5-HT(1A), 5-HT(2A), and 5-HT(2C) receptor mRNA modulation by antidepressant treatment in the chronic mild stress model of depression: sex differences exposed.

It is well established that women experience major depression at roughly twice the rate of men. Interestingly, accumulating clinical and experimental evidence shows that the responsiveness of males and females to antidepressant pharmacotherapy, and particularly to tricyclic antidepressants (TCAs), is sex-differentiated. Herein, we investigated whether exposure of male and female rats to the chronic mild stress (CMS) model of depression, as well as treatment with the TCA clomipramine may affect serotonergic receptors' (5-HTRs) mRNA expression in a sex-dependent manner. Male and female rats were subjected to CMS for 4 weeks and during the next 4 weeks they concurrently received clomipramine treatment (10 mg/ml/kg). CMS and clomipramine's effects on 5-HT(1A)R, 5-HT(2A)R, and 5-HT(2C)R mRNA expression were assessed by in situ hybridization histochemistry in selected subfields of the hippocampus and in the lateral orbitofrontal cortex (OFC), two regions implicated in the pathophysiology of major depression. CMS and clomipramine treatment induced sex-differentiated effects on rats' hedonic status and enhanced 5-HT(1A)R mRNA expression in the cornu ammonis 1 (CA1) hippocampal region of male rats. Additionally, CMS attenuated 5-HT(1A)R mRNA expression in the OFC of male rats and clomipramine reversed this effect. Moreover, 5-HT(2A)R mRNA levels in the OFC were enhanced in females but decreased in males, while clomipramine reversed this effect only in females. CMS increased 5-HT2CR mRNA expression in the CA4 region of both sexes and this effect was attenuated by clomipramine. Present data exposed that both CMS and clomipramine treatment may induce sex-differentiated and region-distinctive effects on 5-HTRs mRNA expression and further implicate the serotonergic system in the manifestation of sexually dimorphic neurobehavioral responses to stress.

Neurochemical and behavioral alterations in an inflammatory model of depression: sex differences exposed.

It is firmly established that women experience major depression (MD) at roughly twice the rate of men and that dysregulation of the immune system is associated with the appearance and course of this condition. In the present study, we sought to identify whether "sickness behavior", an inflammatory model of MD, is characterized by sexual dimorphism by focusing on both neurochemical and behavioral responses. Therefore, we investigated the serotonergic and dopaminergic activity of various brain regions implicated in the pathophysiology of affective disorders (hypothalamus, hippocampus, prefrontal cortex, amygdala and striatum) in response to a mild lipopolysaccharide (LPS) challenge, in rats of both sexes. According to our results, at 2 h post-LPS administration (100 microg/kg i.p.), the neurochemical substrate was primarily altered in female rats with the serotonergic function being markedly enhanced in all brain regions examined. Dopaminergic activation following immune system sensitization with LPS was not apparent in male rats and only modest in female rats with the exception of striatum. LPS administration also affected sickness-associated behaviors to a different extent in male and female rats, as assessed in the forced swim test (FST), the hot plate test (HPT) and the open-field arena. LPS-treated female rats coped better with the stressful FST procedure, as evidenced by an increase in swimming duration. The effects of LPS treatment appeared to be more robust in male rats, as far as suppression of locomotor activity is concerned, while the antinociceptive properties of LPS were evident in both sexes though showing sex-dependent kinetics. Moreover, when traditional measures of sickness (i.e. sucrose consumption, social exploration, food intake) were assessed, males and females appeared to be similarly affected, except for food intake. These data are the first to demonstrate that the serotonergic system is affected to a greater extent in female rats at 2 h post-LPS administration and further contribute to our understanding regarding sexual dimorphism upon sickness establishment.

On dorsal/ventral-specific genes in the iris during lens regeneration.

Recent experiments on gene expression during lens regeneration in adult newts have revealed that both the regeneration-competent dorsal iris and the regeneration-incompetent ventral iris are quite active in expressing important regulatory genes. In this paper we outline some of the issues pertaining to this dorsal/ventral specificity and identity.

A unique aged human retinal pigmented epithelial cell line useful for studying lens differentiation in vitro.

