diplopodia4 (B) and wild-type (C) embryos at seven days of incubation. Note the broad, spatulate foot-plate in the mutant. (D, E) The hind limbs of diplopodia4 (B) and wild-type (C) embryo at 11 days of incubation. Note the extra and bifurcated digits in the mutant. The skeletons of these two hind limbs are shown in C and D in Fig. 35.7. (F) A polydactylous hind limb developed when limb-bud mesenchyme from a diplopodia4 embryo of H.H. 20 was combined with limb-bud epithelium from an H.H. 21 wild-type embryo. (G) A normal hind limb developed when limb-bud mesenchyme from a wild-type embryo of H.H. 20 was combined with limb-bud epithelium from an H.H. 21 diplopodia4 mutant embryo. The results shown in F and G demonstrate that the limb-bud epithelium not the mesenchyme is defective in diplopodia4 embryos. Modified from MacCabe et al. (1975).

If the PNZ – or the equivalent region from hind-limb buds – is grafted below the AER, the AER regresses and disappears as expected. However, surprising at the time, perhaps even now, a new AER forms immediately preaxial to the graft and a limb grows out from this preaxial location. Paradoxically, although the PNZ does not produce AEMF, it seems capable of inducing AEMF from mesenchymal cells immediately preaxial to it. Perhaps this explains the normal post-axial location of the AEMF and asymmetry of the AER, whose position is partially governed by the location of the PNZ. Since the AER is normally thicker at the posterior margin, this zone may play a role during normal limb development, a suggestion consistent with the observation that grafting a portion of the PNZ beneath the epithelium results in preaxial thickening of the AER.16 All well and good, but except for changes in cell death, surprisingly, removing the PNZ does not interfere with normal limb development. Therefore, if the PNZ does influence the production of AEMF in ovo, its action must be rapid, early in development and/or based on a longlasting message(s) passed to adjacent mesenchyme.

In summary, limb mesoderm: ● ● ●

● ●

specifies limb type as wing or leg; responds to the AER by proliferation and outgrowth; maintains or supports formation or regeneration of an AER; transmits AEMF largely in a proximo-distal direction; forms the skeleton in a proximo-distal sequence (see below).

SPECIFICITY OF LIMB-BUD EPITHELIUM Limb mesenchyme can maintain a second ridge in limb epithelium (see above and Zwilling, 1956a). Can limb mesenchyme elicit a ridge from non-limb epithelium? Zwilling (1964) tested this possibility by grafting flank epithelium to a limb bud from which the epithelium had been removed after the normal stage of initiation of the AER. The limb failed to develop further. Therefore, either the ability to initiate an AER is time specific or flank

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Figure 35.7 Limb development in diplopodia4, a polydactylous mutant of domestic fowl. (A, B) The skeleton of the wings of diplopodia4 (A) and wild-type (B) chick embryos at 11 days of incubation to show the polydactyly in the mutant. (B, C) The skeleton of the hind limbs of a diplopodia4 (C) and a wild-type (D) chick embryo at 11 days of incubation to show the polydactyly in the mutant. C and D are the same hind limbs shown in D and E in Fig. 35.6. Modified from MacCabe et al. (1975).

ectoderm is non-responsive. Could younger mesoderm induce flank ectoderm to produce a ridge? Zwilling (1964) dissociated limb mesenchyme into single cells, reaggregated them into a pellet, wrapped them in limb or flank epithelium and grafted the package to the CAM. The mesenchyme only formed cartilage when associated with limb epithelium. Therefore, limb epithelium is both site and time specific. Other experiments involving the response of flank ectoderm to limb mesoderm were discussed under ‘Ectodermal responsiveness’ above, including evidence for the time of onset of this specificity. Once flank ectoderm of a future limb field has been in contact with presumptive limb mesoderm for a time it ‘becomes’ limb ectoderm; ectoderm not having this contact loses the ability to respond to limb mesoderm and can no longer become limb ectoderm. Timing of positioning of limb buds along the axis is thereby specified (see Table 35.1). There is, however, an earlier action of limb-bud epithelium that occurs immediately before and as the AER is forming. This is a requirement on an epithelial signal for limb-bud mesenchyme to chondrify. This is not the patterning interaction that occurs later in development, but rather a differentiation signal akin to those discussed

