Epithelial–Mesenchymal Interactions Initiate Skeletogenesis

Chapter 18

Epithelial Mesenchymal Interactions Initiate Skeletogenesis Extrapolating from a single trout

ideas are easy but data are hard.

Whether skeletal mesenchyme is neural crest or mesodermally derived, and whether skeleton is limb, vertebral, craniofacial or visceral, differentiation begins after interactions with embryonic epitheliaA. Such interactions, introduced at the end of the previous chapter (see Figure 17.11) and known as epithelial mesenchymal interactions (Figure 13.8 and Figure 18.1), may take place: G

G

G

before onset of mesenchymal cell migration such as occurs between neural crest cells (NCCs) and cranial epithelium adjacent to the neural tube (below), and between limb-bud mesenchyme and flank epithelium, as discussed in Chapter 35; during migration as occurs between pharyngeal endoderm and the mesenchyme destined to form the visceral arch skeleton in amphibians (Figure 18.2, and see below); or after migration, when skeletogenic mesenchyme is at its final location. Examples are interaction between mandibular or maxillary arch epithelium and mesenchyme that will form mandibular and maxillary arch bones in birds (below), and interactions between notochord and/or ventral spinal cord and sclerotomal mesenchyme to form vertebral cartilage in tetrapods, discussed in Chapter 41.

These epithelial mesenchymal interactions serve at least four functions (Figure 18.3), as follows: 1. they localise skeletogenic mesenchyme within embryos;

A. Remember that mesoderm and ectoderms are germ layers, and mesenchyme and epithelia are types of cellular organisation, either as meshworks of unconnected cells (mesenchyme) in an extracellular matrix or as a layer(s) of connected cells sitting on an extracellular basement membrane. Note that I do use a shortcut when I use the term ‘pharyngeal endoderm’ for what strictly should be referred to as pharyngeal endodermally derived epithelium. Bones and Cartilage. DOI: http://dx.doi.org/10.1016/B978-0-12-416678-3.00018-5 © 2015 Elsevier Inc. All rights reserved.

2. they provide the signals for condensation of skeletogenic mesenchyme, which allows 3. differentiation of cartilage, bone (or dentine or enamel); and 4. sets the fundamental number of progenitor cells for the skeletal element. Epithelial mesenchymal interactions serve these four functions in the development of almost all the cells and tissues of vertebrate embryos: heart, kidneys, glands, liver, lungs, alimentary canal, skin and skin derivatives such as hair, feathers and scales (Box 2.4). A list of some interactions associated with skeletogenesis in various skeletal elements and taxa is provided in Table 18.1. I also introduce the finding that such interactions are reciprocal: signalling from mesenchyme or ectomesenchyme maintains epithelial cell proliferation and so maintains a positive feedback loop for continued production of skeletogenic signals from the epithelium. Mesenchymal cells lose their dependence on epithelial signals and epithelial cells become insensitive to mesenchymal signalling at the same time during development1. In this chapter, I document the existence and importance of these interactions by outlining their role in initiating chondrogenesis and osteogenesis of the neural crest derived mesenchyme that forms the mandibular skeleton in tetrapods and fish. To summarise a considerable body of evidence, all craniofacial membrane bones and cartilages examined in chick embryos require an epithelial interaction before they can form. All these interactions are listed in Table 18.2. Those interactions required for chondro- or osteogenesis in the lower and upper jaws (mandibles and maxillae) of chick and mouse embryos are discussed in some depth in this chapter. I also address whether the lateral line or neuromasts may play a similar role in fish larvae or in urodeles, and how studies in which different germ layers are combined 299

300 PART | VI Embryonic Origins

(B)

Epithelium Basement membrane

(A)

Mesenchyme (C)

(D) Responding cell

FIGURE 18.3 Four phases of cartilage development. (A) Origination of chondrogenic mesenchyme. (B) Epithelial mesenchymal interaction. (C) Condensation. (D) Differentiation. Condensation and differentiation are both multistep processes.

FIGURE 18.1 Three ways in which epithelium signals to mesenchyme shown diagrammatically as three epithelial cells on their basement membrane and adjacent mesenchymal cells. Interaction may be (i) by epithelial release of a diffusible molecule (top); (ii) by interaction of mesenchymal cells with a product deposited by the epithelium into the basement membrane (middle), a mode requiring close interaction between mesenchymal cells and the basement membrane; or (iii) by direct cell-to-cell communication between epithelial and mesenchymal cells, a mode requiring close interaction between the two cell types, local dissolution of basement membrane and junctional connections between the two cell types.

FIGURE 18.2 Teeth and cartilage differentiate when cranial neural crest from a Mexican axolotl neurula is combined with pharyngeal endoderm and maintained in organ culture. See Figures 17.6 and 18.4 for additional information.

TABLE 18.1 Osteogenic Epithelial Mesenchymal Interactions in Embryonic Development and Regenerationa Skeletal Element/ System

Inductive Epithelium

Osteogenic Mesenchymal/ Ectomesenchymal Cellsb

Scleral ossicles in chick eye

Scleral epithelium

Scleral ectomesenchyme

Ear capsular bone

Otic placode

Otic capsule ectomesenchyme

Mandible

Mandibular epithelium

Mandibular ectomesenchyme

Maxilla

Maxillary epithelium

Maxillary ectomesenchyme

Visceral arches

Pharyngeal endoderm

Visceral arch ectomesenchyme

Limb bones

AER (limb bud)

Limb-bud mesenchyme

Tooth

Oral epithelium

Odontogenic/osteogenic ectomesenchyme of jaws

Regenerating urodele limbs

Wound epithelium

Blastema derived by dedifferentiation of limb cells

Regenerating antlers in deer

Antler bud epithelium

Frontal bone pedicle mesenchyme

a

Based on H. C. Anderson (1990). I use the terms mesenchyme and ectomesenchyme here to distinguish between mesodermally and neural crest derived mesenchyme, respectively. b

Chapter | 18 Epithelial Mesenchymal Interactions Initiate Skeletogenesis 301

TABLE 18.2 Tissue Interactions that Evoke Chondrogenesis or Osteogenesis from Craniofacial Mesenchyme and Ectomesenchyme in Embryonic Chicksa Interacting Epithelium/ Epithelia(s)

H.H. Stages (hrs/days of Incubation) When the Interaction Takes Place

Meckel’s cartilage (E)

Cranial epithelium

9 10 (30 35 h)

Frontal (M)c

Prosencephalon, mesencephalon

13 15 (48 53 h)

Occipital (M)

Rhombencephalon, neural tube

13 15 (48 53 h)

Parietal (M)

Mesen- and rhombencephalon

13 15 (48 53 h)

Squamosal (M)

Mesencephalon

13 17 (48 60 h)

Basisphenoid (M)

Rhombencephalon, notochord

13 17 (48 60 h)

Parasphenoid (M)

Notochord

13 17 (38 60 h)

Scleral cartilage (E)

Pigmented retinal epithelium

14 18 (50 66 h)

Maxillary membrane bone (E)

Mandibular epithelium

18 22 (60 h 3.5 days)

Mandibular membrane bones (E)

Mandibular epithelium

18 24 (60 h 4 days)

Otic capsule cartilage (E)

Otic vesicle

18 27 (60 h 5.25 days)

Palatal bones (E)

Palatal epithelium

? 25 (? 4.5 days)

Frontal (E)c

Cranial epithelium

22 30 (3.5 7 days)

Scleral ossicles (M)d

Scleral papillae

30 36 (7 10 days)

Skeletal Elementb

a Note the clustering of interactions at H.H. stages 13 15 and13 17 between brain/notochord and mesodermally derived mesenchyme initiating posterior skull bones. Ectomesenchymal skeletal elements (other than Meckel’s cartilage) are initiated later and after considerably longer interactions with an epithelium. Information may be found in Figure 21.2. Based on studies summarised in Hall (1987d). b (M), from mesodermal mesenchyme; (E) from neural crest derived ectomesenchyme. c Note that the frontal, which is of mesenchymal and ectomesenchymal origin has two separate interactions one typical of other mesenchymal bone, one typical of ectomesenchymal bones. d The scleral ossicles are induced in groups over this 3-day period. See Chapter 21 for more on these ossicles.

and grafted shed light on whether cell cell interactions occur in those teratomas in which cartilage and bone differentiate. Roles for similar interactions in skull, limb and vertebral development are discussed in Chapters 21, 35 and 41.

URODELE AMPHIBIANS: CHONDROGENESIS Given that the nature of the environment encountered during the migration of neural crest cells is diverse and changes with time, we can ask whether cranial neural crest cells are competent to differentiate into skeletal tissues before neurulation when still in the neural plate or neural folds or whether they acquire competence by association with mesodermal mesenchyme, epithelia or extracellular matrix-products during or after their migration, as discussed in Chapter 17. An association between ectodermal or endodermal epithelial thickenings and condensations of ectomesenchymal cells has been commented upon several times as suggestive of tissue tissue interactions2. The early experiment that opened up this avenue of research was the demonstration of the differentiation of cartilage from neural crest cells of the Alpine newt, Triturus alpestris, cocultured with pharyngeal endoderm. No cartilage differentiated from NCCs cultured alone (Epperlein, 1974; Epperlein and Lehmann, 1975). Hans Epperlein’s group demonstrated that for cartilage to differentiate, direct cell-to-cell contact between NCCs and pharyngeal endoderm is necessary. Neural crest cells do not migrate preferentially toward the endoderm there is no chemoattraction and pharyngeal endoderm evoked the differentiation of mesenchyme and cartilage from the NCCs. The action of the pharyngeal endoderm was specific: somatic and otic mesoderm, from which vertebral and otic capsule cartilages arise, respectively, chondrify in response to notochord and nasal epithelium but not in response to pharyngeal endoderm3. Pharyngeal endoderm is also sufficient to evoke chondro- and odontogenesis from neural crest in the Mexican axolotl Ambystoma mexicanum (Figures 18.2 and 18.4). However, we have to be careful of species specificity or species differences. In another urodele, the Spanish ribbed toad Pleurodeles waltl, Corsin (1975a,b) demonstrated that contact with dorsal mesoderm also evokes cartilage from neural crest cells. Corsin tested for effects of hyaluronan (30 µg/ml) or testicular hyaluronidase (2 µg/mL) on isolated neural crest to see whether these extracellular matrix products would be sufficient for chondrogenesis to occur, but they were not4.

