Accurate staging of chick embryonic tissues via deep learning of salient features.

Recent work shows that the developmental potential of progenitor cells in the HH10 chick brain changes rapidly, accompanied by subtle changes in morphology. This demands increased temporal resolution for studies of the brain at this stage, necessitating precise and unbiased staging. Here, we investigated whether we could train a deep convolutional neural network to sub-stage HH10 chick brains using a small dataset of 151 expertly labelled images.

Fgf signalling triggers an intrinsic mesodermal timer that determines the duration of limb patterning.

Complex signalling between the apical ectodermal ridge (AER - a thickening of the distal epithelium) and the mesoderm controls limb patterning along the proximo-distal axis (humerus to digits). However, the essential in vivo requirement for AER-Fgf signalling makes it difficult to understand the exact roles that it fulfils. To overcome this barrier, we developed an amenable ex vivo chick wing tissue explant system that faithfully replicates in vivo parameters.

A neuroepithelial wave of BMP signalling drives anteroposterior specification of the tuberal hypothalamus.

The tuberal hypothalamus controls life-supporting homeostatic processes, but despite its fundamental role, the cells and signalling pathways that specify this unique region of the central nervous system in embryogenesis are poorly characterised. Here, we combine experimental and bioinformatic approaches in the embryonic chick to show that the tuberal hypothalamus is progressively generated from hypothalamic floor plate-like cells. Fate-mapping studies show that a stream of tuberal progenitors develops in the anterior-ventral neural tube as a wave of neuroepithelial-derived BMP signalling sweeps from anterior to posterior through the hypothalamic floor plate.

Retinoic acid influences the timing and scaling of avian wing development.

A fundamental question in biology is how embryonic development is timed between different species. To address this problem, we compared wing development in the quail and the larger chick. We reveal that pattern formation is faster in the quail as determined by the earlier activation of 5'Hox genes, termination of developmental organizers (Shh and Fgf8), and the laying down of the skeleton (Sox9).

Establishing the pattern of the vertebrate limb.

The vertebrate limb continues to serve as an influential model of growth, morphogenesis and pattern formation. With this Review, we aim to give an up-to-date picture of how a population of undifferentiated cells develops into the complex pattern of the limb. Focussing largely on mouse and chick studies, we concentrate on the positioning of the limbs, the formation of the limb bud, the establishment of the principal limb axes, the specification of pattern, the integration of pattern formation with growth and the determination of digit number.

Sonic hedgehog specifies flight feather positional information in avian wings.

Classical tissue recombination experiments performed in the chick embryo provide evidence that signals operating during early limb development specify the position and identity of feathers. Here, we show that Sonic hedgehog (Shh) signalling in the embryonic chick wing bud specifies positional information required for the formation of adult flight feathers in a defined spatial and temporal sequence that reflects their different identities. We also reveal that Shh signalling is interpreted into specific patterns of and transcription factor expression, providing evidence of a putative gene regulatory network operating in flight feather patterning.

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

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 ()-expressing polarising region cells of the chick wing bud.

Development of the basal hypothalamus through anisotropic growth.

The adult hypothalamus is subdivided into distinct domains: pre-optic, anterior, tuberal and mammillary. Each domain harbours an array of neurones that act together to regulate homeostasis. The embryonic origins and the development of hypothalamic neurones, however, remain enigmatic.

Polarizing Region Tissue Grafting in the Chick Embryo Limb Bud.

The polarizing region of the developing limb bud is an important organizing center that is involved in anteroposterior (thumb to little finger) patterning and has three main functions that are now considered to depend on the secreted protein Sonic hedgehog (Shh). These are (1) specifying anteroposterior positional values by autocrine and graded paracrine signaling; (2) promoting growth in adjacent mesenchyme; (3) maintaining the distal epithelium that is essential for limb outgrowth by induction of a factor in adjacent mesenchyme. The polarizing region was identified using classical tissue grafting techniques in chicken embryos.

An intrinsic cell cycle timer terminates limb bud outgrowth.

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.

The chick limb: embryology, genetics and teratology.

The chick embryo has a long history in investigations of vertebrate limb development because of the ease with which its limbs can be experimentally manipulated. Early studies elucidated the fundamental embryology of the limb and identified the key signalling regions that govern its development. The chick limb became a leading model for exploring the concept of positional information and understanding how patterns of differentiated cells and tissues develop in vertebrate embryos.

progenitors give rise to the chick hypothalamus by rostral and caudal growth and differentiation.

Classical descriptions of the hypothalamus divide it into three rostro-caudal domains but little is known about their embryonic origins. To investigate this, we performed targeted fate-mapping, molecular characterisation and cell cycle analyses in the embryonic chick. Presumptive hypothalamic cells derive from the rostral diencephalic ventral midline, lie above the prechordal mesendoderm and express progenitors undergo anisotropic growth: those displaced rostrally differentiate into anterior cells, then those displaced caudally differentiate into mammillary cells.

Evolution of antero-posterior patterning of the limb: Insights from the chick.