Figure 35.8 Polydactylous hind-limb buds of mutant mouse embryos. (A) Normal hind-limb buds on either side of the tail of a wild-type (
/
) embryo of 15 days of gestation. (B) Additional hind-limb buds in an embryo of 15 days of gestation that is heterozygous (Ds/
) for the mutation disorganization (Ds). (C) Preaxial polydactyly (right) in a hind-limb bud from a 15-day-old Ds/
embryo. (D) A hind-limb bud from an 18-day-old Ds/
embryo with more than double the normal number of digits. Modified from Ede (1980).

The Limb Field and the AER for the induction of ectopic bone (Chapter 12), jaws (Chapter 18) and skull bones and cartilages (Chapter 21). Gumpel-Pinot (1980) established a series of transfilter tissue interactions involving limb epithelium and mesenchyme to investigate whether initial chondrogenesis from limb mesenchyme required an epithelial signal. She investigated the time immediately before and after initial appearance of the AER, i.e. H.H. 14–18. As you can see from the results in Table 35.4 an epithelial interaction is required until H.H. 18 if mesenchyme is to chondrify. Using SEM analysis of the tissue recombinations, Gumpel-Pinot demonstrated mesenchymal cell processes penetrating the pore of the Nuclepore filters used to separate epithelium from mesenchyme. The percentage of cultures forming cartilage increased with the porosity of the filters over the range 0.2–0.8 m porosity. Subsequently, Gumpel-Pinot (1981, 1982) showed that proximity but not direct contact of epithelium and mesenchyme was required for the interaction to occur. Mesenchymal cell processes crossed the Nuclepore filter within 15 minutes and deposited an ECM onto the filter (Table 35.5). She noted that the epithelial–mesenchymal interaction preceded condensation of limb mesenchyme; we now know that such interactions are required for condensation (Fig. 18.3, and see Chapter 18). Around the same time it was shown that avian limb-bud epithelium establishes a peripheral non-chondrogenic area of avascular mesenchyme, characterized by flattened fibroblastic cells through a diffusible factor that

Table 35.4 Incidence (% of cultures) of chondrogenesis from limb mesenchyme maintained in the presence or absence of limb-bud epitheliuma H.H. stage of limb tissues

Mesenchyme cultured transfilter to epithelium

Mesenchyme cultured alone

14 15–16 17 18

60 58 81 100

0 6 57 100

a

Based on data in Gumpel-Pinot (1980).

crosses 25-m but not 150-m Millipore filters. Limb-bud epithelium inhibits chondrogenesis from limb mesenchyme in collagen gel cultures via a diffusible factor that can travel up to 200 m through the gel or that acts in gels preconditioned with ectoderm. Consistent with Gumpel-Pinot’s demonstration of the requirement of pre-limb-bud mesenchyme for epithelial signals to become chondrogenic, limb-bud territory epithelium from H.H. 15 stimulates chondrogenesis, while limb-bud epithelium of H.H. 23/24 inhibits chondrogenesis.17

SPECIFICITY OF DISTAL LIMB MESENCHYME The mesenchyme that responds to an AER in ovo is the most distal mesenchyme immediately subjacent to the AER. Can more proximal limb-bud mesenchyme or nonlimb-bud mesenchyme respond to the AER? In the mid- to late-1950s, Saunders and his colleagues transplanted proximal prospective hind-limb (thigh) mesenchyme adjacent to the AER of chick wing buds at various times between H.H. 18 and 27. Their question was whether transplanted proximal mesenchyme would produce proximal or distal limb structures – or no structures at all – and whether those structures would be typical of the hind limb (the source of the mesenchyme) or of the wing (the source of the AER and the donor site). Proximal mesenchyme did respond, provided that it was taken from embryos younger than H.H. 24 and provided that it was in contact with the AER. The transplanted ‘proximal’ mesenchyme forms distal skeletal elements, but those are distal hind-limb structures, not wing (Table 35.6). Proximal mesenchyme responds by producing skeletal elements appropriate to its new position relative to the AER, but retains its limb-type specificity by producing toes rather than wing digits. Leaving a barrier of distal mesenchyme between the AER and the transplant prevents the transplanted mesenchyme from responding. The influence of the AER is local.18 Amprino and Bonetti (1964) also grafted proximal mesenchyme beneath the most distal limb mesenchyme in embryos of H.H. 25 and 26. Even though they left