AVIAN MANDIBLES: CHONDROGENESIS AND OSTEOGENESIS An epithelial mesenchymal interaction is also required for NCCs of chick embryos to chondrify as Meckel’s cartilage. Indeed, all the cartilages and bones of the chick head, whether mesodermal or neural crest in origin, require interactions, either the brain or notochord (mesodermal elements) or with an epithelium (Table 18.2).

302 PART | VI Embryonic Origins

(A)

muscles arise) and an epithelial covering. In anuran and urodele amphibians, interaction between these mesenchymal cells and pharyngeal endoderm is required for Meckel’s cartilage to form (Figure 18.2).

Meckel’s Cartilage

(B)

In line with the results from urodele amphibians, we investigated whether an interaction with pharyngeal endoderm was required for chick embryos to initiate Meckel’s cartilage development. Rather, what Hall and Tremaine (1977) and Bee and Thorogood (1980) found was that Meckel’s cartilage in chick embryos depends for its differentiation on a much earlier interaction of NCCs with cranial epithelium, which takes place as the cells migrate away from the neural tube. In embryonic chicks, Meckel’s cartilage arises after premigratory NCCs interact with cranial epithelium adjacent to the neural tube over a 5-h period between H.H. 9 and 10 (Table 18.2). The primary evidence is that (i) premigratory NCCs cultured in isolation fail to chondrify, and that (ii) premigratory NCCs taken from embryos of H.H. 9 differentiate into chondroblasts in response to H.H.10 cranial nonneural epithelium adjacent to the neural tube. Thus, to initiate Meckel’s cartilage the foundation of the lower jaws in all vertebrates in chick embryos, premigratory NCCs interact with cranial epithelia, while in anuran and urodele amphibians, postmigratory NCCs interact with pharyngeal endodermal epithelium. While endoderm is not required to initiate Meckelian chondrogenesis in chick embryos, foregut endoderm does play a role in patterning the visceral arches and hyoid cartilages (Ruhin et al., 2003).

Molecular Mechanisms FIGURE 18.4 Histological sections of two teeth (A, B) formed when rostral trunk neural crest (TNC) from neurula-stage embryos of the Mexican axolotl Ambystoma mexicanum is recombined with pharyngeal endoderm and maintained as an organ culture. See Figure 17.6 for details of the fate map of odontogenic neural crest and see Figure 1.10 for naturally occurring teeth in the axolotl.

As discussed in Chapter 17, the cartilaginous (Meckel’s cartilage) and bony mandibular skeletons, along with membrane bones of the upper jaw and skull, are derived from midbrain-level (mesencephalic) neural crest. In chick embryos, NCCs emerge from the mesencephalon at the five-somite stage (H.H. 8.5, which is 30 h of incubation). The last cells leave when embryos have 10 pairs of somites (H.H. 10 m which is 35 h of incubation). Intact mandibular arches are composed of ectomesenchyme (from which skeletal tissues form), a mesodermally derived mesenchymal core (from which

A series of studies by Mina Mina from the University of Connecticut revealed some of the molecular changes and genetic signals associated with initiation of chondrogenesis in embryonic chick mandibles. G

G

An examination of the temporal and spatial patterns of expression of mRNA for type I and type II collagen and core protein revealed low levels for type II collagen throughout the mandible at H.H. 15, increasing at H.H. 25 when mRNA for core protein was first expressed. mRNA for type I collagen was also first expressed at H.H. 15, increasing strongly at H.H. 28 29. An examination of stage-specific chondrogenic potential beginning at H.H. 16 (a stage from which chondrogenesis can be obtained) revealed that mRNA for type II collagen increased fivefold immediately before chondrogenesis, and revealed epithelial inhibition of chondrogenesis.

Chapter | 18 Epithelial Mesenchymal Interactions Initiate Skeletogenesis 303

G

G

G

G

Earlier studies demonstrating that the epithelium promotes cell proliferation and suppresses chondrogenesis were confirmed. Msx1 was demonstrated in growth centres in the distal mesial mesenchyme, Msx2 in adjacent distal mesial epithelium. Both were associated with programmed cell death, with epithelial up-regulation of mRNA for Msx1. Spatial and temporal regulation of mandibular development by Fgfs and Bmps was demonstrated. Restriction of Prx1 and Prx2 to medial mandibular mesenchyme through interactions involving Endothelin15 was demonstrated.

Regulation of Msx1 by Bmp2 or Bmp4, an important genetic pathway in these interactions, is outlined in Box 13.3. Additional information is available for the roles of Fgfs and Hoxa genes. Fgfs Exposing chick premigratory cranial NCCs to Fgf2 or Fgf4 in vitro demonstrates that low concentrations (0.1 1 ng/mL Fgf2) promote NCC proliferation, while higher concentrations (10 ng/mL) promote differentiation of cartilage and bone. With prolonged culture, both intramembranous and endochondral ossification ensue (Thorogood et al., 1998; Sarkar et al., 2001). Thorogood and his colleagues concluded that the tissue interactions may be mediated by Fgf bound to heparan sulphate proteoglycan, which is interesting when we consider that different epithelia elicit cartilage and bone at different times during development of cranial neural crest cells premigration for chondrogenesis and postmigration for osteogenesis (Table 18.2). Premigratory neural crest cells from chick embryos also produces cartilage in response to H.H. 24 maxillary arch epithelium (which normally induces maxillary mesenchyme to initiate osteogenesis; see below) and to H.H. 24 pigmented retinal epithelium (which normally induces scleral cartilage between H.H. 14 and 18; discussed further in Chapter 21). A fate map of the chondro- and osteogenic regions of the mandibular arches of chick embryos was determined by Hall (1982b). Ectopic application of Bmp2 or Bmp4 (Box 13.3) alters expression of Msx1 and extends expression of Fgf4 in distal mandibular epithelium, leading to formation of a bifurcated Meckel’s cartilage. In their overview of Bmp signalling in craniofacial development, Nie et al. (2006) include a most useful table of craniofacial phenotypes in mice from 18 studies in which genes for Bmp or for Bmp receptors were knocked out6. In embryonic chick visceral arches, Fgf8 is expressed in second-arch epithelium, Bmp7 in visceral clefts and arches from H.H. 12 onward and Bmp4 in the distal tips

of the arches from H.H. 18 onward. Fgf8 in mandibular arch primordia defines the maxillo-mandibular region through epithelial mesenchymal interactions that activate Hox genes. Bmp4 in ventral arch epithelium limits the anterior expression of Fgf8, while ectopic Bmp4 decreases mandibular arch development. As discussed in Chapter 44, mandibular mesenchymal Bmp4 regulates interaction with mandibular epithelium to regulate mesenchymal production of Bmp4 and osteogenesis of the lower jaw (Merrill et al., 2008). Chick mandibular epithelial Bmp7 increases proliferation and cell death, and levels of Msx1, Msx2 and Bmp4 in lateral mandibular mesenchyme. Exogenous Bmp7 elicits the development of ectopic mandibular and maxillary elements. Egf enhances cell proliferation but not gene expression, implicating Bmp7 as an independent part of the signalling system in chick mandibular mesenchyme, discussed in Box 15.17. Hoxa Genes Plant et al. (2000) implanted retinoic acid soaked beads into the midbrain hindbrain junction in chick embryos of H.H. 9, which is before onset of emigration of cranial neural crest cells. Within 1 day, expression boundaries of Hoxa1 and Hoxb1 were shifted anteriorly to the level of the mesencephalon; Msx2 was slightly down-regulated in the hindbrain but the onset of expression in the facial processes was normal. Two ectopic cartilages formed: (i) a sheet of cartilage ventral and lateral to the quadrate; and (ii) an accessory rod of cartilage from the side of Meckel’s cartilage, morphologically reminiscent of the retroarticular process in the first arch domain (Meckel’s is in the second arch). Initially, the quadrate was often displaced laterally and fused to the retroarticular process. Labelling cranial neural crest cells with DiI demonstrated that a subpopulation of cells from rhombomere four (r4) of the hindbrain that normally migrate into the second arch, migrate into the wrong arch, maintain their morphogenetic identity, and form second arch structures in the first arch, reminiscent of the study by Noden (1983) using chick embryos as discussed in Chapter 17.

