Defining the Earliest Transcriptional Steps of Chondrogenic Progenitor Specification during the Formation of the Digits in the Embryonic Limb

The characterization of genes involved in the formation of cartilage is of key importance to improve cell-based cartilage regenerative therapies. Here, we have developed a suitable experimental model to identify precocious chondrogenic events in vivo by inducing an ectopic digit in the developing embryo. In this model, only 12 hr after the implantation of a Tgfβ bead, in the absence of increased cell proliferation, cartilage forms in undifferentiated interdigital mesoderm and in the course of development, becomes a structurally and morphologically normal digit. Systematic quantitative PCR expression analysis, together with other experimental approaches allowed us to establish 3 successive periods preceding the formation of cartilage. The “pre-condensation stage”, occurring within the first 3 hr of treatment, is characterized by the activation of connective tissue identity transcriptional factors (such as Sox9 and Scleraxis) and secreted factors (such as Activin A and the matricellular proteins CCN-1 and CCN-2) and the downregulation of the galectin CG-8. Next, the “condensation stage” is characterized by intense activation of Smad 1/5/8 BMP-signaling and increased expression of extracellular matrix components. During this period, the CCN matricellular proteins promote the expression of extracellular matrix and cell adhesion components. The third period, designated the “pre-cartilage period”, precedes the formation of molecularly identifiable cartilage by 2–3 hr and is characterized by the intensification of Sox 9 gene expression, along with the stimulation of other pro-chondrogenic transcription factors, such as HifIa. In summary, this work establishes a temporal hierarchy in the regulation of pro-chondrogenic genes preceding cartilage differentiation and provides new insights into the relative roles of secreted factors and cytoskeletal regulators that direct the first steps of this process in vivo

Lineage and pluripotentiality of epithelial precursor cells in developing chicken skin

How do epithelial cells in developing skin accommodate the constantly growing embryo? Where do cells in skin appendages come from? Are they derivatives of a single appendage stem cell, or are they polyclonal? Here we analyze these issues in developing chicken skin using a replication-defective virus carrying β-galactosidase and DiI microinjections. The results demonstrate that in early skin, epithelial cells labelled near the spine show a parallel linear stripe distribution pattern that is perpendicular to the midline of the trunk. This is similar to the human lines of Blaschko, a linear pattern on the skin, which many skin nevoid or acquired disorders follow. In later skin, feather buds form and contain a mixture of labeled and unlabeled cells, attesting to their polyclonal origin. When cells are traced for shorter time intervals, the labeled progeny appear to follow certain rules. The degree of cell dispersion and mixing increases with a longer incubation period between the time of labeling and detection. The spatial maturation sequence of skin appendages is not regulated by the order in which epithelial cells are generated. Epithelial cells at this developmental stage are pluripotent and competent to respond to new signals to assume appropriate fates according to their micro-environment. The results suggest that local interactions act upon the originally linearly deposited pluripotential epithelial cells to form skin appendages.link_to_subscribed_fulltex

Self-organization of periodic patterns by dissociated feather mesenchymal cells and the regulation of size, number and spacing of primordia

Periodic patterning is a fundamental organizing process in biology. Using a feather reconstitution assay, we traced back to the initial stage of the patterning process. Cells started from an equivalent state and self-organized into a periodic pattern without previous cues or sequential propagation. When different numbers of dissociated mesenchymal cells were confronted with a piece of same-sized epithelium, the size of feather primordia remained constant, not the number or interbud spacing, suggesting size determination is intrinsic to dissociated cells. Increasing bone morphogenetic protein (BMP) receptor expression in mesenchymal cells decreased the size of primordia while antagonizing the BMP pathway with Noggin increased the size of primordia. A threshold number of mesenchymal cells with a basal level of adhesion molecules such as NCAM were sufficient to trigger the patterning process. The process is best visualized by the progressive restriction of β-catenin transcripts in the epidermis. Therefore, feather size, number and spacing are modulated through the available morphogen ligands and receptors in the system.link_to_subscribed_fulltex

Sonic hedgehog signaling pathway in vertebrate epithelial appendage morphogenesis: Perspectives in development and evolution

