Dual origins of the prechordal cranium in the chicken embryo

The prechordal cranium, or the anterior half of the neurocranial base, is a key structure for understanding the development and evolution of the vertebrate cranium, but its embryonic configuration is not well understood. It arises initially as a pair of cartilaginous rods, the trabeculae, which have been thought to fuse later into a single central stem called the trabecula communis (TC). Involvement of another element, the intertrabecula, has also been suggested to occur rostral to the trabecular rods and form the medial region of the prechordal cranium. Here, we examined the origin of the avian prechordal cranium, especially the TC, by observing the craniogenic and precraniogenic stages of chicken embryos using molecular markers, and by focal labeling of the ectomesenchyme forming the prechordal cranium. Subsequent to formation of the paired trabeculae, a cartilaginous mass appeared at the midline to connect their anterior ends. During this midline cartilage formation, we did not observe any progressive medial expansion of the trabeculae. The cartilages consisted of premandibular ectomesenchyme derived from the cranial neural crest. This was further divided anteroposteriorly into two portions, derived from two neural crest cell streams rostral and caudal to the optic vesicle, called preoptic and postoptic neural crest cells, respectively. Fate-mapping analysis elucidated that the postoptic neural crest cells were distributed exclusively in the lateroposterior part of the prechordal cranium corresponding to the trabeculae, whereas the preoptic stream of cells occupied the middle anterior part, differentiating into a cartilage mass corresponding to the intertrabecula. These results suggest that the central stem of the prechordal cranium of gnathostomes is composed of two kinds of distinct cartilaginous modules: a pair of trabeculae and a median intertrabecula, each derived from neural crest cells populating distinct places of the craniofacial primordia through specific migratory pathways.     

Reassessment of Isl1 and Nkx2-5 cardiac fate maps using a Gata4-based reporter of Cre activity

Isl1 and Nkx2-5-expressing cardiovascular progenitors play pivotal roles in cardiogenesis. Previously reported Cre-based fate-mapping studies showed that Isl1 progenitors contribute predominantly to the derivatives of the second heart field, and Nkx2-5 progenitors contributed mainly to the cardiomyocyte lineage. However, partial recombination of Cre reporter genes can complicate interpretation of Cre fate-mapping experiments. We found that a Gata4-based Cre-activated reporter was recombined by Isl1^Cre and Nkx2-5^Cre in a substantially broader domain than previously reported using standard Cre-activated reporters. The expanded Isl1 and Nkx2-5 cardiac fate maps were remarkably similar, and included extensive contributions to cardiomyocyte, endocardial, and smooth muscle lineages in all four cardiac chambers. These data indicate that Isl1 is expressed in progenitors of both primary and secondary heart fields, and that Nkx2-5 is expressed in progenitors of cardiac endothelium and smooth muscle, in addition to cardiomyocytes. These results have important implications for our understanding of cardiac lineage diversification in vivo, and for the interpretation of Cre-based fate maps.     

Patterning of the heart field in the chick

In human development, it is postulated based on histological sections, that the cardiogenic mesoderm rotates 180° with the pericardial cavity. This is also thought to be the case in mouse development where gene expression data suggests that the progenitors of the right ventricle and outflow tract invert their position with respect to the progenitors of the atria and left ventricle. However, the inversion in both cases is inferred and has never been shown directly. We have used 3D reconstructions and cell tracing in chick embryos to show that the cardiogenic mesoderm is organized such that the lateralmost cells are incorporated into the cardiac inflow (atria and left ventricle) while medially placed cells are incorporated into the cardiac outflow (right ventricle and outflow tract). This happens because the cardiogenic mesoderm is inverted. The inversion is concomitant with movement of the anterior intestinal portal which rolls caudally to form the foregut pocket. The bilateral cranial cardiogenic fields fold medially and ventrally and fuse. After heart looping the seam made by ventral fusion will become the greater curvature of the heart loop. The caudal border of the cardiogenic mesoderm which ends up dorsally coincides with the inner curvature. Physical ablation of selected areas of the cardiogenic mesoderm based on this new fate map confirmed these results and, in addition, showed that the right and left atria arise from the right and left heart fields. The inversion and the new fate map account for several unexplained observations and provide a unified concept of heart fields and heart tube formation for avians and mammals.     

