How the positional values along the proximo-distal axis (stylopod-zeugopod-autopod) of the limb are specified is intensely debated. Early work suggested that cells intrinsically change their proximo-distal positional values by measuring time. Recently, however, it is suggested that instructive extrinsic signals from the trunk and apical ectodermal ridge specify the stylopod and zeugopod/autopod, respectively. Here, we show that the zeugopod and autopod are specified by an intrinsic timing mechanism. By grafting green fluorescent protein-expressing cells from early to late chick wing buds, we demonstrate that distal mesenchyme cells intrinsically time Hoxa13 expression, cell cycle parameters and the duration of the overlying apical ectodermal ridge. In addition, we reveal that cell affinities intrinsically change in the distal mesenchyme, which we suggest results in a gradient of positional values along the proximo-distal axis. We propose a complete model in which a switch from extrinsic signalling to intrinsic timing patterns the vertebrate limb.

How time is measured is an enduring issue in developmental biology. Classical models of somitogenesis and limb development implicated intrinsic cell cycle clocks, but their existence remains controversial. Here we show that an intrinsic cell cycle clock in polarizing region cells of the chick limb bud times the duration of Sonic hedgehog (Shh) expression, which encodes the morphogen specifying digit pattern across the antero-posterior axis (thumb to little finger). Timing by this clock starts when polarizing region cells fall out of range of retinoic acid signalling. We found that timing of Shh transcription by the cell cycle clock can be reset, thus revealing an embryonic form of self-renewal. In contrast, antero-posterior positional values cannot be reset, suggesting that this may be an important constraint on digit regeneration. Our findings provide the first evidence for an intrinsic cell cycle timer controlling duration and patterning activity of a major embryonic signalling centre.

Highlights d Early hypothalamic development was profiled in chick using scRNA-seq and HCR d Hypothalamic cells are induced from prethalamic-like cells d Distinct tuberal, mammillary, and paraventricular progenitor populations emerge later d Prethalamic-like progenitor-derived follistatin inhibits hypothalamic specification

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.

A fundamental question is how proliferation and growth are timed during embryogenesis. Although it has been suggested that the cell cycle could be a timer, the underlying mechanisms remain elusive. Here we describe a cell cycle timer that operates in Sonic hedgehog (Shh)-expressing polarising region cells of the chick wing bud. Our data are consistent with Shh signalling stimulating polarising region cell proliferation via Cyclin D2, and then inhibiting proliferation via a Bmp2-p27kip1 pathway. When Shh signalling is blocked, polarising region cells over-proliferate and form an additional digit, which can be prevented by applying Bmp2 or by inhibiting D cyclin activity. In addition, Bmp2 also restores posterior digit identity in the absence of Shh signalling, thus indicating that it specifies antero-posterior (thumb to little finger) positional values. Our results reveal how an autoregulatory cell cycle timer integrates growth and specification and are widely applicable to many tissues.