Vertebrate epithelial appendages are elaborate topological transformations of flat epithelia into complex organs that either protrude out of external (integument) and internal (oral cavity, gut) epithelia, or invaginate into the surrounding mesenchyme. Although they have specific structures and diverse functions, most epithelial appendages share similar developmental stages, including induction, morphogenesis, differentiation and cycling. The roles of the SHH pathway are analyzed in exemplary organs including feather, hair, tooth, tongue papilla, lung and foregut. SHH is not essential for induction and differentiation, but is involved heavily in morphogenetic processes including cell proliferation (size regulation), branching morphogenesis, mesenchymal condensation, fate determination (segmentation), polarizing activities and so on. Through differential activation of these processes by SHH in a spatiotemporal-specific fashion, organs of different shape and size are laid down. During evolution, new links of developmental pathways may occur and novel forms of epithelial appendages may emerge, upon which evolutionary selections can act. Sites of major variations have progressed from the body plan to the limb plan to the epithelial appendage plan. With its powerful morphogenetic activities, the SHH pathway would likely continue to play a major role in the evolution of novel epithelial appendages.link_to_subscribed_fulltex

Local inhibitory action of BMPs and their relationships with activators in feather formation: Implications for periodic patterning

The formation of periodic patterns is fundamental in biology. Theoretical models describing these phenomena have been proposed for feather patterning; however, no molecular candidates have been identified. Here we show that the feather tract is initiated by a continuous stripe of Shh, Fgf- 4, and Ptc expression in the epithelium, which then segregates into discrete feather primordia that are more strongly Shh and Fgf-4 positive. The primordia also become Bmp-2 and Bmp-4 positive. Bead-mediated delivery of BMPs inhibits local feather formation in contrast with the activators, SHH and FGF4, which induce feather formation. Both FGF-4 and SHH induce local expression of Bmp-4, while BMP-4 suppresses local expression of both. FGF-4 also induces Shh. Based on these findings, we propose a model that involves (1) homogeneously distributed global activators that define the field, (2) a position-dependent activator of competence that propagates across the field, and (3) local activators and inhibitors triggered in sites of individual primordia that act in a reaction-diffusion mechanism. A computer simulation model for feather pattern formation is also presented.link_to_subscribed_fulltex

Integument pattern formation involves genetic and epigenetic controls: Feather arrays simulated by digital hormone models

Pattern formation is a fundamental morphogenetic process. Models based on genetic and epigenetic control have been proposed but remain controversial. Here we use feather morphogenesis for further evaluation. Adhesion molecules and/or signaling molecules were first expressed homogenously in feather tracts (restrictive mode, appear earlier) or directly in bud of inter-bud regions (de novo mode, appear later). They either activate or inhibit bud formation, but paradoxically colocalize in the bud. Using feather bud reconstitution, we showed that completely dissociated cells can reform periodic patterns without reference to previous positional codes. The patterning process has the characteristics of being self-organizing, dynamic and plastic. The final pattern is an equilibrium state reached by competition, and the number and size of buds can be altered based on cell number and activator/inhibitor ratio, respectively. We developed a Digital Hormone Model which consists of (1) competent cells without identity that move randomly in a space, (2) extracellular signaling hormones which diffuse by a reaction-diffusion mechanism and activate or inhibit cell adhesion, and (3) cells which respond with topological stochastic actions manifested as changes in cell adhesion. Based on probability, the results are cell clusters arranged in dots or stripes. Thus genetic control provides combinational molecular information which defines the properties of the cells but not the final pattern. Epigenetic control governs interactions among cells and their environment based on physical-chemical rules (such as those described in the Digital Hormone Model). Complex integument patterning is the sum of these two components of control and that is why integument patterns are usually similar but non-identical. These principles may be shared by other pattern formation processes such as barb ridge formation, fingerprints, pigmentation patterning, etc. The Digital Hormone Model can also be applied to swarming robot navigation, reaching intelligent automata and representing a self-re-configurable type of control rather than a follow-the-instruction type of control.link_to_subscribed_fulltex