The Corneal Epithelial Barrier and Its Developmental Role in Isolating Corneal Epithelial and Conjunctival Cells From One Another.

Purpose: During development, the corneal epithelium (CE) and the conjunctiva are derived from the surface ectoderm. Here we have examined how, during development, the cells of these two issues become isolated from each other. Methods: Epithelia from the anterior eyes of chicken embryos were labeled with the fluorescent, lipophilic dye, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI). DiI was placed on the epithelial surface of the developing anterior eye and its diffusion was monitored by fluorescence microscopy. Concomitant morphologic changes in the surface cells of these epithelial were examined by scanning electron microscopy. Immunofluorescence was used to analyze the expression of cytokeratin K3, ZO-1, N-cadherin and Connexin-43 and the function of gap junctions was analyzed using a cut-loading with the fluorescent dye rhodamine-dextran. Results: Prior to embryonic day 8 (E8), DiI placed on the surface of the CE spreads throughout all the epithelial cells of the anterior eye. When older eyes were similarly labeled, dye diffusion was restricted to the CE. Similarly, diffusion of DiI placed on the conjunctival surface after E8 was restricted to the conjunctiva. Scanning electron microscopy showed that developmentally (1) physical separations progressively form between the cells of the CE and those of the conjunctiva, and (2) by E8 these separations form a ring that completely encompasses the cornea. The functional restriction of gap junctions between these tissues did not occur until E14. Conclusions: During ocular development, a barrier to the diffusion of DiI forms between the contiguous CE and conjunctiva prior to the differential expression of gap junctions within these tissues.

Nuclear ferritin mediated regulation of JNK signaling in corneal epithelial cells.

Corneal epithelial (CE) cells are exposed to environmental insults (e.g., UV-irradiation), yet they suffer little damage. Our previous studies suggest that chicken CE cells have a novel form of protection involving having ferritin in a nuclear location where it can bind to DNA and sequester free iron. Here we describe another potential nuclear ferritin-mediated protective mechanism: the down-regulation of the JNK signaling pathway. The JNK pathway has been shown by others to promote apoptosis in response to cell damage and also to be activated in CE cell lines following exposure to UV radiation. Here we show in COS7 reporter cell lines that the expression of ferritin in a nuclear localization significantly down-regulates the JNK pathway (p = 5.7 × 10(-6)), but has no effect on the NFkB or the Erk pathways. In organ cultures of embryonic chicken corneas, we observed that inhibiting the synthesis of nuclear ferritin in CE cells, using the iron-chelating molecule deferoxamine, led to an increase in JNK signaling, as measured by phospho-JNK levels compared to CE cells with nuclear ferritin. Furthermore, the chemical inhibition of the JNK pathway using the molecule AS601245 decreased the production of nuclear ferritin. Taken together, these observations suggest that in CE cells a feedback-loop exists in which JNK signaling increases the production of nuclear ferritin and, in turn, nuclear ferritin decreases the activity of the JNK signaling pathway.

Developmental regulation of trigeminal TRPA1 by the cornea.

PURPOSE: The cornea is densely innervated with nociceptive nerves that detect deleterious stimuli at the ocular surface and transduce these stimuli as sensations of pain. Thus, nociception is a major factor involved in preventing damage to corneal tissues. One class of molecules that is thought to be involved in detecting such stimuli is the transient receptor potential (TRP) family of ion channels. However, little is known about the acquisition of these channels during corneal development. Therefore, the present study examined the developmental acquisition of these receptors and elucidated certain parameters involved in this acquisition. METHODS: Quantitative RT-PCR was used to measure the expression of genes including TRPA and Ret in vivo. In vitro cocultures between cornea and the ophthalmic lobe of the trigeminal ganglion were used to test interactions between nerves and corneas along with recombinant proteins. RESULTS: TRPA1 mRNA showed a progressive temporal increase in the ophthalmic lobe of the trigeminal ganglion in vivo during embryonic development. In vitro, TRPA1 expression was significantly increased in the ganglion when cocultured with cornea, compared to ganglia cultured alone. Similarly, the addition of exogenous neurotrophin-3 (NT3) protein to cultured ganglia increased the expression of TRPA1 more than 100-fold. Addition of NT3 and neurturin synergistically increased TRPA1 expression in embryonic day (E)8 ganglia, but this effect was lost at E12. At E8, Ret+ nonpeptidergic neurons are specified in the trigeminal ganglion. CONCLUSIONS: Corneal-derived factors increase TRPA1 expression in trigeminal nonpeptidergic neurons during their embryonic specification.