Mandibular Bones The mandibular skeleton consists of Meckel’s cartilage as a central element surrounded by membrane bones. The classic study on the development of the mandibular skeleton for any vertebrate is the organ-culture investigation of embryonic chick mandibular arches undertaken by Jacobson and Fell (1941), in which three centres condensations (Chapter 19) were identified within each half of the mandibular arch (each mandible): an osteogenic, a chondrogenic and a myogenic centre. Hall

304 PART | VI Embryonic Origins

TABLE 18.3 Results of Organ Culture of Mandibular Mesenchyme after Removing the Mandibular Epithelium Using Enzymatic Digestiona H.H. Stage (Hours or Days of Incubation) at Which Mandibular Epithelium Was Removed

Per cent of Cultures Producing Membrane Boneb

16 18 (55 66 h)

0 (0/17)

20 (3.25 days)

0 (0/15)

21 22 (3.25 3.5 days)

0 (0/31)

23 (3.75 4.0 days)

0 (0/26)

24 25 (4.0 4.5 days)

100 (71/71)

a

Isolated mandibular mesenchyme forms membrane bones only if epithelium is present until H.H. 23. Based on Tyler and Hall (*1997) and Hall (1978b). b Cartilage forms in all of these cultures. Its formation is independent of epithelial influences. The timing of membrane bone formation is similar when mandibular mesenchyme is grafted to the CAM rather than organ cultured.

(1982b) mapped out the in ovo location of the chondroand osteogenic regions of the mandibular arches. Jacobson and Fell concluded that the skeletogenic centres arose within the mandible; they knew nothing of neural crest cell contributions. Each centre was situated close by a transitory thickening of the buccal epithelium. When maintained in isolation in vitro, each centre formed only one differentiated end product bone, cartilage or muscle and so each was interpreted as a separate lineage of committed cells. All our basic ideas about mandibular development arise from this pioneering study. We know now, however, that skeletogenic cells develop from ectomesenchyme that has migrated into the mandibular arches, and that myogenic cells are derived from local head mesoderm. NCCs first reach the regions of the embryo that will become the mandibular arches at H.H. 15 or 53 h of incubation (Le Lie`vre and Le Douarin, 1975). I say ‘regions that will become the mandibular arches’ because no arches or other facial processes maxillary, nasal, frontal exist until ectomesenchymal cells create them. Indeed and as discussed below crest-derived mesenchyme promotes proliferation of arch epithelial cells, which, in turn, promotes mesenchymal survival and outgrowth of the facial processes; Hall (1982b) and Langille (1994b) describe the distribution of osteogenic and chondrogenic cells in mandibular arches of chick embryos. Initially, the mandibular skeleton of avian embryos indeed of all vertebrate embryos consists of Meckel’s cartilage, which begins to chondrify at 5 days of incubation (H.H. 26). An endochondral bone, the articular, develops in the retroarticular process of Meckel’s cartilage at 14 days of incubation (H.H. 40). The balance of Meckel’s cartilage in avian embryos persists as a rod of cartilage that becomes surrounded by six membrane

FIGURE 18.5 The left and right mandibular arches from H.H. 22 Chick Embryo placed on a circle of black Millipore Filter in preparation for CAM-grafting.

bones, whose ossification commences at 7 days of incubation (H.H. 31). Having shown that Meckel’s cartilage in chick embryos arises only after an interaction with cranial non neural crest epithelium, we set out to ask whether bone formation in the mandible required one or more epithelial mesenchymal interactions. Mary Tyler the first of a wonderful series of postdoctoral fellows I have been privileged to have in my laboratory demonstrated that the mesenchyme that will form the membrane bones that invest Meckel’s cartilage must interact with mandibular epithelium for osteogenesis to begin (see Table 18.3 and Tyler and Hall, *1977). Neither Meckel’s cartilage nor the articular bone depends on interactions with mandibular arch epithelium to differentiate. The articular an endochondral element with substantial subperiosteal bone depends for its initiation on hypertrophy of the chondrocytes of the retroarticular process and transformation of the perichondrium to a periosteum. No other parts of Meckel’s cartilage hypertrophy in birds. In the initial studies, and as a control for the trypsin and pancreatin enzymatic digestion used to facilitate separating epithelium from mesenchyme (below), Mary Tyler took intact mandibular arches before either cartilage or bone had differentiated and established them in organ culture or grafted them to the CAM as outlined in Box 12.3. These control cultures and grafts were established to ensure that cartilage and bone would differentiate normally under these artificial conditions, which indeed they do (Figures 18.5 and 18.6). Experimental cultures or grafts consisted of mandibular mesenchyme and ectomesenchyme from which the mandibular epithelial ‘jacket’ had been removed. I refer to such tissue as isolated mesenchyme, even though it consists of mesenchyme and ectomesenchyme; mesenchyme forms a central core of myogenic cells. Anything

Chapter | 18 Epithelial Mesenchymal Interactions Initiate Skeletogenesis 305

(A)

FIGURE 18.6 Normal morphogenesis and growth of Meckel’s cartilage and adjacent membrane bones has taken place in this graft of mandibular mesenchyme from an H.H. 22 embryonic chick combined with maxillary arch epithelium and maintained as a CAM-graft.

else would be cumbersome and require referring to tissue as isolated ectomesenchyme, isolated somitic mesenchyme, isolated somatic mesenchyme, isolated lateralplate mesenchyme and so forth. So, bear in mind that the term ‘isolated mesenchyme’ is shorthand and does not reveal the source of the mesenchyme. The context, however, should (does?). In ovo, osteogenesis of these mandibular bones begins at 7 days of incubation. No bone forms from isolated mesenchyme if mandibular epithelium is removed before H.H. 24 (4.5 days of incubation). Osteogenesis proceeds normally, however, if the mandibular epithelium is removed after H.H. 24, which means that mandibular epithelium has to be present until H.H. 23 (4 days of incubation) for osteogenesis to begin at H.H. 31 (Table 18.3; and see additional images in Figures 13.8, 18.7 and 18.8). Osteogenesis of mandibular (lower jaw) membrane bones in chick embryos therefore requires an interaction between postmigratory ectomesenchyme and mandibular epithelium that takes place between 60 h and 4.0 days of incubation once the mesenchyme is within the mandibular arches (Table 18.2). Osteogenesis of maxillary (upper jaw) membrane bones in chick embryos begins after a later interaction between postmigratory ectomesenchyme and maxillary epithelium that takes place between 60 h and 3.5 days of incubation once the mesenchyme is within the maxillary arches (Table 18.2, and see section below). The similarity in timing is because mesenchyme for both arches accumulates at the base of the mandibular and maxillary processes as a single condensation where the interaction with epithelia takes place (Dunlop and Hall, *1995, and see below). You can imagine how mutations that affect this common condensation could direct more or fewer cells into one or other of the facial

(B)

FIGURE 18.7 Two examples of bone development in culture. (A) Bone that developed from embryonic chick maxillary mesenchyme (H.H. 30). (B) Bone that differentiated from embryonic mouse mandibular mesenchyme (10 days of gestation). Matrix products were deposited into the Millipore filter substrate (below).

FIGURE 18.8 Meckel’s cartilage and membrane bone formed from H. H. 22 embryonic chick mandibular mesenchyme combined with wingbud epithelium and CAM-grafted.

processes or how evolutionary divergence in jaw sizes could operate through this cell population, as indeed has been documented through analysis of the function of Dlx genes in these cells (see below).

306 PART | VI Embryonic Origins

If I may classify by exclusion invertebrates being a fine precedent in all nonmammalian vertebrates, multiple membrane bones develop around Meckel’s cartilage. The most phylogenetically conserved of these bones are the dentary, angular, surangular and splenial. The majority if not all of these bones persist into adult life, either separately or in various stages of fusion. In mammals and, in various intermediate stages, in therapsids portions of what would be Meckel’s cartilage in other vertebrates transform into the three middle ear ossicles the malleus, incus and stapes, introduced in Chapter 13 and discussed further below leaving the dentary as the single bone of mammalian lower jaw, as can be seen in Figures 13.3 13.6.

TABLE 18.4 Results of Initiation of Osteogenesis in Organ Culture of Maxillary Arch (Mesenchyme and Epithelium) and Isolated Maxillary Mesenchymea

H.H. Stage (Hours/Days) of Tissue When Cultured

Per cent of Cultures Producing Cultured Membrane Bone after Seven Days In Vitro

Intact Maxillary Arch H.H. 17 18 (60 66 h)

100 (8/8)

H.H. 23 (3.75 4 days)

100 (8/8)

H.H. 26 (5 days)

100 (6/6)

H.H. 29 (6 6.5 days)

100 (7/7)

Isolated Maxillary Mesenchyme

Maxillary Bones

H.H. 17 19 (60 h 3 days)

0 (0/8)

The membrane bones of vertebrate lower jaws develop in close association with the perichondrial surface of Meckel’s cartilage. Other membrane bones at other sites do not develop in proximity to the primary cartilaginous skeleton. In pioneering discussions, Marshall Urist (1965, 1970) proposed that such bones are induced to form by interactions between potentially osteogenic mesenchyme and fibrous connective tissues, although he had no experimental evidence to support this hypothesis8. Two membrane bones the quadratojugal (QJ) and the jugal differentiate from mesenchyme in the maxillary (upper jaw) region of embryonic chicks at 7 days of incubation. Their differentiation follows interaction with maxillary epithelium. Neither develops in association with the primary cartilaginous skeleton. Consequently, we can study their development without any of the confounding effects of chondrogenesis in adjacent mesenchyme, as could (and has been proposed to) occur in mandibular arch mesenchyme (following section). The QJ and jugal are derived from mesencephalic neural crest cells. For osteogenesis to begin in chick embryos during the seventh day of incubation, mandibular mesenchyme must interact with mandibular epithelium until H.H. 23. Similarly, osteogenic QJ mesenchyme requires that maxillary epithelium be present until H.H. 23 for intramembranous ossification to be initiated at H.H. 31 or 7 days of incubation (Table 18.4). As indicated above, the similarity in timing is because preosteogenic mesenchyme for both arches accumulates at the base of the mandibular and maxillary processes as a single condensation where the interaction with epithelia takes place (Dunlop and Hall, *1995). Development of the bones of the palate in chick embryos also requires an epithelial mesenchymal interaction9. Barx1, a mouse homeodomain transcription factor, is expressed in the mesenchyme of the first and second visceral arches from 10.5 days of gestation on. In chick

