Marco conceptual

3.1 INTRODUCTION

In this study, connexin expression and functional gap junctional communication were examined during a particular window in development when epidermal histogenesis has just commenced, and an instructive epidermis, in this case the apical ectodermal ridge

(AER), is also present. This permitted simultaneous analysis of gap junctional

communication between epithelial cells in functionally different tissue types: instructive and non-instructive epidermis

Ectoderm that does not receive signals from the Spemann organizer during gastrulation becomes epidermis. It has been demonstrated that epidermal specification depends on local signalling and that neural tissue forms when this communication is blocked. A putative epidermal inducer and neural inhibitor is the activin relative BMP-4 (Wilson and Hemmati-Brivanlou, 1995).

Reciprocal epithelial-mesenchymal interactions are thought to be a universal

phenomenon regulating epidermal and mesenchymal growth and differentiation (e.g. see Smola et af, 1993). During embryonic development, some regions of the epidermis form specialized instructive epithelia which are thought to play a specific role in establishing the correct outgrowth and morphogenesis of the mesenchyme which underlies it; examples include the interactions between the apical ectodermal ridge (AER) and mesenchyme of the limb bud, epidermis and mesoderm of the facial primordium and odontogenic placode epithelium amd mesenchyme of the presumptive jaws

Epidermal histogenesis occurs throughout gestation and begins when the single-cell- layered surface ectoderm begins to differentiate into a peridermal and epidermal

(Stratum (S) basale) bilayer at about El 2. The periderm forms a single layer of surface cells (Nakamura and Yasuda, 1979) which persists throughout gestation in rats

(Bonneville, 1968), whilst the underlying epidermis develops into a complex stratified squamous epithelium (E15-E20). This highly organized epithelial structure results from progressive differentiation and stratification of epidermal tissue. The S. basale undergoes epidermal stratification giving rise to an intermediate layer, the S.

intermedium, which forms between the basal layer and periderm at E l 4-El 7. The S.intermedium is a transient layer and by E l 8 has subsequently differentiated into spiny cells of the S. spinosum. At this time some of the spiny cells further differentiate into cells containing keratohyalin granules to form another layer, the S. granulosum. Finally, as birth approaches, a superficial comified layer, the S. comeum, forms under the periderm and the periderm is subsequently shed. The main stages comprising rat epidermal histogenesis are illustrated diagramatically in figure 3 .1, but for a more detailed description see Hanson, 1947.

In the vertebrate embryo, somatopleural cells in the limb-forming region are released from the mesodermal layer and undergo outgrowth from the flank mesoderm,

ultimately forming the limb bud. Briefly, limb bud patterning is greatly influenced by an area of mesenchyme at the limb bud posterior margin called the 'zone of polarizing activity' (ZPA) which defines structures of the limb bud in the anteroposterior (A-P) axis. A second signal from the AER maintains, at the tip of the limb bud, an area of undifferentiated cells called the progress zone which is acted on by the ZPA giving rise to limb bud patterning in a proximodistal (P-D) sequence.

The initial induction and maintenance of the AER appears to be a result of underlying mesodermal interactions with flank epidermis in regions which will form the limb bud. In rat the AER is a thickened layer of stratified cuboidal epithelial cells at the tip of the developing limb bud. It appears to be approximately 80-100|im (approx. 10 cells) in thickness (P-D plane) and width (D-V plane). The AER is thought to exert its effects

Fig. 3.1. Stages in rat epidermal histogenesis STAGE A(E8-E11) T i. r UNILAYERED ECTODERIS MESENCHYME STAGE B (E12-E14) T * ^ ‘ PERIDERM S.BASALE STAGE C (E14-E17) STAGE D (E18-E20) PERIDERM S.INTERMEDIUM S.BASALE MESENCHYME SCORNEUM S.GRANULOSUM S. SPINOSUM S.BASALE

* S.Comeum forms beneath the peridermal layer at E20 and the periderm is subsequently shed.

by releasing certain instructive signalling molecules to the underlying mesoderm. Members o f the fibroblast growth factor (FGF) family such as FGF-2 ( Cohn et al.,

1995), FGF-4 (Niswander and Martin, 1993; Cohn et al., 1995) and FGF-1 (Cohn et al., 1995), appear to mimic the actions of the AER. However, FGF-4 is believed to be the endogenous FGF since its mRNA is localized to the posterior AER (Vogel and Tickle, 1993). It has recently been demonstrated that sonic hedgehog {Shh) is the endogenous polarizing signal and that FGF-4 is regulated by Shh and vice-versa, establishing a positive feedback loop between the AER and ZPA (see Niswander et al., 1994). Figure 3 .2a-b are photographs showing the different stages in limb bud

development which were studied and figure 3.2c shows a bright-field image of a whole hindlimb bud. The structure of the limb bud is shown schematically in figure 3 .3.

Fig. 3.2. Illustrates the different embryonic stages used to examine connexin expression in the limb bud.

A. Different stages in limb bud development shown in whole embryos at E l 1, E12 and E13. Scale bar=2mm.

B. Different stages in limb bud development shown in eviscerated embryos at E l l , E 12 and E l 3 pinned out on a sylgard-coated culture dish.

The hindlimb bud is often very difficult to see in E l l preparations as it curls under the body. Scale bar=2mm.

F=forelimb, H=hindlimb.

C. An individual hindlimb bud visualized free-floating in culture medium (A) and lightly compressed under a coverslip to reveal the AER (B). (A) Scale bar=200pm (B) Scale bar= 100|im.

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Fig. 3.3. Structure of the limb bud