Graded potential of neural crest to form cornea, sensory neurons and cartilage along the rostrocaudal axis

Neural crest cells arising from different rostrocaudal axial levels form different sets of derivatives as diverse as ganglia, cartilage and cornea. These variations may be due to intrinsic properties of the cell populations, different environmental factors encountered during migration or some combination thereof. We test the relative roles of intrinsic versus extrinsic factors by challenging the developmental potential of cardiac and trunk neural crest cells via transplantation into an ectopic midbrain environment. We then assess long-term survival and differentiation into diverse derivatives, including cornea, trigeminal ganglion and branchial arch cartilage. Despite their ability to migrate to the periocular region, neither cardiac nor trunk neural crest contribute appropriately to the cornea, with cardiac crest cells often forming ectopic masses on the corneal surface. Similarly, the potential of trunk and cardiac neural crest to form somatosensory neurons in the trigeminal ganglion was significantly reduced compared with control midbrain grafts. Cardiac neural crest exhibited a reduced capacity to form cartilage, contributing only nominally to Meckle's cartilage, whereas trunk neural crest formed no cartilage after transplantation, even when grafted directly into the first branchial arch. These results suggest that neural crest cells along the rostrocaudal axis display a graded loss in developmental potential to form somatosensory neurons and cartilage even after transplantation to a permissive environment. Hox gene expression was transiently maintained in the cardiac neural tube and neural crest at 12 hours post-transplantation to the midbrain, but was subsequently downregulated. This suggests that long-term differences in Hox gene expression cannot account for rostrocaudal differences in developmental potential of neural crest populations in this case

Embryonic Origin of Avian Corneal Sensory Nerves

AbstractSensory nerves play a vital role in maintaining corneal transparency. They originate in the trigeminal ganglion, which is derived from two embryonic cell populations (cranial neural crest and ectodermal placode). Nonetheless, it is unclear whether corneal nerves arise from neural crest, from placode, or from both. Quail–chick chimeras and species-specific antibodies allowed tracing quail-derived neural crest or placode cells during trigeminal ganglion and corneal development, and after ablation of either neural crest or placode. Neural crest chimeras showed quail nuclei in the proximal part of the trigeminal ganglion, and quail nerves in the pericorneal nerve ring and in the cornea. In sharp contrast, placode chimeras showed quail nuclei in the distal part of the trigeminal ganglion, but no quail nerves in the cornea or in the pericorneal nerve ring. Quail placode-derived nerves were present, however, in the eyelids. Neural crest ablation between stages 8 and 9 resulted in diminished trigeminal ganglia and absence of corneal innervation. Ablation of placode after stage 11 resulted in loss of the ophthalmic branch of the trigeminal ganglion and reduced corneal innervation. Noninnervated corneas still became transparent. These results indicate for the first time that although both neural crest and placode contribute to the trigeminal ganglion, corneal innervation is entirely neural crest-derived. Nonetheless, proper corneal innervation requires presence of both cell types in the embryonic trigeminal ganglion. Also, complete lack of innervation has no discernible effect on development of corneal transparency or cell densities

Lens-derived Semaphorin3A regulates sensory innervation of the cornea

The cornea, one of the most highly innervated tissues of the body, is innervated by trigeminal sensory afferents. During development, axons are initially repelled at the corneal margin, resulting in the formation of a circumferential nerve ring. The nature and source of guidance molecules that regulate this process remain a mystery. Here, we show that the lens, which immediately underlies the cornea, repels trigeminal axons in vivo and in vitro. Lens ablation results in premature, disorganized corneal innervation and disruption of the nerve ring and ventral plexus. We show that Semaphorin3A (Sema3A) is expressed in the lens epithelium and its receptor Neuropilin-1 (Npn1) is expressed in the trigeminal ganglion during cornea development. Inhibition of Sema3A signaling abrogates axon repulsion by the lens and cornea in vitro and phenocopies lens removal in vivo. These results demonstrate that lens-derived Sema3A mediates initial repulsion of trigeminal sensory axons from the cornea and is necessary for the proper formation of the nerve ring and positioning of the ventral plexus in the choroid fissure

Human Fetal Keratocytes Have Multipotent Characteristics in the Developing Avian Embryo