Developmental guidance of embryonic corneal innervation: roles of Semaphorin3A and Slit2.

The cornea is one of the most densely innervated structures of the body. In the developing chicken embryo, nerves from the ophthalmic trigeminal ganglion (OTG) innervate the cornea in a series of spatially and temporally regulated events. However, little is known concerning the signals that regulate these events. Here we have examined the involvement of the axon guidance molecules Semaphorin3A and Slit2, and their respective receptors, Neuropilin-1 and Robo2. Expression analyses of early corneas suggest an involvement of both Semaphorin3A and Slit2 in preventing nerves from entering the corneal stroma until the proper time (i.e., they serve as negative regulators), and analyses of their receptors support this conclusion. At later stages of development the expression of Semaphorin3A is again consistent with its serving as a negative regulator-this time for nerves entering the corneal epithelium. However, expression analyses of Robo2 at this stage raised the possibility that Slit2 had switched from a negative regulator to a positive regulator. In support of such a switch, functional analyses-by addition of recombinant Slit2 protein or immunoneutralization with a Slit2 antibody-showed that at an early stage Slit2 negatively regulates the outgrowth of nerves from the OTG, whereas at the later stage it positively regulated the growth of nerves by increasing nerve branching within the corneal epithelium.

Developmental corneal innervation: interactions between nerves and specialized apical corneal epithelial cells.

PURPOSE: The corneal epithelium is one of the most highly innervated structures in the body, and proper innervation is necessary for corneal maintenance and sensation. However, little is known about how these nerves function and how innervation occurs developmentally. The authors have examined certain aspects of corneal innervation in the developing chicken embryo. METHODS: DiI was used to determine the source of the neurons responsible for innervating the cornea. Immunohistochemistry, electron microscopy, and immunoelectron microscopy were used to examine corneal innervation and the relationships that develop between nerves and corneal epithelial cells. RESULTS: Corneal nerves in the embryonic chicken originate entirely from the ophthalmic lobe of the trigeminal ganglion. Within the cornea the nerves interact with apical corneal epithelial (ACE) cells to form specialized structures that are synapse-like because they contain accumulations of vesicles and have the SV2 synaptic vesicle protein. These ACE cells themselves have unique characteristics, including transient expression of the neuronal isoform of class III beta-tubulin and formation of extensive intercellular channels and clefts that contain these specialized synapse-like structures and nerves; in addition, they are mitotically active. Given that these ACE cells react with a monoclonal antibody against this neuronal isoform of beta-tubulin (the TuJ-1 antibody), we have termed them TuJ-1(+)ACE cells. CONCLUSIONS: During avian corneal development the nerves make close associations with a specialized type of ACE cell. There they form synapse-like structures, suggesting that not all nerves within the CE terminate as free nerve endings.

Developmental regulation of the nuclear ferritoid-ferritin complex of avian corneal epithelial cells: roles of systemic factors and thyroxine.

Previously we observed that avian corneal epithelial cells protect their DNA from oxidative damage by having the iron-sequestering molecule ferritin - normally cytoplasmic - in a nuclear location. This localization involves a developmentally-regulated ferritin-like protein - ferritoid - that initially serves as the nuclear transporter, and then as a component of a ferritoid-ferritin complex that is half the size of a typical ferritin and binds to DNA. We also observed that developmentally, the synthesis of ferritin and ferritoid are regulated coordinately - with ferritin being predominantly translational and ferritoid transcriptional. In the present study we examined whether the mechanism(s) involved in this regulation reside within the cornea itself, or alternatively involve a systemic factor(s). For this, we explanted embryonic corneas of one age to the chorioallantoic membrane (CAM) of host embryos of a different age - all prior to the initiation of ferritin synthesis. Consistent with systemic regulation, the explants initiated the synthesis of both ferritin and ferritoid in concert with that of the host. We then examined whether this systemic regulation might involve thyroxine - a hormone with broad developmental effects. Employing corneal organ cultures, we observed that thyroxine initiated the synthesis of both components in a manner similar to that which occurs in vivo (i.e. ferritin was translational and ferritoid transcriptional).

Phosphorylation regulates the ferritoid-ferritin interaction and nuclear transport.