The evolution of developmental mechanisms

Over the past two to three decades, developmental biology has demonstrated that all multicellular organisms in the animal kingdom share many of the same molecular building blocks and many of the same regulatory genetic pathways. Yet we still do not understand how the various organisms use these molecules and pathways to assume all the forms we know today. Evolutionary developmental biology tackles this problem by comparing the development of one organism to another and comparing the genes involved and gene functions to understand what makes one organism different from another. In this review, we revisit a set of seven concepts defined by Lewis Wolpert (fate maps, asymmetric division, induction, competence, positional information, determination, and lateral inhibition) that describe the characters of many developmental systems and supplement them with three additional concepts (developmental genomics, genetic redundancy, and genetic networks). We will discuss examples of comparative developmental studies where these concepts have guided observations on the advent of a developmental novelty. Finally, we identify a set of evolutionary frameworks, such as developmental constraints, cooption, duplication, parallel and convergent evolution, and homoplasy, to adequately describe the evolutionary properties of developmental systems.     

Intrinsic differences among spatially distinct neural crest stem cells in terms of migratory properties, fate determination, and ability to colonize the enteric nervous system

We have systematically examined the developmental potential of neural crest stem cells from the enteric nervous system (gut NCSCs) in vivo to evaluate their potential use in cellular therapy for Hirschsprung disease and to assess differences in the properties of postmigratory NCSCs from different regions of the developing peripheral nervous system (PNS). When transplanted into developing chicks, flow-cytometrically purified gut NCSCs and sciatic nerve NCSCs exhibited intrinsic differences in migratory potential and neurogenic capacity throughout the developing PNS. Most strikingly, gut NCSCs migrated into the developing gut and formed enteric neurons, while sciatic nerve NCSCs failed to migrate into the gut or to make enteric neurons, even when transplanted into the gut wall. Enteric potential is therefore not a general property of NCSCs. Gut NCSCs also formed cholinergic neurons in parasympathetic ganglia, but rarely formed noradrenergic sympathetic neurons or sensory neurons. Supporting the potential for autologous transplants in Hirschsprung disease, we observed that Endothelin receptor B (Ednrb)-deficient gut NCSCs engrafted and formed neurons as efficiently in the Ednrb-deficient hindgut as did wild-type NCSCs. These results demonstrate intrinsic differences in the migratory properties and developmental potentials of regionally distinct NCSCs, indicating that it is critical to match the physiological properties of neural stem cells to the goals of proposed cell therapies.     

Phox2b function in the enteric nervous system is conserved in zebrafish and is sox10-dependent

Zebrafish lacking functional sox10 have defects in non-ectomesenchymal neural crest derivatives including the enteric nervous system (ENS) and as such provide an animal model for human Waardenburg Syndrome IV. Here, we characterize zebrafish phox2b as a functionally conserved marker of the developing ENS. We show that morpholino-mediated knockdown of Phox2b generates fish modeling Hirschsprung disease. Using markers, including phox2b, we investigate the ontogeny of the sox10 ENS phenotype. As previously shown for melanophore development, ENS progenitor fate specification fails in these mutant fish. However, in addition, we trace back the sox10 mutant ENS defect to an even earlier time point, finding that most neural crest cells fail to migrate ventrally to the gut primordium.     