Table 35.5 Activity of mesenchymal cells from chick wing buds as they travel across a Nuclepore filter with pore sizes of 0.6–0.8 ma

Table 35.6 Results of typical experiments grafting proximal hind limb-bud mesenchyme subjacent to the AER of the wing buda

Elapsed timeb

Mesenchymal cell activity

15 min

Appearance of cell processes on the other surface of the filter, some projecting into the filter More cell processes and filopodia (some as small as 0.1–0.2 m in diameter) cross the filter Filter surface covered with processes; maximum coverage between 2 and 4 hours

Donor age (H.H. stage)

Per cent (N) of grafts producing distal foot structures

17–19 20–22 23–25b 26–27b

86 (74/86) 62 (24/39) 29 (15/51) 0 (0/33)

30 min 1–4 hours

a a

Based on data from Gumpel-Pinot (1981). b Time after epithelium and mesenchyme established transfilter.

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Based on data in Saunders et al. (1959). The ability of proximal limb mesoderm to produce distal skeletal structures is lost between H.H. stages 25 and 26. b

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15–20 rows of mesenchymal cells at the tip, they still obtained distal structures from the transplant, and so concluded that the proximal mesenchyme did not have to be in contact with the AER. At such a late developmental stage, however, the graft may have received the epithelial message secondarily from already-determined distal mesenchyme with which it had come into contact before being transplanted. Indeed, the experiment does not necessarily rule out epithelial involvement at earlier stages.

THE TEMPORAL COMPONENT Rubin and Saunders (1972) examined temporal aspects of interaction within early limb buds by combining mesenchyme from younger (H.H. 18–20) with epithelium from older (H.H. 23–25) embryos, and mesenchyme from older with epithelium from younger embryos. Regardless of age, epithelium elicited complete limbs from limb mesenchyme when tissue recombinants were grown as flank grafts. Epithelium from the older embryos was just as capable of eliciting limb development as epithelium from younger embryos. Rubin and Saunders proposed that signals from the epithelium – presumably from the AER – are constant over time, and that any proximo-distal sequence within the limb bud must be a property of limb mesenchyme or earlier limb-field mesoderm. A decade later it was confirmed that flank epithelium of embryos of H.H. 15 and 20 could respond to ridge induction. The AER finally loses its ability to induce limb outgrowth during H.H. 29 (Table 35.7). Interestingly, the loss of inductive ability is neither accelerated by combining limb epithelium with mesenchyme from older embryos nor slowed down by combining epithelium with mesenchyme from younger embryos.19

A MECHANICAL ROLE FOR THE EPITHELIUM? Amprino and his colleagues produced evidence that they thought contradicted what, by the mid-1960s, was known Table 35.7 Results of experiments to test the time dependency of the ability of the AER to induce limb-bud mesenchyme to grow and produce the skeleton of the limba,b Age of embryo providing Limb-bud ectoderm (H.H. stage)

Per cent (N) of grafts forming wings

26 to early 29 29 30

78 (36/46) 18 (4/22) 0 (0/9)

a Ectoderm from the limb buds of embryos of various ages was recombined with limb-bud mesoderm from embryos of H.H. 18–20 and maintained as a flank graft. Based on data in Rubin and Saunders (1972). b This ability to induce limb-bud mesoderm is lost between H.H. 29 and 30.