Ferritin is an iron-sequestering protein that is generally cytoplasmic; however, our previous studies have shown that in avian corneal epithelial (CE) cells ferritin is nuclear. We have also observed that this nuclear localization involves a tissue-specific nuclear transporter that we have termed ferritoid, and that nuclear ferritin protects DNA from oxidative damage. Recently we have determined that ferritoid functions not only as a nuclear transporter, but also, within the nucleus, it remains associated with ferritin as a heteropolymeric complex. This ferritoid-ferritin complex has unique properties such as being half the size of a typical ferritin molecule and showing preferential binding to DNA. It is likely that the association between ferritoid and ferritin is involved both in the nuclear transport of ferritin and in determining certain of the properties of the complex; therefore, we have been examining the mechanisms involved in regulating the association of these two components. As the ferritoid sequence contains six putative phosphorylation sites, we have examined here whether phosphorylation is one such mechanism. We have determined that ferritoid in the nuclear ferritoid-ferritin complexes is phosphorylated, and that inhibition of this phosphorylation, using inhibitors of PKC, prevents its interaction with ferritin. Furthermore, in an experimental model system in which the nuclear transport of ferritin normally occurs (i.e., the co-transfection of COS-1 cells with full length constructs for ferritin and ferritoid), when phosphorylation sites in ferritoid are mutated, the interaction between ferritoid and ferritin is inhibited, as is the nuclear transport of ferritin.

Nuclear ferritin: a ferritoid-ferritin complex in corneal epithelial cells.

PURPOSE: Ferritin is an iron storage protein that is generally cytoplasmic. However, in embryonic avian corneal epithelial (CE) cells, the authors previously observed that the ferritin was predominantly nuclear. They also obtained evidence that this ferritin protects DNA from oxidative damage by UV light and hydrogen peroxide and that the nuclear localization involves a tissue-specific nuclear transporter, termed ferritoid. In the present investigation, the authors have determined additional properties of the nuclear ferritoid-ferritin complexes. METHODS: For biochemical characterization, a combination of molecular sieve chromatography, immunoblotting, and nuclear-cytoplasmic fractionation was used; DNA binding was analyzed by electrophoretic mobility shift assay. RESULTS: The CE nuclear ferritin complex has characteristics that differentiate it from a "typical" cytoplasmic ferritin, including the presence of ferritin and ferritoid subunits; a molecular weight of approximately 260 kDa, which is approximately half that of cytoplasmic ferritin; its iron content, which is below our limits of detection; and its ability to bind to DNA. CONCLUSIONS: Within CE cell nuclei, ferritin and ferritoid are coassembled into stable complex(es) present in embryonic and adult corneas. Thus, ferritoid not only serves transiently as a nuclear transporter for ferritin, it remains as a component of a unique ferritoid-ferritin nuclear complex.

Corneal epithelial nuclear ferritin: developmental regulation of ferritin and its nuclear transporter ferritoid.

The corneal epithelium is exposed to reactive oxygen species that are potentially deleterious to nuclear DNA. However, our previous studies show that corneal epithelial cells have a novel, developmentally regulated mechanism for protection from such damage that involves having the iron-sequestering molecule, ferritin, in the nucleus. Nuclear localization of ferritin is achieved through the action of a tissue-specific nuclear transporter, ferritoid, which is itself a ferritin family member. Here, we show that during development ferritoid appears before ferritin. At this time, ferritoid is cytoplasmic, suggesting that its nuclear transport function requires an interaction with ferritin. To examine the developmental regulation of these two interacting components, cultured corneas were treated with the iron chelator deferoxamine. The results show that, while iron-mediated translational regulation is involved in the synthesis of both molecules, ferritoid is also transcriptionally regulated, demonstrating that these family members--whose functions depend upon one another--are regulated differently.

Identification of unique molecular subdomains in the perichondrium and periosteum and their role in regulating gene expression in the underlying chondrocytes.

Developing cartilaginous and ossified skeletal anlagen is encapsulated within a membranous sheath of flattened, elongated cells called, respectively, the perichondrium and the periosteum. These periskeletal tissues are organized in distinct morphological layers that have been proposed to support distinct functions. Classical experiments, particularly those using an in vitro organ culture system, demonstrated that these tissues play important roles in regulating the differentiation of the subjacent skeletal elements. However, there has been a lack of molecular markers that would allow analysis of these interactions. To understand the molecular bases for the roles played by the periskeletal tissues, we generated microarrays from perichondrium and periosteum cDNA libraries and used them to compare the gene expression profiles of these two tissues. In situ hybridization analysis of genes identified on the microarrays revealed many unique markers for these tissues and demonstrated that the histologically distinct layers of the perichondrium and periosteum are associated with distinct molecular expression domains. Moreover our marker analysis identified new domains that had not been previously recognized as distinct within these tissues as well as a previously uncharacterized molecular domain along the lateral edges of the adjacent developing cartilage that experimental analysis showed to be dependent upon the perichondrium.

