DIAGNÓSTICO DE LA SEGURIDAD, LA VIOLENCIA Y EL DELITO EN EL PERÚ

a) Expression patterns of BMP-2 and 4 in the limb:

As well as their role in inducing bone formation, it has also been found that, in mice, the Bmp genes, especially Bmp-2 and 4, are expressed in the developing limb, long before bone formation.

Work in Brigid Hogan's laboratory (Lyons et al., 1990; Jones et a i, 1991) had previ­ ously shown that, in the mouse, BMP-2 and 4 are expressed in the AER and mesoderm in early limb development. The chick cDNAs of BMP-2 and 4 were cloned in our laboratory and their mRNA expression has been localised to several different areas of the developing chick limb by in situ hybridisation (Francis et al, 1994) (Figure 1.20). Bmp-2 transcripts are not detectable in the wing area until stage 16/17, and then, be­ tween stages 17 and 26, Bmp-2 expression is restricted to the AER and a posterior region of the mesoderm, which co-localises with the polarising region. Later, at stages 25 and 26, there is also an additional domain of expression in the anterior proximal part of the limb bud; and then at later stages, it is expressed in the interdigital necrotic zone and around the cartilage elements.

The pattern of Bmp-4 expression is very similar to that of Bmp-2 in the AER, but transcripts are present throughout the wing mesenchyme at stages 16 to 17, and then expression becomes progressively restricted between stages 19 and 24, so that tran­ scripts are found in two regions at the anterior and posterior margins of the limb bud, and in the Progress Zone. Bmp-4 also is expressed interdigitally and around the carti­ lage elements at later stages.

BMP-4 expression overlaps that of BMP-2 in some areas so there may be some co­ operation between BMP-2 and 4, possibly involving the formation of heterodimers.

The distribution of Bmp-2 and Bmp-4 transcripts in the developing limb suggests that BMP-2 and BMP-4 could play important roles in limb development. Both genes are expressed in the AER, and could be involved in maintaining the cells in the Progress Zone as undifferentiated cells (Lyons et a l, 1990; Jones et a l, 1991; Niswander and

Martin, 1993). Equally, both genes are expressed in chondrogenic regions in the later bud, suggesting a role in cartilage differentiation and morphogenesis (Lyons et al,

1989). This raises the possibility that, like the related dpp gene in Drosophila, which has been shown to act at several different stages in development (Ferguson and Ander­ son, 1992; Heberlein et al, 1993), the BMPs may be necessary for both initial pattern­ ing and for later skeletal morphogenesis in the limbs.

b) Interactions of BMPs with Retinoic Acid and H ox Gene Expression:

The co-localisation of BMP-2 expression with the ZPA also raises the possibility that BMP-2 may be one of the molecules involved in the specification of the antero­ posterior axis, in the polarising region signalling pathway.

For example, if a RA-soaked bead is implanted into the limb such that is duplicates the ZPA activity and causes digit duplication, a mirror-image ectopic domain of BMP-2 expression forms as well (Francis et al, 1994) (Figure 1.21). The Bmp-2 gene is there­ fore probably part of the response component of the polarising region signalling path­ way, but BMP-2 is not itself a polarising molecule, as beads soaked in recombinant human BMP-2 protein, at concentrations of 0.2 to 2 mg ml \ do not cause the forma­ tion of additional digits (Francis et al, 1994).

RA treatment also causes the formation of an ectopic region of Hox-dlS expression at approximately the same time and place as BMP-2 (See section 1.6 above; Figure 1.21). The normal domain of posterior expression of BMP-2 also overlaps considerably with that of Hox-dl3 (Izpisua-Belmonte et al, 1991). This overlap is maintained throughout limb development and ectopic expression of Bmp-2 can induce Hoxd-11 and d-13 expression in the developing chick limb (Duprez et al, in press).

There is evidence that other members of the BMP family can activate homeobox genes too: DPP, synthesised by mesoderm, activates expression of the homeotic gene, labial, in neighbouring ectoderm in Drosophila (Panganiban et al, 1990a), and, in developing teeth, BMP-2 and 4 can induce Msx-1 expression, a homeobox-containing gene.

A,

m !

