An intrinsic cell cycle timer terminates limb bud outgrowth

Abstract

The longstanding view of how proliferative outgrowth terminates following the patterning phase of limb development involves the breakdown of reciprocal extrinsic signalling between the distal mesenchyme and the overlying epithelium (e-m signalling). However, by grafting distal mesenchyme cells from late stage chick wing buds to the epithelial environment of younger wing buds, we show that this mechanism is not required. RNA sequencing reveals that distal mesenchyme cells complete proliferative outgrowth by an intrinsic cell cycle timer in the presence of e-m signalling. In this process, e-m signalling is required permissively to allow the intrinsic cell cycle timer to run its course. We provide evidence that a temporal switch from BMP antagonism to BMP signalling controls the intrinsic cell cycle timer during limb outgrowth. Our findings have general implications for other patterning systems in which extrinsic signals and intrinsic timers are integrated.

Evolution of the avian digital pattern

“…Furthermore, in mouse embryos, inhibiting BMP signalling throughout limb buds by overexpressing Grem1 between E10.5 and E11.5 leads to the elongation of digits as well as the formation of both pre- and post-axial polydactylys 28 . In the limb buds, proliferation of mesenchymal cells terminates after the downregulation of Grem1 , and subsequently these cells undergo chondrogenic differentiation 17,29 . It is more likely that the avian digital pattern is regulated by the well-documented SHH/Gremlin1/AER-FGF feedback loops, in which Gremlin1 is the critical node linking each signalling module 10,14–17 , and both intraspecific and interspecific variation in the digital pattern can be recognized as the expression pattern of Grem1 in early limb buds.…”

An autoregulatory cell cycle timer integrates growth and specification in chick wing digit development

“…Cell cycle timers have been predicted in several developmental contexts (Newport and Kirschner, 1982; Durand and Raff, 2000; Burton et al, 1999; Primmett et al, 1989; Lewis, 1975). Recently, we provided evidence that a progressive increase in Bmp signalling is responsible for the slowing of the cell cycle (Pickering et al, 2018), which is linked to the acquisition of proximo-distal positional values in the distal mesenchyme of the chick wing bud (humerus to digit tips) (Saiz-Lopez et al, 2015). It is tempting to speculate that this could involve a D cyclin-dependent kinase inhibitor acting downstream of Bmp signalling, although it is unlikely to be p27 kip1 , which is only expressed in myogenic cells at later stages of chick wing development.…”

Establishing the pattern of the vertebrate limb

“…In the mouse, Grem1 expression is induced by Bmp4 and Shh signalling in the early bud (Benazet et al, 2009), and recent work in the chick (Pickering and Towers, 2016) and the mouse (Zhu et al, 2020 preprint) has shown that Grem1 expression becomes independent of Shh signalling following the early period of digit identity specification. In addition, grafts of distal mesoderm made between young and old chick wing buds have shown that the decline in the rate at which proliferation terminates proximo-distal outgrowth during the patterning phase is intrinsically controlled (Saiz-Lopez et al, 2015, and is associated with a progressive increase in Bmp signalling that overcomes Grem1 inhibition (Pickering et al, 2018) (Fig. 5).…”