as the Saunders-Zwilling model of epithelial–mesenchymal interaction. Their view did not gain wide support, although one of their studies supporting a biomechanical role for limb epithelium did gain some acceptance. Using wing and hind-limb buds from embryos between H.H. 18 and 25 (3–5 days of incubation), Amprino and Ambrosi (1973) placed chips of dyed agar immediately beneath the AER or at various depths within the distal mesenchyme. By monitoring subsequent limb outgrowth in ovo they observed a proximo-distal sliding of the entire epithelial cover, moving the AER progressively more and more distally. ‘Sliding’ ceased if the epithelium was pinned dorso-ventrally, growth of the limb bud stopped, and distal deficiencies arose. Such results had been seen before, but after removing or destroying the AER. The AER was not injured in the study; rather, movement of the entire limb epithelium was stopped. Amprino and Ambrosi proposed that the entire limb epithelium – a coherent sheet of epithelium, which we could regard as a compartment (Box 35.4) – plays a role in modeling mesenchymal outgrowth. They did not say – indeed, could not resolve from their experimental design – whether epithelial movement governs mesenchymal growth or mesenchymal growth governs epithelial movement.20 The notion that epithelial sliding largely maintains the AER is in line with the low mitotic activity in the AER. Box 35.4 Compartments Compartments are important features of the development of some invertebrate embryos (Drosophila being the most studied), and have been invoked in stochastic models of cell differentiation (MacLean and Hall, 1987). The proposal by Amprino and Ambrosi (1973) that the epithelium of the limb functions as a coherent sheet in directing mesenchymal cell movement essentially treats the epithelium as a compartment. Altabef et al. (1997) identified dorsal and ventral ectodermal compartments associated with developing chick limb buds, claiming them as the first non-neural compartments in vertebrates (but see text for ectomeres). No equivalent mesodermal compartments were found, indicating primacy of limb ectoderm. The future AER was fate-mapped as scattered over the dorsal and ventral ectoderm. On the basis of labeling with DiI or Lucifer yellow and expression patterns of genes such as Shh, Wnt and Bmp, the sclerotome has also been postulated as consisting of compartments.a Nowicki and Burke (2000) transplanted segmental plate in chick embryos and identified two compartments: (i) a dorsal compartment of somitic mesoderm that retains its Hox gene code when transplanted along the axis, and (ii) a ventral compartment of somitic and lateral plate mesoderm that adapts to the Hox code appropriate to the level to which it is transplanted. Here compartmentalization is associated with (based on?) independent paraxial and lateral-plate mesoderm Hox codes. a See Bagnell (1992), Bagnell et al. (1992) and Brand-Saberi et al. (1996) for the DiI and Lucifer yellow labeling, Christ et al. (1998) for the gene expression patterns, and Brand-Saberi and Christ (2000) for a review of the development and evolution of somitic cell lineages/compartments.

The Limb Field and the AER Mitosis is slight at H.H. 17 when the ridge arises and ceases by H.H. 21. Epithelial cells adjacent to the ridge do divide and could provide a mechanism to cause, secondarily, piling-up of cells into a ridge.21 The experimental analysis by Amprino and Ambrosi shows that mesenchymal outgrowth is greater distally than proximally, and that during growth, superficial blood vessels maintain a constant distance from the epithelium as a consequence of ongoing vascular reconstruction. They considered the possibility that pressure exerted by the vascular tissue might be important for mesenchymal growth. As Amprino (1974) summarized the situation: the pressure of the vascularized mesoderm is greater along the proximo-distal axis than along other axes, exerting a stress on the ectoderm that is balanced by division and sliding of ectoderm, i.e. control is initially mesodermal. Later in limb-bud development, regression (or exclusion) of blood vessels from the core mesenchyme precedes chondrogenesis (Hallmann et al., 1987). The proposal that mesenchymal growth might be greater distally raises the question of the mechanism of that growth. Amprino found no evidence for cell migration – which would bring additional cells into the limb bud – or cellular hypertrophy, the enlargement of cells seen as chondrocytes mature. Unless pressure from the superficial blood vessels is sufficient in itself – as Amprino thought it might be – he was left with the possibility of a proximo-distal gradient in cell division within the mesenchyme. After reviewing the available data, Amprino and colleagues emphasized the lack of evidence for any significant gradient but concluded that some slight gradient must be operative. Interestingly, and as discussed above, a proliferation gradient must be incorporated into computer models if they are to simulate limb outgrowth with any accuracy.