Perichondrial-mediated TGF-beta regulation of cartilage growth in avian long bone development.

We previously observed using cultured tibiotarsal long-bone rudiments from which the perichondrium (PC) and periosteum (PO) was removed that the PC regulates cartilage growth by the secretion of soluble negative regulatory factors. This regulation is "precise" in that it compensates exactly for removal of the endogenous PC and is mediated through at least three independent mechanisms, one of which involves a response to TGF-beta. PC cell cultures treated with 2 ng/ml TGF-beta1 produced a conditioned medium which when added to PC/PO-free organ cultures effected precise regulation of cartilage growth. In the present study, we have investigated the possibility that TGF-beta itself might be the negative regulator which is produced by the PC cells in response to their treatment with TGF-beta1. Using a TGF-beta responsive reporter assay, we determined that PC cell cultures, when treated with 2 ng/ml or greater exogenous TGF-beta1, produce 300 pg/ml of active TGF-beta. Then we observed that this concentration (300 pg/ml) of active TGF-beta1, when added to PC/PO-free tibiotarsal organ cultures, effected precise regulation of cartilage growth, whereas concentrations of TGF-beta1 either greater or less than 300 pg/ml produced abnormally small cartilages. These results suggest that one mechanism by which the PC effects normal cartilage growth is through the production of a precisely regulated amount of TGF-beta which the PC produces in response to treatment with exogenous TGF-beta itself.

Identification and characterization of a previously undetected region between the perichondrium and periosteum of the developing avian limb.

In developing long bones, the growing cartilage and bone are surrounded by the fibrous perichondrium (PC) and periosteum (PO), respectively, which provide cells for the appositional growth (i.e., growth in diameter) of these tissues. Also during the longitudinal growth of a bone, the cartilage is continuously replaced by bony tissue, giving rise to the widely held assumption that the PC concomitantly gives rise to the PO. Except for this morphological correlate, however, no evidence exists for a direct conversion of PC cells to PO cells, and our observations presented here question this assumption. Instead, we have obtained evidence suggesting that a previously undescribed region exists between the PC and PO. This region, termed the border region (BR), has several unique characteristics which distinguish it from either the PC or PO, including (1) its lack of being determined to differentiate as either cartilage or bone, (2) its ability to preferentially elicit the invasion of blood vessels, and (3) its ability to undergo preferential growth.

SP3/SP1 transcription activity regulates specific expression of collagen type X in hypertrophic chondrocytes.

Previously, we have shown that two non-canonical specificity protein (SP)-binding sites within the proximal promoter (nucleotide (nt) -139 to +5) of the chicken Col10a1 gene are involved in conferring tissue-specific expression of type X collagen to hypertrophic chondrocytes. In the present study, we examined the role of SP3/SP1 transcription factors in the regulation of the Col10a1 promoter. The SP3/SP1 ratio is higher in hypertrophic versus non-hypertrophic chondrocytes, due to the significant decrease in SP1 in hypertrophic cells detected by real-time PCR and Western blot analyses. Functional analyses by transfection-mediated overexpression of SP1 and SP3 suggest that SP1 inhibits the Col10a1 promoter. This effect is negated by an interaction with SP3 in hypertrophic chondrocytes. Additionally, mutation analysis showed that the 40-bp intervening sequence (nt -115 to -75) is required for expression of the Col10a1 gene. In this sequence, a binding site for Dlx5/6 transcription factors (nt -99 to -87) retards a protein specific for hypertrophic chondrocytes in electrophoretic mobility shift assay. Endogenous levels of Dlx5 are 3-fold higher in hypertrophic versus non-hypertrophic cells by real-time PCR analysis, and overexpression of Dlx5 in non-hypertrophic chondrocytes activates the proximal Col10a1 promoter 3-fold. These results indicate that the SP3/SP1 ratio and Dlx5 are important regulators of the proximal Col10a1 promoter in hypertrophic cartilage and suggest that interactions between SP3 and SP1 regulate expression of different types of collagen during chondrocyte differentiation.

Nuclear ferritin in corneal epithelial cells: tissue-specific nuclear transport and protection from UV-damage.