H.H. 21 (3.25 days)

0 (0/6)

H.H. 23 (3.75 4 days)

0 (0/6)

H.H. 26 (5 days)

100 (5/5)

H.H. 29 (6 6.5 days)

100 (7/7)

H.H. 32 (7.5 days)

100 (8/8)

a

Based on Hall (1978b) and Tyler (1978).

maxillary primordia, Barx1 has an expression pattern that is complementary to Msx1. Epithelial signals are required to up-regulate Barx1, although Fgf8 can substitute for epithelial signalling. Bmp4 down-regulates Barx1 and antagonises Fgf8, establishing a feedback loop: epithelial Fgf8 up-regulates Barx1, and Bmp4 down-regulates Barx1 by inhibiting Fgf8 (Tissier-Seta et al., 1995; Barlow et al., 1999). A patterning role for Barx1 is reinforced from studies on mouse tooth development, in which Barx1 determines a molariform tooth type. Barx1 is lost exclusively from the molar teeth at 16.5 days of gestation. Barx1 in tooth primordia is inhibited by Bmp4, restricting Barx1 expression to proximal premolar mesenchyme at 10 days of gestation. Inhibiting Bmp4 with Noggin elicits ectopic expression of Barx1 in distal incisor mesenchyme and transformation of the incisor to a molariform tooth (A. S. Tucker et al., 1998c).

Ruling Out a Role for Meckel’s Cartilage in Mandibular Bone Formation Because cartilage always develops in cultures of mandibular mesenchyme from all ages, it is possible that Meckel’s cartilage itself, and not mandibular epithelium, influences mandibular ectomesenchyme to ossify. Indeed, it had been claimed, for example by Frommer and Margolies (1971) from studies with mouse embryos, that

Chapter | 18 Epithelial Mesenchymal Interactions Initiate Skeletogenesis 307

proximity of the membrane bones (or the single dentary bone in mammals) to Meckel’s cartilage is presumptive evidence for an inductive interaction between the two skeletal elements. However, mouse mandibular mesenchyme forms bone in the absence of Meckel’s cartilage (Hall, *1980c; MacDonald and Hall, *2001). Further, in our cultures and grafts of chick tissues, bone often formed some distance from Meckel’s cartilage. Other membrane bones, such as the maxilla or quadratojugal of the upper jaws, develop in isolation from cartilage in ovo, and, as discussed above, upper jaw (maxillary) mesenchyme interacts with maxillary epithelium before it can ossify10.

MAMMALIAN MANDIBULAR SKELETON Considerable information on epithelial involvement during the differentiation of mammalian (mostly mouse) mandibular ectomesenchyme is available. Osteogenesis occurs in cultured mandibular mesenchyme from mouse embryos of 13 16 days of gestation. However, since these are rather late stages of skeletogenesis when compared with chick mandibular development, cell-to-cell interactions may already have taken place. For instance, mandibular mesenchyme isolated from embryos of 12 days of gestation can chondrify (osteogenesis was not mentioned in this study). The implication is that if an epithelial mesenchymal interaction is required for Meckelian chondrogenesis, it has taken place by 12 days. A thickening of the oral epithelium associated with osteogenesis in the adjacent ectomesenchyme was observed in CAM-grafts of fetal rat mandibular processes, reminiscent of the observation made on chick mandibles in culture by Fell and Jacobson 30 years before11. Such experiments and observations are consistent with interaction between the epithelium and the mandibular ectomesenchyme in mammals, but they do not prove it. Subsequent studies in my laboratory demonstrated a requirement for epithelium and the timing of the epithelial mesenchymal interaction of dentary induction in mouse embryos; an example is depicted in Figure 17.5 (Hall, 1980b,c; Miyake et al., 1996a,*b, 1997*a,b; MacDonald and Hall, *2001). Cartilage and bone, along with other tissues such as teeth that require epithelial mesenchymal interactions to develop in situ (Figures 6.8, 12.8 and 18.3), commonly develop in teratomas, which are encapsulated tumours that contain tissues normally derived from one or more germ layers. Given the normal strict requirement for epithelial mesenchymal interactions, and the proximity of cartilage and bones to epithelia within teratomas, we have to wonder whether epithelial mesenchymal interactions take place in teratomas, a possibility entertained by a number of us and outlined in Box 18.1.

Molecular Mechanisms The roles played by members of several families of molecules in murine mandibular development are known. Many play similar roles in chondrogenesis, osteogenesis and tooth formation, indicating substantial evolutionary conservation of what we might call ‘differentiation signal pathways’. I discuss two here, Endothelin1 and members of the distal-less (Dlx) gene family. Others are introduced in the following section on teleost fish jaw development.

Endothelin1 (Edn1) Endothelin1 (Edn1) a vasoactive peptide in vascular endothelial cells that regulates blood pressure and its receptors play important roles in craniofacial development. At 9.5 days of gestation in mice, receptors are localised in osteogenic mesenchyme. Edn is localised in mandibular epithelium and deep mesenchyme12. T. Thomas et al. (1998) identified a signal cascade from EdndHand-Msx1 that regulates development of visceral arch mesenchyme (see Box 13.3 for Msx genes). dHand, a helix loop helix protein, is down-regulated in Edn2/2 mice in which the first and second visceral arches are smaller than in wild typeB mice (the mice have small mandibles), and the third and fourth arches fail to form. Expression in limb buds is normal. Regulation by Edn is specific to the craniofacial skeleton. An inappropriate environment for early specification or emigration of NCCs is created within the neural tube of Edn12/2 mice, which die soon after birth. At 9.5 days of gestation, homozygote mutants lack any neural tissue at what would be the normal midbrain hindbrain boundary, a defect equivalent to that seen in Wnt112/2 embryos, and one that disrupts the population of neural crest cells that normally arises from that region (Wurst et al., 1994). On the other hand, in embryos lacking the Endothelin-A receptor (Ednra), migration of NCCs to the arches and their proliferation in the mandibular arch is normal. However, Ednra2/2 embryos display enhanced cell death in both mandibular mesenchyme and epithelium between 9.5 and 10.5 days of gestation (Abe et al., 2007). Although neural crest derived cells are present in the arches that do form they fail to express Msx1. The cascade is (i) epithelial triggering of Endothelin1; which (ii) regulates mesenchymal dHand; which in turn (iii) regulates mesenchymal Msx1 in the distal visceral arches. Jumping ahead to zebrafish, whose mandibular development is discussed below, these have two Endothelin type-A receptor genes, Ednra1 and Ednra2. Both receptor genes are expressed in neural crest cells and both regulate the

B. In this context and in similar contexts in later chapters, ‘wild type’ refers to embryos that do not have the mutation in the particular gene under study.

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BOX 18.1 Teratomas As noted in the text, cartilage, bone, teeth and other tissues that require epithelial mesenchymal interactions to develop in situ (Figures 6.8, 12.8 and 18.3) commonly develop in teratomas. Given the normal strict requirement for epithelial mesenchymal interactions discussed in the text, and the proximity of cartilage and bones to epithelia within teratomas, we have to wonder whether such interactions take place in teratomasa. Germ-Layer Combinations Differentiation in some teratomas is restricted to neuroectodermal tissues (Damjanov et al., 1973; Illmensee and Stevens, 1979), in others to specific germ layers or to germ layer combinations that facilitate cell interactions. The contribution of specific germ layers to the formation of cartilage and bone in teratomas has been addressed most thoroughly by Levak-Svajger and Svajger, who used enzymes to separate rat embryonic shields into germ layers, which they then established as renal grafts or organ cultures; see LevakSvajger and Svajger (*1979) for an overview of their studies. The major findings are that single germ layers can produce a teratoma, and that cartilage and bone form only when ectoderm and mesoderm, or endoderm and mesoderm (remember these are germ layers; footnote A) are grafted together. For example, respiratory epithelium 1 mesoderm-cartilage, while oesophageal, stomach or intestinal epithelium 1 mesoderm-muscleb. These were among the first studies to imply inductive interactions between germ layers in mammalian embryos. Cartilage and bone also develop when blastocysts are cultivated in vitro or after ova are grafted into the testes (Stevens, 1968; Hogan and Tilly, 1977). At the molecular level we have interesting

patterning of the skeleton of the lower jaw and the jaw joint, serving partially redundant roles: knockdown of Ednra1 results in fusions of upper and lower jaw cartilages; knockdown of Ednra1 and 2 results in failure of the lower jaw to develop. Mandibular arch epithelium is a primary source of Edn1. Ednra1 is expressed in an Edn1-dependent manner, which is consistent with (i) autoregulation within the epithelium and (ii) with epithelial signalling to arch mesenchyme both being required both being required to pattern cartilage development (Nair et al., 2006). In murine limb development (Chapter 38), dHand regulates the Shh pathway to establish the anterior posterior limb axis. Clouthier et al. (2000) identified Ednra in neural crest cells destined for the visceral arches and determined that Edn acts on postmigratory neural crest cells to up-regulate a number of transcription factors (Dlx2, Dlx3, dHand, eHand) but not Prx1, Hoxa2 or cellular retinoic acid binding protein (Crabp1). A subsequent study from this laboratory demonstrated that Edn1 patterns the lower jaw and participates with Hox genes to pattern the most posterior visceral arches (Clouthier et al.,