NOTES 1. Wallace (1981), p. 224. 2. Trollope (1999), p. 157. 3. See Dworkin et al. (2001) and Atallah et al. (2004) for the studies on Drosophila morphogenetic fields, and see Hall and Wake (1999) and Hall (1998a, 2004a) for how larval density can affect developmental processes in individual larvae. 4. See Kieny (1960), Reuss and Saunders (1965) and Saunders and Reuss (1974) for the experiments inducing ectopic AERs and limbs. See Carrington and Fallon (1984a,b, 1986, 1988) and Wilson and Hinchliffe (1985) for the timing of the responsiveness of flank ectoderm and for recombinations between wingless and wild-type embryos. See Abbott (1975) and Hall (1983d, 2000d) for the utility of the tissuerecombination approach for studies within and between wild type or mutant individuals of the same species, and between species or classes (mouse–chick, reptile–chick). 5. See Dhouailly and Kieny (1972) and Searls and Zwilling (1964) for these two studies. Normally, tail development is

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initiated by the tail equivalent of an AER, a ventral ectodermal (epithelial) ridge (VER); see Chapter 43. See Cairns and Saunders (1954), Zwilling (1955) and Saunders et al. (1957, 1958) for specification of limb type. See Fallon (2002) for an interview with John Saunders, including how he, Saunders, came to investigate the AER. Kelley (1973) has traced identification of the ridge back to studies by Kölliker in 1879 and by Balfour in 1885. Kelley (1975) and Kieny and Fallon (1976) published an informative paper on the ultrastructure of the AER of human limb buds. Some general reviews in which cell proliferation is considered are Faber (1971), Zwilling (1972), Searls (1973a,b) and Hinchliffe and Johnson (1980, 1983). See Janners and Searls (1970) and Searls and Janners (1971) for mitotic rate between H.H. 16 and 22. See Lewis (1975) for the data upon which this discussion is based, including data for the level of mitotic activity at each stage, and see Paulsen and Finch (1977) for the lectin data. See Ede et al. (1977b) for the gradient, and Kieny (1975) for blocking mitosis. This gradient is reversed in talpid3. See Amprino (1965), Hornbruch and Wolpert (1970), Amprino and Ambrosi (1973) and Summerbell (1974b, 1977a) for possible physical constraints of the ectoderm. See Summerbell and Wolpert (1973) for the precision of skeletal growth. See Zwilling (1956b, 1974) and Zwilling and Hansborough (1956) for the original experiments, and Chapter 36 for a discussion of them. See Calandra and MacCabe (1978) and MacCabe et al. (1977) for the cell-free extract and molecular weight analyses, and Kieny (1975) and W. J. Scott et al. (1977) for the mitotic inhibitors. See MacCabe and Abbott (1974) and MacCabe et al. (1975) for the mouse mutants. Gasseling and Saunders (1964) first described the PNZ in wing buds – see the summary by Saunders and Gasseling (1968) – and did the transplant studies, which were confirmed by Summerbell (1974a). See Solursh (1984) for the non-chondrogenic zone, and Solursh et al. (1981, 1984) and Solursh and Reiter (1988) for epithelial inhibition/stimulation of chondrogenesis. See Saunders et al. (1955, 1957, 1959) for transplantation of proximal mesenchyme to distal sites in the limb bud, Amprino (1964, 1968) for transplantation to the more proximal sites, and Amprino (1984) for an evaluation of these studies. See Carrington and Fallon (1984b, 1986, 1988) for the later studies, and see Fallon (2002) for an interview with John Saunders. See Amprino (1965, 1975a, 1977b, 1978) for his data and views against the generally accepted model of limb outgrowth. Coffin-Collins and Hall (1989) and Hall and Coffin-Collins (1990) demonstrated regulation of mitotic activity of craniofacial prechondrogenic epithelia and mesenchyme by Egf. See Amprino (1974) for the mitotic data, and Errick and Saunders (1976) and Saunders et al. (1976) for a contrary view. Mitotic rates are similarly low in mandibular-arch epithelia (Hall and Coffin-Collins, 1990).