We have identified the heavy chain of ferritin as a developmentally regulated nuclear protein of embryonic chicken corneal epithelial cells. The nuclear ferritin is assembled into a supramolecular form that is indistinguishable from the cytoplasmic form of ferritin found in other cell types. Thus it most likely has iron-sequestering capabilities. Free iron, via the Fenton reaction, is known to exacerbate UV-induced and other oxidative damage to cellular components, including DNA. Since corneal epithelial cells are constantly exposed to UV light, we hypothesized that the nuclear ferritin might protect the DNA of these cells from free radical damage. To test this possibility, primary cultures of cells from corneal epithelium and other tissues were UV irradiated, and damage to DNA was detected by an in situ 3'-end labeling assay. Consistent with the hypothesis, corneal epithelial cells with nuclear ferritin had significantly less DNA breakage than the other cells types examined. However, when the expression of nuclear ferritin was inhibited the cells now became much more susceptible to UV-induced DNA damage. Since ferritin is normally cytoplasmic, corneal epithelial cells must have a mechanism that effects its nuclear localization. We have determined that this involves a nuclear transport molecule which binds to ferritin and carries it into the nucleus. This transporter, which we have termed ferritoid for its similarity to ferritin, has at least two domains. One domain is ferritin-like and is responsible for binding the ferritin; the other domain contains a nuclear localization signal that is responsible for effecting the nuclear transport. Therefore, it seems that corneal epithelial cells have evolved a novel, nuclear ferritin-based mechanism for protecting their DNA against UV damage. In addition, since ferritoid is structurally similar to ferritin, it may represent an example of a nuclear transporter that evolved from the molecule it transports (i.e., ferritin).

Cellular invasion of the chicken corneal stroma during development: regulation by multiple matrix metalloproteases and the lens.

Avian corneal development requires cellular invasion into the acellular matrix of the primary stroma. Previous results show that this invasion is preceded by the removal of the fibril-associated type IX collagen, which possibly stabilizes matrices through interfibrillar cross-bridges secured by covalent crosslinks. In the present study, we provide evidence for the expression of three matrix metalloproteinases (MMPs) in early corneas, two of which act cooperatively to selectively remove type IX collagen in situ. In organ cultures, MMP inhibitors (either TIMP-2 or a synthetic inhibitor) resulted in arrested development, in which collagen IX persisted, and the stroma remained compact and acellular. We also show that blocking covalent crosslinking of collagen allows for cellular invasion to occur, even when the removal of type IX collagen is prevented. Thus, one factor regulating corneal invasion is the physical structure of the matrix, which can be modified by either selective proteolysis or reducing interfibrillar cross-bridges. We also detected another level of regulation of cellular invasion involving inhibition by the underlying lens. This block, which seems to influence invasive behavior independently of matrix modification, is a transient event that is released in ovo just before invasion proceeds.

Chondrocyte-derived transglutaminase promotes maturation of preosteoblasts in periosteal bone.

During endochondral development, elongation of the bone collar occurs coordinately with growth of the underlying cartilaginous growth plate. Transglutaminases (TGases) are upregulated in hypertrophic chondrocytes, and correlative evidence suggests a relationship between these enzymes and mineralization. To examine whether TGases are involved in regulating mineralization/osteogenesis during bone development, we devised a coculture system in which one cellular component (characterized as preosteoblastic) is derived from the nonmineralized region of the bone, and the other cellular component is hypertrophic chondrocytes. In these cocultures, mineralization is extensive, with the preosteoblasts producing the mineralized matrix, and the chondrocytes regulating this process. Secreted regulators are involved, as conditioned medium from chondrocytes induces mineralization in preosteoblasts, but not vice versa. One factor is TGase. In the cocultures, inhibition of TGase reduces mineralization, and addition of the enzyme enhances it. Exogenous TGase also induces markers of osteoblastic differentiation (i.e., bone sialoprotein and osteocalcin) in the preosteoblasts, suggesting their differentiation into osteoblasts. Two possible signaling pathways may be affected by TGase and result in increased mineralization (i.e., TGF-beta and protein kinase A pathways). Addition of exogenous TGF-beta2 to the cocultures increases mineralization; though, when mineralization is induced by TGase, there is no detectible elevation of TGF-beta, suggesting that these two factors stimulate osteogenesis by different pathways. However, an interrelationship seems to exist between TGase and PKA-dependent signaling. When mineralization of the cocultures is stimulated through the addition of TGase, a concomitant reduction (50%) in PKA activity occurs. Consistent with this observation, addition of an activator of PKA (cyclic AMP) to the cultures inhibits matrix mineralization, while known inhibitors of PKA (H-89 and a peptide inhibitor) cause an increase in mineralization. Thus, at least one mechanism of TGase stimulation probably involves inhibition of the PKA-mediated signaling.