Bmp- 2

Hoxd -13

_Bmp-4

F igu re 1.21: Ectopic activation o f Bmp-2 and Hoxd-13 genes following RA treatment. Ectopic expression o f A. Bmp-2, B. Hoxd-13, and C. Bmp-4 is shown by the arrow in each panel.

homologue of the msh (muscle segment homeobox) gene of Drosophila (Vainio et al, 1993). In limb buds stripped of the AER and cultured (Niswander and Martin, 1993a and 1993b), BMP-2 and FGF-4 co-ordinately regulate the expression of Evx~l, the murine homologue of the Drosophila even-skipped homeotic gene, which is normally expressed in the posterior Progress Region mesenchyme shortly after formation of the AER (Bastian and Gmss, 1990). BMP-2 on its own will not stimulate ectopic anterior expression of Evx-1, but 10 ng/ml of BMP-2 + lOOng/ml FGF-4 will switch on ectopic Evx-1, whereas in the presence of 100 ng/ml BMP-2 + 1 0 ng/ml FGF-4, no expression is seen. In this culture system, BMP-2 has also been shown to affect outgrowth of the limb, in concert with FGF: FGF stimulates outgrowth of the limb, and BMP-2 inhibits outgrowth (Niswander and Martin, 1993b). At 50 ng/ml of applied BMP-2, Evx-1 expression is activated, but outgrowth of the limb bud is inhibited.

BMP-2 could also function in the polarising region signalling pathway in concert with BMP-4 or 7. The Bmp-2 domain overlaps with that of Bmp-4 and Bmp-7 in the poste­ rior mesenchyme of normal limb buds, and in the RA-treated limbs, the ectopic domain of Bmp-2 also overlaps with the anterior expression domain of Bmp-4 (Francis-West et al, 1994). Members of the TGF-P family, including BMPs, have been shown to form both homodimers and heterodimers (Sampath et al., 1990). BMP-2 and 4 and 7 might therefore act as homo- and/or heterodimers in the developing limb. This introduces further complexity because homodimers and heterodimers can have opposing effects, as in the case of the inhibins (Hsueh et al., 1987). However, the distribution of Bmp-2 and 4 transcripts may not accurately reflect the distribution of active protein, since there is the possibility of further control at the levels of translation, secretion and prote­ olytic activation after secretion, and no specific antibodies against individual BMPs have been made (reviewed by Wozney, 1989).

RA has also been shown to affect Bmp gene expression in cell lines. In F9 cells induced to differentiate with RA, Vgr-2 and Bmp-4 expression levels decrease, and Bmp-2 and Bmp-6 levels increase (Jones et al, 1992). In embryonic stem cells and carcinoma cells induced to differentiate with RA, endogenous BMP-2 levels increase 11-fold and BMP-4 decrease 12-fold (Rogers et al, 1992). TGF-P 1 and P2 expression also increases in these cells, but pi receptor levels decrease dramatically (Weima et al, 1989; Mum­ mery et al, 1990). In the C3H-10T1/2 mouse cell line, treatment with RA increases the

incidence of osteoblast differentiation. These cells normally express Bmp-2 and Bmp-4, and low levels of Bmp-6: upon RA treatment, Bmp-2 and 4 expression decreases tran­ siently (4 days) and Bmp-6 expression increases (Gazit et al, 1993). Recombinant BMP-2 can also stimulate osteoblastic maturation and inhibit myogenic differentiation in rat osteoblast cell lines (Yamaguchi et al, 1991).

c) Similarities with Insect Models of Development:

Insights into the possible roles of Bmp-2 and 4 in vertebrates may also be gained by looking at the roles of their Drosophila homolog, dpp.

dpp has been shown to act as a morphogen in at least two systems in development in Drosophila: the eye, and the developing gut, as well as in specifying dorsal ectodermal cell fates (Heberlein et al, 1993).

In the retina in Drosophila, a wave of differentiation passes across the eye disc over a 2 day period, hh has been shown to induce dpp expression, which appears to be a primary mediator of movement of the furrow, marking differentiation (Heberlein et al, 1993). Both of these genes act as diffusible signals in this process: differentiating cells poste­ rior to the furrow produce HH protein which diffuses anteriorly and mediates induction of dpp expression in the furrow (Heberlein et al, 1993).

dpp is also expressed in parasegments 4 and 7 of the embryonic visceral mesoderm (Hursh et al, 1993). Using an antibody specific to DPP (Panganiban et al, 1990b), dpp was found to be secreted and to influence the expression of different homeobox genes involved in the interaction between the outer mesodermal and inner endodermal layers of the midgut. DPP and wingless act in adjacent parasegments as the link between the mesoderm and the endoderm layers (Immerglück et al, 1990; Panganiban et al, 1990a; Hursh et al, 1993; Staehling-Hampton et al, 1994a). It is thought that Ultrabithorax expression in the mesoderm of parasegment 7, directly or indirectly switches on dpp expression (Reuter et al, 1990), DPP protein then moves locally to the endodermal layer where it elicits expression of the homeobox labial, in collaboration with wingless. Therefore dpp functions apparently both upstream and downstream of homeobox

genes. In addition, Ubx and abd-A homeotic genes and dpp are needed to form the second midgut constriction (Capovilla et al, 1994). Capovilla et al (1994) isolated an enhancer element in dpp controlling this expression, which contains binding sites for UBX and ABD-A. UBX directly switches on DPP activity, and ABD-A represses it (Capovilla et al, 1994).