data on the role of Sox9 in chondrogenesis within teratomas. Chimaeric mice generated using Sox92/2 embryonic stem cells exclude any Sox92/2 cells from the cartilages that form, and, furthermore, cartilage fails to form in teratomas derived from embryonic stem cells from these chimaeras (Bi et al., 1999). The discovery that ectodermal cells from head-fold stage embryos produced bone and cartilage prompted the conclusions that presumptive ‘ectoderm’ contains other presumptive germ layers, a conclusion based on the expectation that only mesoderm would produce skeletal tissues. We now know that neural crest primordia were within the ectoderm (Hall, *2009). Although formation of cartilage or bone in the stomach is rare, Ohtsuki et al. (1987) reported a case of metaplastic bone formation in a stomach polyp. Bone formation followed transformation of the stomach epithelium to a cancerous state, suggesting that the altered epithelium induced the bone. Kumasa et al. (1990) examined ectopic bone formation in nine tumours, which they interpreted as having arisen by metaplastic transformation of the stroma under the inductive influences of epithelia within the tumours. Wight and Duff (1985) assumed that ectopic pulmonary cartilage and bone in newly hatched chicks arose from misplaced germ cells (Figure 17.8, and see the discussion of such ectopic cartilages in Box 17.2). a. See Damjanov and Solter (1974), Skreb et al. (*1976), O’Hare (1978), Hall (1987d, 1994c) and Maclean and Hall (1987) for overviews of the developmental aspects of teratomas, including epithelial mesenchymal interactions. b. See Levak-Svajger and Svajger (*1979), Svajger and Levak-Svajger (1976) and Skreb et al. (*1976) for the initial studies on germ layer contribution to teratomas.

2010, and see Gitton et al., 2010 for an overview Edn1 and Dlx genes [see below] in craniofacial patterning). The normal centre of expression of Hoxa1 is in rhombomeres 4 7 (r4 r7) at 7.5 8.5 days of gestation, which is before NCC migration begins. Disrupting Hoxa1 (Hox 1.6) results in defects in the normal rostral domain of expression of the gene. Homozygotes for Hoxa1 exhibit delayed closure of the neural tube, lack cranial nerves and ganglia, and have inner ear malformations. Skeletal malformations are seen in elements that arise from paraxial mesoderm, while the visceral arches are normal (Lufkin et al., 1991). With further investigation, members of the Dlx family of genes were shown to play critical roles in patterning mandibular development and other regions of the craniofacial skeleton.

The Dlx and Msx Gene Families and Murine Craniofacial Development Distal-less (Dlx) and Msx multigene gene families of transcriptional activators and repressors, respectively, are

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FIGURE 18.9 Craniofacial skeletal abnormalities in mice in which one of Dlx1, 2 or 5 was knocked out, depicted as lateral views of the skeleton. Changes consist of shape changes (shown as defective bones, light shading) and the formation of additional bones (darker shading). (A) Morphogenesis of the squamosal (Sq) and stapes (St) is abnormal when Dlx1 is knocked out. (B) Skull morphogenesis is even more abnormal and additional bones develop when Dlx2 is knocked out. (C) Abnormalities in the lower jaw occur when Dlx5 is knocked out. See the text for further details. D, dentary; Hy, hyoid arch skeleton; Ma, malleus; Mc, Meckel’s cartilage; Mx, maxilla. Modified from Merlo et al. (2000).

expressed in overlapping yet distinct domains during division, differentiation, patterning and morphogenesis of various tissues such as the visceral arches and craniofacial skeleton in which epithelial mesenchymal interactions or cell death occur. Some functions of Msx genes are discussed in Box 13.3. A similar outline for some of the six Dlx genes is provided in Figure 18.9 and in the discussion below13.

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FIGURE 18.10 Expression of Dlx genes in the mandibular arch of embryonic mice of 10.5 days of gestation. (A) A diagrammatic lateral view to summarise expression of the genes Dlx 1 7 in developing median nasal (mn) and maxillary (mx) processes, and in the mandibular arch (md). (B) In this frontal (oral) view of the mandibular (above) and second visceral arches (below), Dlx2 is expressed in the left and right mandibular arches and along one surface of the second arch. (C) In this frontal view of the developing brain (above) and arches (below), Dlx3 is expressed along the lateral borders of the first and second arches and in the antero-medial portion of the first (mandibular) arch. (D) Dlx6 is expressed in the mandibular arch (above), weakly in the maxillary arch and strongly in the otic vesicle (OV). (E) Dlx5 has extension expression in mandibular and maxillary arches. hb, hindbrain. Adapted from X. Zhao et al. (2000)

Dlx1 and Dlx2 are essential for development of the proximal regions of the first and second arches in mice. Null mutation of Dlx2 results in failure of forebrain development and changes in the proximal portions of the first and second arch skeletons (Qiu et al., 1997). Z. Zhao et al. (2000) examined the six Dlx genes in developing murine dentition and mandibular processes, finding that at 10.5 days of gestation all six genes are expressed in mandibular mesenchyme with Dlx3 expression in mesial epithelium (Figure 18.10). Dlx2 is expressed in the proximal mesenchyme and distal epithelium of the first visceral arch. Loss of function of Dlx1 or Dlx2 results in failure of formation of the upper molars but does not affect development of the lower molars, probably because of functional redundancy with Dlx5; although Dlx2, 3, 4 and 5 genes are active during pharyngeal tooth development in zebrafish, neither Dlx1a nor Dlx6a is expressed (Borday-Birraux et al., 2006).

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B. L. Thomas et al. (2000), who also asked whether Dlx1 or Dlx2 pattern skeletal mesenchyme in the first arch, found that mesenchymal and epithelial expression of Dlx2 in the first arch is differentially regulated: G

G

Bmp4 is coexpressed with Dlx2 in distal epithelium, where it regulates expression of epithelial Dlx2; Fgf8 is expressed in proximal mesenchyme (Figure 13.7), up-regulates mesenchymal expression of Dlx2 and downregulates epithelial Dlx2.

With the growing knowledge concerning the role during development of the degradation of mRNA by microRNAs (miRNAs), which are 20- to 22-nucleotidelong sequences of noncoding RNA, post-transcriptional regulation of genes such as Dlx2 in epithelial mesenchymal interactions initiating skeletogenesis in the craniofacial skeleton are becoming more precisely understood. Over 600 miRNAs have been identified in the neural tube of mouse embryos alone. Each miRNA regulates a number of genes and there are multiple copies of each miRNA in the genome. Sheehy et al. (2010) took advantage of a mouse mutant lacking the RNAse III enzyme Dicer. Dicer mutants fail to develop major features that are neural crest in origin: they lack craniofacial skeleton, dorsal root ganglia and thymus, and septation of the cardiac outflow tract fails to occur. Expression of Dlx2 in the first visceral arch is reduced in Dicer mutants. One particular miRNA, miR-452, is enriched in neural crest cells and can rescue Dlx2 expression in the first visceral arch. In the model proposed by Sheehy and colleagues, miR-452 blocks Wnt5a in arch mesenchyme, which in turn blocks the pathway Shh-Fgf8 in arch epithelium and so blocks epithelial Fgf8 signalling to Dlx2 in arch mesenchyme. In tracheal chondrocytes, miR-125b and miR-30a/c maintain Snail1 at a low level. As Snail1 inhibits Acan (the aggrecan gene) and Col2a1, these miRNAs enhance chondrogenesis. Dicer1 knockout derepresses Snail1, lowers formation of aggregan and collagen type II and so suppresses chondrogenesis (Gradus et al., 2011). Turning briefly to chick embryos, misexpression of Dlx2 and Dlx5 in craniofacial mesenchyme results in formation of ectopic bone and cartilage in the upper jaw. Ability to respond is limited to a subset of craniofacial mesenchymal cells in which Dlx elicits ectopic condensations (Gordon et al., 2010). Dlx5 and Dlx6 are expressed in condensations for membrane bones and in periostea around cartilage models in endochondral ossification (Figure 18.11). Dlx5 exhibits stage-specific expression in mouse calvarial osteoblasts, repressing osteocalcin with onset of mineralisation. Dlx5 is inducible by Bmp4 in mouse embryos, in fractures and in MC3T3-E1 cells (in which osteoblast markers are up-regulated and deposition of extracellular matrix is

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FIGURE 18.11 Expression of Dlx5 in murine limb and craniofacial development. (A) Expression in the AER and distal mesenchyme of a limb bud at 10.5 days of gestation. (B) Expression in distal digital mesenchyme and in the perichondria of more proximal skeletal elements in the forelimb at 14.5 days of gestation. (C) Expression in visceral arches one and two not in three and in the otic vesicle (arrow) at 9.5 days of gestation. (D) Strong expression in maxillary and mandibular arches and in the vestibular organ (arrowhead). Modified from Merlo et al. (2000).

enhanced), demonstrating the sequence Bmp4-Dlx5osteogenesis. (In their overview of Bmp signalling in craniofacial development, Nie et al. (2006) include a most useful table of craniofacial phenotypes in mice from 18 studies in which genes for Bmp or for Bmp receptors have been knocked out.) Analysis of Col2a1-Dlx5 transgenic mouse embryos reveals that Dlx5 and 6 function autonomously in regulating chondrocyte hypertrophy and are functionally equivalent14. An earlier role for Dlx5 and Dlx6 in mouse embryogenesis was revealed when Heude et al. (2010) demonstrated that, although neither gene is expressed in jaw muscles, if either gene is inactivated then jaw muscles fail to form. Cranial neural crest cells migrating to the first visceral arch provide the Dlx signalling required for myogenic precursors to differentiate, although extrapolating these results to the origin of the vertebrate head is a stretch.

