Role of the apical ectodermal ridge in shaping the limb bud

In mice, the apical ectodermal ridge is first evident around 11.5 dpc as a thickening of the epithelium running antero-posteriorly along the distal edge of the buds (Carter, 1954; Wanek et al, 1989). Loss of limb structures in vertebrate mutants are sometimes associated with a premature loss or lack of the apical ectodermal ridge. Mutations in the limb deformity (Id) gene disrupt differentiation of the apical ectodermal ridge and lead to the loss of distal limb structures (Zeller et ai., 1989), and lack of an apical ectodermal ridge in the mouse mutant legless (/g/)» association with an increase in mesenchymal cell death, leads to severe loss of hindlimb structures distal to the femur (Singh et aL,

1991). Induction and maintenance of the apical ectodermal ridge requires reciprocal interactions between the ectoderm and underlying mesenchyme. Ridge defects may therefore arise due to intrinsic defects in the ectoderm, or as a secondary effect of defects in the underlying mesenchyme. The H d mutation affects HoxalSy which is usually

expressed in the distal limb bud mesenchyme, and the presence of ridge defects in Hd/Hd mutants implies that these defects may be a secondary effect of the H d mutation.

4.2 Aims of this Chapter

The first section of this chapter details the distribution of mesenchymal cell death in developing limbs of Hd/Hd, Hd/+, and +/+ embryos at the developmental stages when morphological alterations in limb bud size and shape are first observed. The alterations in limb bud size and shape suggests that the amount of tissue and/or space available for digital development is reduced in Hd mutant limbs. The limb skeleton develops from mesenchymal condensations that ferm in the developing limb in a proximo-to-distal manner, such that proximal skeletal elements are specified prior to more distal ones. The Hd/Hd adult phenotype suggests that specification of proximal skeletal elements of the limb occurs normally, but that the specification of more distal elements are affected by the mutation. The second section of this chapter analyses the appearance of cartilage

rudiments in developing Hd/Hd, Hd/+ and +/+ limbs, and the results show that loss of digits is associated with lack of formation of mesenchymal condensations that give rise to digits. This suggests that the ability of Hd/Hd undifferentiated mesenchymal cells to undergo chondrogenic differentiation is altered. In order to test this, the third section of this chapter analyses the ability of undifferentiated mesenchymal cells to produce cartilage in vitro culture. TGFp, FGF2 and FGF4 are known to increase levels of chondrogenesis in culture, and the ability of Hd/Hd mesenchymal cells to respond to these inductive signals were tested. Finally, observations made under the light microscope suggests that Hd/Hd embryos may have an abnormal apical ectodermal ridge, and so the final section of this chapter discusses the results obtained from scanning e l e c t m i c r o s c o p y and ^ histological sections of Hd/Hd, Hd/+, and +/+ limbs. Results of this chapter are then

discussed in relation to what they tell us about the nature of the Hd mutation and the putative role of Hox genes during normal vertebrate limb development

4.3 Results

4.3.1 M esenchym al cell death in the developing limbs of H d m u tan ts

The early phase of mesenchymal cell death normally observed in developing limbs that is thought to influence size and shape of the developing limb bud, was analysed in Hd/Hd, Hd/+, and +/+ limbs at 11.5 -12.5 dpc by staining with vital dye Nile Blue Sulphate (NBS). A t 11.5 dpc, both forelimbs and hindlimbs of Hd/Hd embryos (11 embryos analysed) are less paddle-shaped than their +/+/Hd/+ littermates (Figure 4.1A and B). In addition, these limb buds possess an opaque patch of cells at the distal tip that appears very white in comparison to the rest of the limb bud. This is visible under the light microscope (compare A and B in Figure 4.1). NBS staining of these Hd/Hd embryos reveals that both forelimbs and hindlimbs have a large area of strong staining in posterior mesenchyme at the distal tip (Figure 4.2C and D). This strong staining represents

mesenchymal cell death, and correlates with the position of the opaque patch of cells. This massive distal mesenchymal cell death is not detected in either the forelimbs or hindlimbs of 11.5 dpc +/+/Hd/+ embryos (twenty-four embryos analysed; Figure 4.2 A and B).

A t 12.5 dpc, Hd/Hd limbs are clearly not paddle-shaped, and have a more pointed morphology than their +/+ and Hd/-\- littermates (Figure 4.1 C and D). In

addition, the opaque patch of cells first observed at 11.5 dpc, is still present at the distal tip of the limbs buds (Figure 4.1 D). This is not present in +/+ or Hd/+ limb buds (Figure 4.1 C). Hd/Hd limb buds show punctate mesenchymal cell death was detected throughout the developing hand and footplates of Hd/Hd embryos (6 embryos analysed), with strong NBS staining in the anterior and posterior distal mesenchyme (Figure 4.2 G, H, and K). There is also strong staining in the apical ectodermal ridge (Figure 4.2 K). A t this stage, developing limbs of Hd/+ and +/+ embryos exhibit two small regions of cell death in anterior and posterior mesenchyme of developing hand and footplates (Figure 4.2 E and F). However, in contrast to the pattern of cell death in +/+ footplates, the footplates of Hd/+ embryos which are reduced along the distal-anterior border (see chapter 3), have an increase in the anterior region of mesenchymal cell death (Figure 4.2 F). Cell death is detected in the apical ectodermal ridge of both +/+ and Hd/+ limbs at this stage (Figure

4.2 I and J)These regions of mesenchymal cell death in +/+ limbs have been previously identified as the anterior and posterior necrotic zones respectively (Milaire, 1992).

A t 14.5 dpc when mterdigital cell death is occurring, Hd/+ limbs have a strongly staining region of ceU death along the anterior border of the developing feet in addition to interdigital cell death (Figure 4.2 L and M). Mice showing the Hd/Hd limb phenotype at this stage have not been analysed.

Figure 4.1

Photographs of the hindlimbs of +/+/Hd/+, +/+ and Hd/Hd embryos at 11.5 and 12.5

dpc showing the shape of the hindlimb buds and the opaque patch of cells in H d /H d ^ ^ h buds that colocahse to regions where mesenchymal cell death is detected. (A): ventral view of a.+/+/Hd/+ 11.5 dpc hindlimb bud; (B): ventral view of d. Hd/Hd 11.5 dpc hindlimb bud indicating the region of cells that have a very white and opaque appearance (white arrows) in comparison to the rest of the bud and the appearance of +/+/Hd/+ buds; (C): dorsal view of a+/+ 12.5 dpc hindlimb bud; (D): ventral view of a Hd/Hd

12.5 dpc hindlimb bud indicating the region of cells that have a very white and opaque