The developing limbs of chicken embryos have served as pioneering models for understanding pattern formation for over a century. The ease with which chick wing and leg buds can be experimentally manipulated, while the embryo is still in the egg, has resulted in the discovery of important developmental organisers, and subsequently, the signals that they produce. Sonic hedgehog (Shh) is produced by mesenchyme cells of the polarizing region at the posterior margin of the limb bud and specifies positional values across the antero-posterior axis (the axis running from the thumb to the little finger).

Transcriptional changes in chick wing bud polarization induced by retinoic acid.

Background: Retinoic acid is implicated in the induction of the gene encoding Sonic hedgehog (Shh) that specifies anteroposterior positional values and promotes growth of the developing limb bud. However, because retinoic acid is involved in limb initiation, it has been difficult to determine if it could have additional roles in anteroposterior patterning. To investigate this, we implanted retinoic acid-soaked beads to the anterior margin of the chick wing bud and performed microarray analyses prior to onset of Shh expression.

Sonic Hedgehog Signaling in Limb Development.

The gene encoding the secreted protein Sonic hedgehog (Shh) is expressed in the polarizing region (or zone of polarizing activity), a small group of mesenchyme cells at the posterior margin of the vertebrate limb bud. Detailed analyses have revealed that Shh has the properties of the long sought after polarizing region morphogen that specifies positional values across the antero-posterior axis (e.g.

Intrinsic properties of limb bud cells can be differentially reset.

An intrinsic timing mechanism specifies the positional values of the zeugopod (i.e. radius/ulna) and then autopod (i.

The origins, scaling and loss of tetrapod digits.

Many of the great morphologists of the nineteenth century marvelled at similarities between the limbs of diverse species, and Charles Darwin noted these homologies as significant supporting evidence for descent with modification from a common ancestor. Sir Richard Owen also took great care to highlight each of the elements of the forelimb and hindlimb in a multitude of species with focused attention on the homology between the hoof of the horse and the middle digit of man. The ensuing decades brought about a convergence of palaeontology, experimental embryology and molecular biology to lend further support to the homologies of tetrapod limbs and their developmental origins.

Inhibition of Shh signalling in the chick wing gives insights into digit patterning and evolution.

In an influential model of pattern formation, a gradient of Sonic hedgehog (Shh) signalling in the chick wing bud specifies cells with three antero-posterior positional values, which give rise to three morphologically different digits by a self-organizing mechanism with Turing-like properties. However, as four of the five digits of the mouse limb are morphologically similar in terms of phalangeal pattern, it has been suggested that self-organization alone could be sufficient. Here, we show that inhibition of Shh signalling at a specific stage of chick wing development results in a pattern of four digits, three of which can have the same number of phalanges.

An intrinsic timer specifies distal structures of the vertebrate limb.

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.

Sonic hedgehog-expressing cells in the developing limb measure time by an intrinsic cell cycle clock.

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).

Mathematical modelling of digit specification by a sonic hedgehog gradient.

Background: The three chick wing digits represent a classical example of a pattern specified by a morphogen gradient. Here we have investigated whether a mathematical model of a Shh gradient can describe the specification of the identities of the three chick wing digits and if it can be applied to limbs with more digits.

Results: We have produced a mathematical model for specification of chick wing digit identities by a Shh gradient that can be extended to the four digits of the chick leg with Shh-producing cells forming a digit.

Gradients of signalling in the developing limb.

The developing limb is one of the first systems where it was proposed that a signalling gradient is involved in pattern formation. This gradient for specifying positional information across the antero-posterior axis is based on Sonic hedgehog signalling from the polarizing region. Recent evidence suggests that Sonic hedgehog signalling also specifies positional information across the antero-posterior axis by a timing mechanism acting in parallel with graded signalling.

Insights into bird wing evolution and digit specification from polarizing region fate maps.

The proposal that birds descended from theropod dinosaurs with digits 2, 3 and 4 was recently given support by short-term fate maps, suggesting that the chick wing polarizing region-a group that Sonic hedgehog-expressing cells-gives rise to digit 4. Here we show using long-term fate maps that Green fluorescent protein-expressing chick wing polarizing region grafts contribute only to soft tissues along the posterior margin of digit 4, supporting fossil data that birds descended from theropods that had digits 1, 2 and 3. In contrast, digit IV of the chick leg with four digits (I-IV) arises from the polarizing region.

3D expression patterns of cell cycle genes in the developing chick wing and comparison with expression patterns of genes implicated in digit specification.

Sonic hedgehog (Shh) signalling controls integrated specification of digit pattern and growth in the chick wing but downstream gene networks remain to be unravelled. We analysed 3D expression patterns of genes encoding cell cycle regulators using Optical Projection Tomography. Hierarchical clustering of spatial matrices of gene expression revealed a dorsal layer of the wing bud, in which almost all genes were expressed, and that genes encoding positive cell cycle regulators had similar expression patterns while those of N-myc and CyclinD2 were distinct but closely related.

Expression of E2F transcription factor family genes during chick wing development.

The E2F family of transcriptional regulators activate or repress gene expression during specific phases of the cell cycle and control various processes including proliferation, apoptosis and differentiation. However, little is known about the developmental roles of E2F transcription factors in higher vertebrates. The chick wing is an excellent system for studying these processes because, in addition to having a rich classical embryology, it is increasingly amenable to molecular and genomic approaches.