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Two studies of Dlx5 knockouts confirm this role in mice and provide information on craniofacial defects, delayed skull ossification, hypomineralisation and ectopic bone in the mandible. Dlx5 is expressed in the visceral arches, otic and olfactory placodes and in all bones. Homozygous Dlx52/2 embryos die at or soon after birth with anomalies involving all visceral arches (especially the proximal portion of the mandibular arches) and sense organs (ears, nose) and delayed and hypomineralisation of the skull bones. Dlx5 and Dlx6 are both expressed in the distal but not in the proximal regions of the embryonic arches (Figure 18.10). Knocking out both genes results in a homeotic transformation of lower to upper jaws. Depew’s group provided a model for the role of nested domains of Dlx genes and the organisation of proximo-distal elements, Dlx1 and 2 specifying proximal and Dlx1, 2, 5 and 6 specifying distal elements15. A homeobox gene, Satb2 (Special AT-rich sequence-binding protein2), which encodes a DNA-binding protein, defects in which are associated with cleft palate, is expressed in the distal mesenchymal domains of both upper and lower jaws under the control of epithelial Fgf8. Loss of function of Satb2 results in defects in the distal (incisor) domain elements only, providing further confirmation of the modular nature of epithelial mesenchymal control of jaw development (Fish et al., 2011). Differential signalling by mandibular and maxillary epithelia was revealed in mouse embryos, primarily from studies of epithelial or mesenchymal control of tooth form, but the signalling is applicable to control of arch mesenchyme. Beginning with the knowledge that homeobox gene expression correlates with molar or incisor development and that epithelium induces homeobox gene expression, C. A. Ferguson et al. (2000) found that all arch mesenchyme can respond to epithelial Fgf8 in embryos younger than 10 days of gestation. By 11 days, mesenchymal expression is independent of the epithelium. Mandibular and maxillary mesenchyme respond differentially; mandibular and maxillary epithelia induce Dlx5 and Dlx2 in mandibular mesenchyme but only Dlx2 in maxillary mesenchyme. Neither epithelium induces Dlx5 in maxillary mesenchyme. Intrinsic differences between mandibular and maxillary mesenchyme are therefore established by 11 days of gestation, which is when inductive interactions with mandibular epithelium take place (Hall, 1980a; MacDonald and Hall, *2001). A note on limb development: Dlx5 is expressed in chick-wing and leg-bud epithelia. Implanting beads soaked in Fgf2 into the flank of chick embryos induces Dlx5 within 12 h. (Beads implanted into the mandibular arches of chick embryos are illustrated in Figures 3.5 and 3.6.) Dlx5 is transiently expressed in the epithelium of limb buds from limbless embryos, suggesting a requirement for Dlx5 to maintain the AER (Ferrari et al., 1999).

Dlx Genes in Shark, Skate and Paddlefish Craniofacial Development To determine whether a nested Dlx-code is a basal feature of jawed vertebrates, Gillis et al. (*2013) examined Dlx expression in embryos of the little skate Leucoraja erinacea and of the small-spotted catshark Scyliorhinus canicula as representatives of two lineages of elasmobranchs, and in embryos of the Mississippi paddlefish Polyodon spathula, which is a basal actinopterygian bony fish16. A nested mesenchymal code of six Dlx genes is present in the visceral and gill arches in embryos of all three species, results that are consistent with serial homology of the visceral and gill arches, and a Dlx code in the earliest gnathostomes. Using fate mapping, Gillis and colleagues further demonstrated that dorsal and ventral skeletal elements in each arch arise from mesenchymal cells expressing the same Dlx code seen in mice and zebrafish. Compagnucci et al. (2013) also described a proximaldistal pattern in expression of Dlx genes in the visceral arches of the small-spotted catshark along with expression patterns of Emx2, Bapx1 and/or Shh in different regions of the visceral arches.

TELEOST MANDIBULAR ARCH SKELETON As we saw for urodele amphibians, interaction with pharyngeal endoderm plays an important role in patterning visceral arch cartilages in the zebrafish. For fish the evidence comes not from experimental manipulation but from a mutant phenotype in the zebrafish, Danio rerio. The mutation is in the gene van gogh (vgo). Mutant embryos lack the entire pharyngeal region. The defect occurs in the late stages of NCC migration, although segmentation of the hindbrain is normal. Although initial emigration of neural crest cells from the neural tube is via the normal streams, the streams fuse peripherally. Because visceral arch segmentation requires signals from endoderm and from neural crest, arch segmentation fails to occur and, as a consequence, segmental cartilages fail to develop; see Piotrowski and Nu¨sslein-Volhard (2000), who also provide data on one-eye, pinhead and Casanova. All three of these mutants lack endoderm; arch segmentation also requires normal endoderm17. Our knowledge of the transcription and growth factors associated with jaw development in teleosts outstrips our understanding of the cellular interactions involved. Nonetheless, an interesting story is emerging, one that indicates conservation of signalling systems between teleosts, sharks and tetrapods, especially genes in the Dlx and Fgf families, Hoxd4 and retinoic acid and Endothelin1, all of which will be familiar to you from earlier discussions. Lower jaws and visceral arches in zebrafish (and, we assume, in other teleosts such as the alewife; Figure 3.8),

312 PART | VI Embryonic Origins

are specified by Endothelin1 (Edn1), which is encoded by the gene sucker (suc), which is expressed in central arch mesoderm and arch epithelium but not in ectomesenchyme. C. T. Miller et al. (2000) concluded that Edn1 expression creates an environment in which neural crest derived skeletogenic mesenchyme is specified.

Dlx Genes Further to the patterning role for Dlx discussed above, only some craniofacial cartilages develop abnormally when zebrafish embryos are treated with retinoic acid. The abnormalities are associated with loss of expression of Dlx, which in zebrafish is normally expressed in hindbrain, neural crest cells and visceral arches. The midbrain, which lacks Dlx, is less affected by retinoic acid treatment (Ellies et al., 1997). As in mice, Dlx genes pattern the visceral arches along their proximo-distal axes in zebrafish embryos. In zebrafish, however, there is a temporal separation of gene expression: Dlx2a (the teleost orthologue of mammalian Dlx2) is expressed in NCCs migrating from the hindbrain (knocking down Dlx2a reduces NCC number); Dlx1a (the teleost ortholog of mammalian Dlx1) is expressed in the developing visceral arches. Morpholino knockout of Dlx1a and Dlx2a results in severe arch cartilage anomalies. Despite the temporal separation of Dlx1a and 2a into cells at different stages in their differentiation toward cartilage, both genes participate in patterning the proximal regions of the visceral arches that form the mandibular and ceratohyal skeletons, as do Dlx5a/6a and Dlx3b/4b. Loss of function of Dlx2a is associated with disrupted expression of Barx1 and loss of goosecoid transcripts from the proximal region of the ceratohyal arch (S. M. Sperber et al., 2008).

Fgfs Fgfs are expressed in the surface epithelium, lateral-line blastemata and otic vesicles of embryos of the Japanese flounder Paralichthys olivaceus. Blocking Fgf in vitro impairs chondrogenesis (T. Suzuki and Kurokawa, 1996). David et al. (2002) found a requirement for endoderm mediated via Fgf3 for cartilage formation in zebrafish, while Albertson and Yelick (2005) showed that Fgf8 is required for symmetrical development of the pharyngeal skeleton, probably by acting through Kupffer’s vesicle, which is the zebrafish homologue of Hensen’s node. Walshe and Mason (2003) showed that an Fgf signal is required for 6 h after initiation of NCC migration for neurocranial and pharyngeal cartilages to develop in zebrafish, and that Fgf3 and Fgf8 together comprise an Fgf-signalling system: G

inhibiting Fgf3 results in complete absence of all cartilages that normally arise from pharyngeal arches three to six;

G

G

inhibiting Fgf8 has minimal and mild effects on chondrogenesis; and inhibiting both Fgf3 and Fgf8 results in complete lack of formation of all pharyngeal cartilages and of almost all the cartilages of the neurocranium.

Fgf10, for which see Box 14.1, was important in the evolution of bi- or multicusped teeth in teleosts; overexpressing Fgf10 in either zebrafish or Mexican tetra results in transformation of unicuspid pharyngeal teeth to bicuspid teeth and the formation of supernumerary teeth. Jackman et al. (2013) concluded that the shared action of Fgf10 on tooth morphogenesis and number constituted an evolutionary constraint. Slight modification of that constraint, they propose, could propose the range of tooth shapes seen in teleosts.