Positive regulation of endochondral cartilage growth by perichondrial and periosteal calcitonin.

Our previous studies showed that during the embryonic development of avian long bones, growth of the cartilaginous component is regulated by multiple factors secreted by the surrounding perichondrium (PC) and periosteum (PO). The activities of these factors--which include both positive and negative regulators--can be detected in conditioned media from PC and PO cell cultures. In the present study, we have obtained evidence suggesting that a positive regulator is the peptide hormone calcitonin (CT). By mass spectrometry of conditioned media, one of the components has a molecular mass of 3.4 kDa, the size of chicken CT. By RT-PCR the tissue and cell cultures contain mRNA for CT, and by immunohistochemistry the cells contain the protein. That the protein is normally secreted is suggested by further immunohistochemical analyses, which show that cells treated with monensin, a compound that blocks exocytosis, contain elevated intracellular CT. Functionally, the addition of CT to organ cultures of long bone rudiments effects increased growth in a manner similar to that of the PC- and PO-conditioned media. Taken together, these data suggest that secretion of CT by the PC and PO effects, in a paracrine manner, positive stimulation of growth in the underlying cartilage.

Ferritoid, a tissue-specific nuclear transport protein for ferritin in corneal epithelial cells.

Previously we reported that ferritin in corneal epithelial (CE) cells is a nuclear protein that protects DNA from UV damage. Since ferritin is normally cytoplasmic, in CE cells, a mechanism must exist that effects its nuclear localization. We have now determined that this involves a nuclear transport molecule we have termed ferritoid. Ferritoid is specific for CE cells and is developmentally regulated. Structurally, ferritoid contains multiple domains, including a functional SV40-type nuclear localization signal and a ferritin-like region of approximately 50% similarity to ferritin itself. This latter domain is likely responsible for the interaction between ferritoid and ferritin detected by co-immunoprecipitation analysis. To test functionally whether ferritoid is capable of transporting ferritin into the nucleus, we performed cotransfections of COS-1 cells with constructs for ferritoid and ferritin. Consistent with the proposed nuclear transport function for ferritoid, co-transfections with full-length constructs for ferritoid and ferritin resulted in a preferential nuclear localization of both molecules; this was not observed when the nuclear localization signal of ferritoid was deleted. Moreover, since ferritoid is structurally similar to ferritin, it may be an example of a nuclear transporter that evolved from the molecule it transports (ferritin).

Multiple mechanisms of perichondrial regulation of cartilage growth.

We previously observed that the perichondrium (PC) and the periosteum (PO) negatively regulate endochondral cartilage growth through secreted factors. Conditioned medium from cultures of PC and PO cells when mixed (PC/PO-conditioned medium) and tested on organ cultures of embryonic chicken tibiotarsi from which the PC and PO have been removed (PC/PO-free cultures) effect negative regulation of growth. Of potential importance, this regulation compensates precisely for removal of the PC and PO, thus mimicking the regulation effected by these tissues in vivo. We have now examined whether two known negative regulators of cartilage growth (retinoic acid [RA] and transforming growth factor-beta1 [TGF-beta1]) act in a manner consistent with this PC/PO-mediated regulation. The results suggest that RA and TGF-beta1, per se, are not the regulators in the PC/PO-conditioned medium. Instead, they show that these two factors each act in regulating cartilage growth through an additional, previously undescribed, negative regulatory mechanism(s) involving the perichondrium. When cultures of perichondrial cells (but not periosteal cells) are treated with either agent, they secrete secondary regulatory factors into their conditioned medium, the action of which is to effect precise negative regulation of cartilage growth when tested on the PC/PO-free organ cultures. This negative regulation through the perichondrium is the only activity detected with TGF-beta1. Whereas, RA shows additional regulation on the cartilage itself. However, this regulation by RA is not "precise" in that it produces abnormally shortened cartilages. Overall, the precise regulation of cartilage growth effected by the action of the perichondrial-derived factor(s) elicited from the perichondrial cells by treatment with either RA or TGF-beta1, when combined with our previous results showing similar--yet clearly different--"precise" regulation by the PC/PO-conditioned medium suggests the existence of multiple mechanisms involving the perichondrium, possibly interrelated or redundant, to ensure the proper growth of endochondral skeletal elements.