The human cornea contains stem cells that can be induced to express markers consistent with multipotency in cell culture; however, there have been no studies demonstrating that human corneal keratocytes are multipotent. The objective of this study is to examine the potential of human fetal keratocytes (HFKs) to differentiate into neural crest-derived tissues when challenged in an embryonic environment. HFKs were injected bilaterally into the cranial mesenchyme adjacent to the neural tube and the periocular mesenchyme in chick embryos at embryonic days 1.5 and 3, respectively. The injected keratocytes were detected by immunofluorescence using the human cell-specific marker, HuNu. HuNu-positive keratocytes injected along the neural crest pathway were localized adjacent to HNK-1-positive migratory host neural crest cells and in the cardiac cushion mesenchyme. The HuNu-positive cells transformed into neural crest derivatives such as smooth muscle in cranial blood vessels, stromal keratocytes, and corneal endothelium. However, they failed to form neurons despite their presence in the condensing trigeminal ganglion. These results show that HFKs retain the ability to differentiate into some neural crest-derived tissues. Their ability to respond to embryonic cues and generate corneal endothelium and stromal keratocytes provides a basis for understanding the feasibility of creating specialized cells for possible use in regenerative medicine

Corneal Plasticity: Characterization of the Multipotentiality of Human Keratocytes

Purpose: To determine the cell properties of adult human corneal keratocytes when challenged in the chick embryonic environment. Methods: Cultured human keratocytes were injected along cranial neural crest migratory pathways in chick embryos. Human keratocytes were also cultured under various conditions and differentiated into either fibroblasts or myofibroblasts, then transplanted into the chick embryo. Migration of the injected cells was determined by immunohistochemistry using human cell-specific markers and markers of crest derivatives. Results: Injected human keratocytes proliferated and migrated ventrally adjacent to host neural crest cells. They contributed to numerous neural crest-derived tissues including cranial blood vessels, ocular tissues, musculature of the mandibular process, and cardiac cushion tissue. Conclusions: Adult human corneal keratocytes that have undergone terminal differentiation can be induced to form cranial neural crest derivatives when grafted into an embryonic environment

Stem-Cell Properties of Human Corneal Keratocytes

Purpose: To determine the stem cell properties of human corneal stromal keratocytes when challenged in the chick embryonic environment. Methods: Stromal keratocytes isolated from human corneas were injected along cranial neural crest migratory pathways and in the periocular mesenchyme in chick embryos. Localization Migration of the injected cells stromal keratocytes was determined at various stages of development by immunohistochemistry using human cell-specific markers. Differentiation of the human keratocytes into other neural crest-derived tissues was determined by immunohistochemistry with tissue cell-specific markers. Results: Human keratocytes injected along cranial neural crest pathways proliferated and migrated ventrally adjacent to host neural crest cells. They contributed to numerous neural crest-derived tissues including cranial blood vessels, ocular tissues, and cardiac cushion tissue mesenchyme. Keratocytes injected into the periocular mesenchyme region contributed to the corneal stroma and endothelial layers. Conclusions: Adult human corneal stromal keratocytes exhibit stem cell characteristics. They can be induced to form cranial neural crest derivatives, including other anterior ocular structures, when grafted into an embryonic environment

Altered Gravity and Early Heart Development in Culture

The macromolecules comprising the cytoskeleton and extracellular matrix of cells may be sensitive to gravitation. Since early development of organs depends on dynamic interactions across cell surfaces, altered gravity may disturb development. We investigated this possibility for heart development. Previous studies showed that the extracellular matrix glycoprotein fibronectin (Fn) is necessary for normal heart development. We cultured precardiac tissue explants in a high aspect ratio bioreactor vessel (HARV) to simulate microgravity. We observed tissue morphology, contraction, and Fn distribution by immunolocalization in HARV rotated and control (lxg) explants, cultured 18 hr. We also measured Fn amount by immunoassay. Explants in HARV were rotated at 6 rpm to achieve continuous freefall. Thirty-five of 37 control, but only 1 of 37 matched rotated explants exhibited contractions. Tissue architecture was identical. Immunolocalization of Fn showed remarkable differences which may be related to the development of contractions. The Fn staining in the HARV explants was less intense in all areas. Areas of linear staining along epithelia were present but shorter, and there was less intercellular staining in both mesenchymal tissue and myocardium. Initial immunoassay results of 5 matched pairs of explants showed a 22% reduction in total tissue Fn in the HARV rotated samples. Our results indicate that altered gravity in the HARV reduced the amount and distribution of Fn, as assessed by two independent criteria. This was correlated with a reduction in the development of contractile activity