Distinct populations of endoderm cells converge to generate the embryonic liver bud and ventral foregut tissues

The location and movement of mammalian gut tissue progenitors, prior to the expression of tissue-specific genes, has been unknown, but this knowledge is essential to identify transitions that lead to cell type specification. To address this, we used vital dyes to label exposed anterior endoderm cells of early somite stage mouse embryos, cultured the embryos into the tissue bud phase of development, and determined the tissue fate of the dye labeled cells. This approach was performed at three embryonic stages that are prior to, or coincident with, foregut tissue patterning (1-3 somites, 4-6 somites, and 7-10 somites). Short-term labeling experiments tracked the movement of tissue progenitor cells during foregut closure. Surprisingly, we found that two distinct types of endoderm-progenitor cells, lateral and medial, arising from three spatially separated embryonic domains, converge to generate the epithelial cells of the liver bud. Whereas the lateral endoderm-progenitors give rise to descendants that are constrained in tissue fate and position along the anterior-posterior axis of the gut, the medial gut endoderm-progenitors give rise to descendants that stream along the anterior-posterior axis at the ventral midline and contribute to multiple gut tissues. The fate map reveals extensive morphogenetic movement of progenitors prior to tissue specification, it permits a detailed analysis of endoderm tissue patterning, and it illustrates that diverse progenitor domains can give rise to individual tissue cell types.     

Mother-daughter precursor cell fate transformation after Cdc2 down-regulation in the Drosophila bristle lineage

The Drosophila bristle lineage is an excellent system in which to study how cell cycle and fate determination are synchronized in invariant cell lineages. In this model, five different cells arise from a single precursor cell, pI, after four asymmetric cell divisions. Cell diversity is achieved by the asymmetric segregation of cell determinants, such as Numb and Neuralized (Neur), resulting in differential activation of the Notch (N) pathway. We show that down-regulation of Cdc2, by over-expressing Tribbles, Dwee1, and Dmyt1 (three negative regulators of Cdc2) or by using thermo-sensitive Cdc2 mutant flies, delayed pI mitosis, and altered the polarity and the number of subsequent cell divisions. These modifications were associated with a mother-daughter cell fate transformation as the pI cell acquired the identity of the secondary precursor cell, pIIb. This type of change in cell identity only occurred when the N signaling pathway was inactive since ectopic N signaling transformed pI to pIIa-progeny fate. These transformations in cell identity suggest that, although synchronized, cell cycle and fate determination are independent phenomena in the bristle lineage.     

A new origin for the maxillary jaw

One conserved feature of craniofacial development is that the first pharyngeal arch has two components, the maxillary and mandibular, which then form the upper and lower jaws, respectively. However, until now, there have been no tests of whether the maxillary cells originate entirely within the first pharyngeal arch or whether they originate in a separate condensation, cranial to the first arch. We therefore constructed a fate map of the pharyngeal arches and environs with a series of dye injections into stage 13-17 chicken embryos. We found that from the earliest stage examined, the major contribution to the maxillary bud is from post-optic mesenchyme with a relatively minor contribution from the maxillo-mandibular cleft. Cells labeled within the first pharyngeal arch contributed exclusively to the mandibular prominence. Gene expression data showed that there were different molecular codes for the cranial and caudal maxillary prominence. Two of the genes examined, Rarbeta (retinoic acid receptor beta) and Bmp4 (bone morphogenetic protein) were expressed in the post-optic mesenchyme and epithelium prior to formation of the maxillary prominence and then were restricted to the cranial half of the maxillary prominence. In order to determine the derivatives of the maxillary prominence, we performed focal injections of CM-DiI into the stage 24 maxillary prominence. Labeled cells contributed to the maxillary, palatine, and jugal bones, but not the other elements of the upper beak, the premaxilla and prenasal cartilage. We also determined that the cranial cells give rise to more distal parts of the upper beak, whereas caudal cells form proximal structures. Grafts of stage 24 maxillary prominences were also analyzed to determine skeletal derivatives and these results concurred with the DiI maps. These early and later fate maps indicate that the maxillary prominence and its skeletal derivatives are not derived from the first pharyngeal arch but rather from a separate maxillary condensation that occurs between the eye and the maxillo-mandibular cleft. These data also suggest that during evolution, recession of the first pharyngeal arch-derived palatoquadrate cartilage to a more proximal position gave way to the bony upper jaw of amniotes.