Hoxd4 and Retinoic Acid As seen from the discussions for chick and mouse embryos, retinoic acid is an important morphogen for limb and craniofacial development, discussed separately below. (Morphogens are discussed further in Box 40.2.) A retinoic acid Hoxd11, d12 and d13 connection has been established in limb development. Retinoic acid is required for Shh, Hoxd12, Hoxd13 and Fgf4 signalling during rat limb-bud growth, but not for initiation of the limb buds; application of retinoic acid at the 45-somite stage suppresses Hoxd13 and Fgf8 and is associated with a flattening of the AER and suppression of limb-bud growth. Applying retinoic acid to the anterior limb-bud mesenchyme of chick wing buds in physiological or pharmacological doses (100 or 333 mg/mL, respectively) elicits expression of Shh and Hoxd11 and induces additional digits from tissue distal to the implanted retinoic acid18. Retinoic acid lies upstream of Hoxd4 genes, a pathway that has been investigated in fin and craniofacial development in teleost fishes. Jaw and pectoral fin malformations are induced in flounder exposed to retinoic acid; exposure of shield-stage embryos for 1 h suppresses development of Meckel’s cartilage (T. Suzuki et al., 2000). Hoxd4 was cloned from the flounder and expression and response to retinoic acid examined. Hoxd4 is expressed in the brain from rhombomere 7 rostrally into the spinal cord, in visceral arches 2 5 and in their cartilages. In the presence of exogenous retinoic acid, the anterior border of expression moves further anteriorly, as it does in tetrapods. Hoxb5 is expressed in gill arch five and in the spinal cord, and may play a role in specification of arch five. Retinoids are required to maintain pharyngeal endodermal expression of a large battery of genes, including Hoxa1, Hoxb1, Pax1, Pax9, Fgf3 and Fgf8 (Wendling et al., 2000). When administered later in embryonic development, retinoic acid depresses Shh and Hoxd4

Chapter | 18 Epithelial Mesenchymal Interactions Initiate Skeletogenesis 313

expression in the visceral arches and induces skeletal malformations (T. Suzuki et al., *1999). At the chondrogenic condensation stage Shh is expressed in pharyngeal endoderm, and mandibular, hyoid and gill primordia, expression subsequently expanding to the posterior endoderm of each arch. Retinoic acid depresses Shh and Hoxd4, changes that result in malformed cartilages with their growth shifted posteriorly. Additional insights on Hox genes and on the cells involved in skeletogenesis in zebrafish lower jaws come from a study by X. Wang et al. (2012) in which the lower jaw was amputated and shown to regenerate. As discussed in Chapter 14, regeneration occurs through the formation of a blastema, which, surprisingly, is derived from two separate cell populations, one composed of Foxi1-positive cells from which skeletal cells arise in the regenerate, and the other composed of Isi1-positive cells from which muscle in the regenerating lower jaw arises. Hoxa2, Hoxa11b and Sox9 are up-regulated in the skeletogenic cells within the blastema, expression is reduced if Foxi1 is knocked down, and Pax3a is up-regulated in the muscle-forming cells.

Mutants With respect to Fgf signalling, discussed earlier, and as demonstrated in Sp8 mutant mice in which the zinc-finger transcription factor Sp8 and its proto-oncogene product Ski have been knocked out, Fgf8 and Fgf17 are expressed in the forebrain signalling centre from which they regulate cell proliferation and decrease apoptosis in neural crest and mesoderm-derived mesenchyme destined to form the facial skeleton. The Sp8 mutant can be rescued by reducing Shh signalling (Kasberg et al., 2013). As discussed in Box 16.2, Zic2a and Zic2b pattern craniofacial cartilages at the condensation stage through earlier action on the induction and migration of neural crest cells and by signalling via the same forebrain signalling centre that expresses Fgf8 and Fgf17 (TeSlaa et al., 2013). Numerous mutations affecting the visceral and mandibular arches in zebrafish are known. Neuhass et al. (1996) identified 48 mutations in 34 loci affecting craniofacial development. For instance, chinless disrupts skeletal fate and interactions between neural crest and muscle. Chinless embryos lack cartilages in all seven visceral arches and so lack Meckel’s cartilage. Secondarily, chinless mutants lack mesodermal muscle, although, interestingly, both cartilage and muscle precursors are present, giving us a clue to the time of action of the mutation19.

LATERAL LINE, NEUROMASTS AND DERMAL BONE The lateral-line system is an extensive network of superficial sensory nerves in teleosts, lampreys, elasmobranchs

FIGURE 18.12 Cross section through a developing lateral line canal of the zebrafish Danio rerio showing the lateral line bone (pink) enclosing the open cavity with the neuromast at the base. Scale bar, 50 µm. Image provided by Jacqueline Webb.

and larval amphibians. Multiple lateral lines on the head follow the sutures of dermal bones, or vice versa. Usually only a single mid-body lateral line is present on the trunk. In teleost fishes this single line is associated with specialised lateral-line scales. Lateral-line nerves are associated with specialised sensory receptor organs or neuromasts that detect mechanical, electrical and chemical signals. Neuromasts are often embedded in a bone, variously known as a canal bone (because it forms a canal for the lateral-line nerve) or a lateral-line bone (Figure 18.12). The tight correlation between neuromasts and the underlying dermal bones is lost in Endothelin-1 knockdown zebrafish embryos (H. Wada et al., 2010). Partly because of the past recognition of the association of cranial lateral lines with bones of the skull, lateral-line nerves and/or neuromasts have been implicated as inducers of dermal bone20.

Hope from a Single Trout The classic experimental study suggesting a relationship among neuromasts, lateral line and dermal-bone development in a teleost fish, the rainbow trout Oncorhynchus mykiss, was undertaken over 74 years ago by MoyThomas (1941). Excising the lateral-line system had no effect on the development of the frontal bones, but the bony ‘gutter’ (the canal bone) associated with the frontal bones failed to form. Moy-Thomas proposed that if the lateral line or neuromasts were developmentally coupled in this ‘higher’ teleost, then neuromasts and dermal bones should also be associated in such ‘primitive’ fishes as the bowfin Amia calva. This turns out to be one of those situations where the idea is easy but the data hard. The vertebrate palaeontologist T. S. Westoll followed up Moy-Thomas’s

314 PART | VI Embryonic Origins

suggestion and described a topographical association between dermal bones and the lateral-line system in Amia and in fossils of other ‘primitive’ fishes (Westoll, 1941). But no experimental studies were performed. Jollie (1981) and in five papers in 1984 (*1984) and Meinke (1982a) examined times of first ossification and the topographical relationship between lateral lines and osteogenesis in salmon (especially Coho Oncorhynchus kisutch, and sockeye O. nerka) and in Lepisosteus and Polypterus. In The Development of the Vertebrate Skull, his masterful summary and analysis of skull development and structure, Gavin de Beer also took up the suggestion from Moy-Thomas and discussed the relationship between dermal bones and lateral-line canals (de Beer, 1937). Some dermal bones in fishes nasals in flatfish, frontalC, intertemporal and postparietal in Amia develop in close association with neuromasts of the lateral-line system. De Beer concluded that this association was a secondary one. This is in part because many dermal bones in fishes have no such association and in part because of the importance he placed on homology. Because homologous bones in other vertebrates arise in the absence of lateralline organs, de Beer accepted that this would be the case for all vertebrates. Later, de Beer came to accept that homologous structures bones in this case could arise by different developmental processes (presence or absence [loss] of induction by neuromasts) and that a causal developmental relationship in one group could be broken in another. Such a situation is inevitable with the bones in question; most tetrapods have neither lateral lines nor neuromasts. Webb (*1989) investigated the distribution of neuromasts and the lateral-line system in teleost fishes, drawing attention to the relationship between neuromasts and dermal bone21. Although the study by Moy-Thomas is a classic in part because it has never been repeated it is seriously flawed. Moy-Thomas removed the lateral-line primordium from one side of a single rainbow trout embryo. Apart from the minimal sample size, a further objection is that the dermal bones of many teleosts develop and regenerate independently of neuromasts. This does not mean that the frontal bone of rainbow trout or its associated lateral-line bone might not be one of the bones in one of the species with a neuromast connection. Amia, however, C. The late Angus Bellairs, an eminent British herpetologist, wrote a novel called The Isle of Sea Lizards in which we find the following: ‘“You know that tome by Gavin de Beer, the late Sir Gavin, I should say?” She shook her head. “Well, nobody reads it these days, but it’s a fascinating book if that’s the way your mind works. But dry, one has to admit. I read it in parallel with Tess [of the D’Urbervilles, by Thomas Hardy], one on each arm of my chair. When Tess made me too weepy, I turned to the timeless serenity of the frontal bones, from fish to man”’ (A. d’A. Bellairs, 1989, p. 93).

would be a better choice of experimental animal, but has not been explored experimentally22. Heterochronic changes may also come into play, as suggested by a study by Smirnov (1990) on the Oriental fire-bellied toad, Bombina orientalis, in which paedomorphic skeletal features include retention of lateral lines in adulthood, decreased cranial ossification, juvenilised teeth and a reduced middle ear. See Hall and Hanken (*1993) and Maglia and Pugener (1998) for skeletal development in Bombina orientalis. Further examples are discussed in Chapter 44. In an extensive analysis of induction of the dermal skeleton in salmon (Salmo), DeVillers (1947, 1965) obtained descriptive and experimental support for neuromast induction of dermal bone. His chief findings are as follows. G

G

Osteogenic cells remain in close association with neuromasts as the neuromasts differentiate. Neuromasts act as aggregation centres for osteogenic cells that form the canal bones, defined by DeVillers and herein as bones developing in association with the lateral-line canal.

DeVillers concluded that canal bones have a dual composition, each bone consisting of a laterosensory or tubular component and a basal or membranous component. Furthermore, he proposed that while both components were induced by neuromasts in salmon, only the tubular component was induced in cyprinid fishes23. In one of the few experimental studies that bears on these issues, Merrilees (1975) took semicircular canals (which are specialised neuromasts) from goldfish Carassius auratus, bisected them and transplanted each into the pocket left after a scale was removed (see Box 2.4 for teleost scales). Normally scales regenerate, but regeneration was inhibited in the presence of the semicircular canals, as was development of any cartilage or bone. Merrilees described a specialised cord of epithelial cells in the lateral-line canals that inhibits osteogenesis and so controls cavitation of the tubular component of the canal bone described above24. Little work has been done since, although in a detailed analysis of scale development, Sire and Arnulf (1990) discuss the role of epithelial mesenchymal interactions and the lateral-line system. A promising exception is a study of lateral line/neuromast patterning and dermal bone or scale development in zebrafish and Japanese medaka by H. Wada et al. (2010). Although still not providing evidence for inductive interactions, this study suggests that ‘the formation and patterning of accessory neuromasts [in zebrafish] are associated with the morphogenesis of the underlying dermal structures’ (p. 589) and that ‘once scales have formed [in Japanese medaka], they determine or constrain the position and budding of neuromasts’ (p. 592).