Developmental origins and evolution of jaws: new interpretation of “maxillary” and “mandibular”

AbstractCartilage of the vertebrate jaw is derived from cranial neural crest cells that migrate to the first pharyngeal arch and form a dorsal “maxillary” and a ventral “mandibular” condensation. It has been assumed that the former gives rise to palatoquadrate and the latter to Meckel's (mandibular) cartilage. In anamniotes, these condensations were thought to form the framework for the bones of the adult jaw and, in amniotes, appear to prefigure the maxillary and mandibular facial prominences. Here, we directly test the contributions of these neural crest condensations in axolotl and chick embryos, as representatives of anamniote and amniote vertebrate groups, using molecular and morphological markers in combination with vital dye labeling of late-migrating cranial neural crest cells. Surprisingly, we find that both palatoquadrate and Meckel's cartilage derive solely from the ventral “mandibular” condensation. In contrast, the dorsal “maxillary” condensation contributes to trabecular cartilage of the neurocranium and forms part of the frontonasal process but does not contribute to jaw joints as previously assumed. These studies reveal the morphogenetic processes by which cranial neural crest cells within the first arch build the primordia for jaw cartilages and anterior cranium

Distinct Roles for Neuropilin1 and Neuropilin2 during Mouse Corneal Innervation

Trigeminal sensory innervation of the cornea is critical for protection and synthesis of neuropeptides required for normal vision. Little is known about axon guidance during mammalian corneal innervation. In contrast to the chick where a pericorneal nerve ring forms via Npn/Sema signaling, mouse corneal axons project directly into the presumptive cornea without initial formation of an analogous nerve ring. Here we show that during development of the mouse cornea, Npn1 is strongly expressed by the trigeminal ganglion whereas Npn2 is expressed at low levels. At the same time Sema3A and Sema3F are expressed in distinct patterns in the ocular tissues. Npn1sema−/− mutant corneas become precociously and aberrantly innervated by nerve bundles that project further into the corneal stroma. In contrast, stromal innervation was not affected in Npn2−/− mutants. The corneal epithelium was prematurely innervated in both Npn1sema−/− and Npn2−/− mutants. These defects were exacerbated in Npn1sema−/−;Npn2−/− double mutants, which in addition showed ectopic innervation of the region between the optic cup and lens vesicle. Collectively, our data show that Sema3A/Npn1 and Sema3F/Npn2 signaling play distinct roles and both are required for proper innervation of the mouse cornea

Recapitulation of normal collagen architecture in embryonic wounded corneas

Wound healing is characterized by cell and extracellular matrix changes mediating cell migration, fibrosis, remodeling and regeneration. We previously demonstrated that chick fetal wound healing shows a regenerative phenotype regarding the cellular and molecular organization of the cornea. However, the chick corneal stromal structure is remarkably complex in the collagen fiber/lamellar organization, involving branching and anastomosing of collagen bundles. It is unknown whether the chick fetal wound healing is capable of recapitulating this developmentally regulated organization pattern. The purpose of this study was to examine the three-dimensional collagen architecture of wounded embryonic corneas, whilst identifying temporal and spatial changes in collagen organization during wound healing. Linear corneal wounds that traversed the epithelial layer, Bowman´s layer, and anterior stroma were generated in chick corneas on embryonic day 7. Irregular thin collagen fibers are present in the wounded cornea during the early phases of wound healing. As wound healing progresses, the collagen organization dramatically changes, acquiring an orthogonal arrangement. Fourier transform analysis affirmed this observation and revealed that adjacent collagen lamellae display an angular displacement progressing from the epithelium layer towards the endothelium. These data indicate that the collagen organization of the wounded embryonic cornea recapitulate the native macrostructure