Chapter | 18 Epithelial Mesenchymal Interactions Initiate Skeletogenesis 315

Modern molecular and genetic studies on the induction of lateral-line bones are minimal. Fgf is expressed in surface epithelium and in the lateral line blastemata in embryos of the Japanese flounder Paralichthys olivaceus (T. Suzuki and Kurokawa, 1996). Similarly, no one has investigated whether similar interactions control the development of sensory canal cartilages in elasmobranchs, although the possibility has been discussed25. Blind cave fish Astyanax mexicanus have more extensive development of other sense organs such as lateral lines than do sighted members of the species. Life in caves is associated with or perhaps results in cryptic speciation in which morphologically similar species (often subspecies) are genetically distinct (Ivanovic et al., 2013). Consequently, skeletal changes may be subtle. Among skeletal changes in the cave fish are additional suborbital bones whose numbers correlate with increased numbers of cranial neuromasts. This association suggests an inductive interaction and is consistent with experimental studies in which ablation of the lens from surface cave fish alters suborbital bone number and shape (Dufton et al., 2012). The number of ossification centres for each infraorbital bone in zebrafish also may relate to nearness of association with lateral-line canals (Chang and Franz-Odendaal, 2014)26.

7.

8.

9.

10.

11.

12.

NOTES 1. See Hall (1982c, 1983b,c,d,e, 1984a, 1989, 1991a,b,c, 1994b, 1995b) for epithelial mesenchymal interactions growth factors and skeletogenesis in normal development, and see Hall (1994c) for such interactions during skeletogenesis in tumours. 2. See Jacobson and Fell (1941), de Beer (1947), Ho¨rstadius (1950), Kingsbury et al. (1953), Holtfreter (1968), Tonegawa (1973), Hall and Ho¨rstadius (1988) and Jabalee et al. (2013) for spatial associations of epithelial thickenings and aggregations of mesenchyme. 3. Association with endodermal epithelia may be relevant to the patterning of neural crest cells in development and evolution and to the evolutionary origin of teeth (Piotrowski and Nu¨sslein-Volhard, 2000; Wendling et al., 2000; Graham and Smith, *2001; Chambers and McGonnell, 2002; David et al., 2002; Matt et al., 2003; Ruhin et al., 2003). 4. See Graveson and Armstrong (1987) for chondrogenesis in the axolotl, Graveson et al. (1997) for evocation of teeth from trunk neural crest in the axolotl. 5. See Mina et al. (1994) for mRNA distributions and for proliferation confirming the earlier studies by Coffin-Collins and Hall (1989) and Hall and Coffin-Collins (1990) Mina et al. (1995) for Msx1 and 2, Mina et al. (2002) for Fgfs and Bmps, and Doufexi and Mina (2008) for Prx1 and Prx2. Also see McGonnell et al. (1998) for Msx1 in expanding facial mesenchyme and Fgf8 in adjacent ectoderm (Figure 13.7). 6. See Hall (*1980c), Kollar and Mina (1991) and MacDonald and Hall (*2001) for epithelial mesenchymal interactions in mouse Meckel’s

13.

14.

15.

16.

17.

18.

19.

cartilage, and Barlow and Francis-West (1997) and Bogardi et al. (2000) for the bifurcated cartilage. See Wall and Hogan (1995) for the expression data and ectopic elements, Y.-H. Wang et al. (1999) for Egf, and Shigetani et al. (2000) and Box 14.1 for Fgf8. Marshall Urist was an orthopaedic surgeon who maintained an enormously active and productive programme of basic research and clinical practice during his career. His pioneering studies on the osteoinductive role of demineralised bone matrix paved the way to the discovery of Bmps as skeletal inducers, for which see Box 12.1 and Urist (1965, 1970, 1991), Urist et al. (1985, 1997), Johnson and Urist (1998). The two papers cited in the text (Urist, 1962, 1970) show the breadth of his appreciation of skeletal biology. His text, Bone Fundamentals of the Physiology of Skeletal Tissues (McLean and Urist, 1968), was a mainstay for many of us for many years. See Le Lie`vre (1974) and Le Lie`vre and Le Douarin (1975) for mapping the upper jaw elements to the neural crest, and Tyler and Koch (1977), Hall (1978b, 1980a,b,c, 1981a,b,c, 1983b,c,d,e) and Tyler (1978) for epithelial requirements. See Tyler and Hall (*1977), Minkoff and Kuntz (1978), CoffinCollins and Hall (1989) and Hall and Coffin-Collins (1990) for mesenchymal influences on epithelial proliferation, and Hall (1978b, *1980c) for heterotopic interactions. See Kollar and Baird (1969) for 13 16 days, Svajger and LevakSvajger (1971) for 12 days, and Tenenbaum et al. (1976) for the epithelial thickenings. Box 12.3 contains additional studies in which mammalian tissues were CAM-grafted. See Barni et al. (1995), Richman and Mitchell (1996) and Clouthier et al. (2000) for endothelin1 expression and role in craniofacial development, and Richman and Mitchell (1996) for a review of knockout mice. See Bendall and Abate-Shen (2000) for a review. Merlo et al. (2000) review the roles of Dlx genes in the craniofacial skeleton and in osteogenesis. See Ryou et al. (1997) and Ducy et al. (2000) for expression of Dlx5 and 6, Miyama et al. (1999) for the link to osteogenesis via Bmp4, and Zhu and Bendall (2009) for Dlx5, 6 functional equivalency during chondrocytes hypertrophy. See Depew et al. (1999) and Acampora et al. (1999) for Dlx5 knockout, Depew et al. (2002) for mice lacking Dlx5 and Dlx6, and Bendall et al. (2003) for the role of Dlx5 in endochondral ossification in chicken and mouse embryos, including its role in condensation of prechondrogenic mesenchyme. A timeline for formation of condensations for the endoskeleton of these two species is now available (Gillis et al., 2012a). Grande and Bemis (1991) provided a detailed assessment of the osteology and phylogeny of paddlefish. C. B. Kimmel et al. (1998) investigated the shaping of the simple columnar visceral arch cartilages in zebrafish and mutations that deform the stacking of chondroblasts into columns. See Power et al. (1999) and Helms et al. (1994) for these two studies with limb buds. Earlier, Morgan et al. (1992) showed that retroviral vectors containing Hoxd11 (Hox4.6) expand the Hoxd11 domain in chick limb buds more anteriorly and initiate posterior limb skeletal patterns in the anterior limb bud in a homeotic transformation. See Piotrowski et al. (1996), Schilling et al. (1996b) and Andreeva et al. (2011) for craniofacial mutants, and Schilling et al. (1996a) for

316 PART | VI Embryonic Origins

chinless. A fish gene named chinless causes us to ask what is a chin and begs Stephen J. Gould’s conclusion that chins are spandrels. 20. See Blaxter (1987), Webb and Shirey (*2003), Pieper et al. (2011), Schlosser (*2010), the special journal issue to which Schlosser (2012) provides an introduction, and Webb (2014a,b) for overviews of the lateral-line system. See Adriaens et al. (1997) for a detailed analysis of canal bone development in a single species, the African sharp tooth catfish, Clarias gariepinus, and see Webb et al. (2014) for a comparison between two species of cichlids from Lake Malawi. See Hanken and Hall (*1993) for relationship between lateral lines and bone induction. See Lannoo (*1988) for neuromasts in anuran and urodele amphibians with comments on caecilians and the possible relationship of neuromasts to bone induction. 21. See de Beer (1937, reissued 1985), especially pp. 6, 489 490 and 508, agenda items ii.6 (p. 513) and iii.10 12 (p. 514), and the preface to the reissue by Hall and Hanken (1985b). See Northcutt (1996), Webb and Shirey (2003), and Schlosser (*2010) for reviews of placodes, neuromasts and lateral line development, and see S. C. Smith et al. (1994) for developmental and evolutionary links between placodal ectoderm and neural crest cells. 22. See Westoll (1941) and Pinganaud-Perrin (1973) for independence of teleost dermal bones from neuromasts, and (Jollie, *1984) for

23.

24.

25.

26.

head development in Amia calva, including relation of lateral-line canals to bones. For development and function of the frontal bones in the cichlid Astatotilapia elegans, see Verraes and Ismail (1980). See Meinke (1986) for the dermal skeleton in lungfish, including neuromasts and the timing of tissue interactions. See the chapters and bibliography of papers on lungfishes in Bemis et al. (1987), and Bartsch (1994) for the development of the cranium in the Queensland lungfish, Neoceratodus. For other discussions of the possibility of such interactions, see Patterson (1977), Schaeffer (1977), Graham-Smith (1978) and Northcutt and Gans (1983). See Patterson (1977) for a general discussion, and see Holmgren (*1943) and Stensio (1947), especially for sharks and rays, including skull and lateral-line development and neural crest origins. See Yamamoto et al. (2003) and Dufton et al. (2012) for the skeletal changes in Astyanax, Strickler et al. (2002), Jeffery et al. (2003) and Franz-Odendaal and Hall (2006a) for an overview of how the eyes are lost and the lateral line and taste buds expanded in this blind cave fish, and Re´taux and Casane (2013) for an overview of adaptations to cave life in various lineages of animals.