Lens regeneration occurs in some urodeles and fish throughout their adult life. Such an event is possible by the transdifferentiation of the pigment epithelial cells (PECs) from the dorsal iris. Studies of this event at the cellular level have been facilitated owing to the ability of PECs to become lens cells even when they are placed in culture, outside of the eye. In fact, PECs possess the capacity for transdifferentiation regardless of the origin of species or age. However, studies at the molecular level are still hindered by the intrinsic problems of primary cultures, namely storage, reproducibility and genetic manipulation. In an attempt to establish an ideal model system for lens transdifferentiation, we have analyzed the ability of a human dedifferentiated PEC line to differentiate into lens. We have found that this cell line can indeed be induced to synthesize crystallin and morphologically differentiate to three-dimensional structures resembling lentoids under controlled treatment in vitro. Gene expression studies also provided important insights into the role of key genes. This human cell line can be used for detailed genetic studies in order to identify the key factors involved in lens transdifferentiation from PECs.

A novel role of complement: mice deficient in the fifth component of complement (C5) exhibit impaired liver regeneration.

Components of innate immunity have recently been implicated in the regulation of developmental processes. Most strikingly, complement factors appear to be involved in limb regeneration in certain urodele species. Prompted by these observations and anticipating a conserved role of complement in mammalian regeneration, we have now investigated the involvement of complement component C5 in liver regeneration, using a murine model of CCl(4)-induced liver toxicity and mice genetically deficient in C5. C5-deficient mice showed severely defective liver regeneration and persistent parenchymal necrosis after exposure to CCl(4.) In addition, these mice showed a marked delay in the re-entry of hepatocytes into the cell cycle (S phase) and diminished mitotic activity, as demonstrated, respectively, by the absence of 5-bromo-2'-deoxyuridine incorporation in hepatocytes, and the rare occurrence of mitoses in the liver parenchyma. Reconstitution of C5-deficient mice with murine C5 or C5a significantly restored hepatocyte regeneration after toxic injury. Furthermore, blockade of the C5a receptor (C5aR) abrogated the ability of hepatocytes to proliferate in response to liver injury, providing a mechanism by which C5 exerts its function, and establishing a critical role for C5aR signaling in the early events leading to hepatocyte proliferation. These results support a novel role for C5 in liver regeneration and strongly implicate the complement system as an important immunoregulatory component of hepatic homeostasis.

Role of retinoic acid in lens regeneration.

Prompted by the actions of retinoids and their receptors in gene regulation, in the developing eye and especially in the lens, we have undertaken a detailed study to examine the effects of retinoids on urodele lens regeneration. First, we examined the effects of exogenous retinoids. It was found that exogenous retinoids had no significant effect on lens regeneration. However, when synthesis of retinoic acid was inhibited by disulfiram, or when the function of the retinoid receptors was impaired by using a RAR antagonist, the process of lens regeneration was dramatically affected. In the majority of the cases, lens regeneration was inhibited and lens morphogenesis was disrupted. In a few cases, we were also able to observe ectopic lens regeneration from places other than the normal site, which is from the dorsal iris. The most spectacular case was the regeneration of a lens from the cornea, an event possible only in premetamorphic frogs. These data show that inhibition of retinoid receptors is paramount for the normal course and distribution of lens regeneration. We have also examined expression of RAR-delta during lens regeneration. This receptor was expressed highly in the regenerating lens only. Therefore, it seems that this receptor is specific for the regeneration process and consequently such expression correlates well with the effects of RAR inhibition observed in our studies.

Regeneration in vertebrates.

One way or another, all species possess the ability to regenerate damaged tissues. The degree of regeneration, however, varies considerably among tissues within a body and among species, with urodeles being the most spectacular. Such differences in regenerative capacity are indicative of specific mechanisms that control the different types of regeneration. In this review the different types of regeneration in vertebrates and their basic characteristics are presented. The major cellular events, such as dedifferentiation and transdifferentiation, which allow complex organ and body part regeneration, are discussed and common molecular mechanisms are pinpointed.

Regulation of Prox 1 during lens regeneration.

PURPOSE: To determine the expression pattern of Prox 1 during the process of lens regeneration in the urodele Notophthalmus viridescens. METHODS: Polymerase chain reaction was performed to amplify a partial newt Prox 1 sequence. In situ hybridization and immunodetection methods were used to detect the Prox 1 mRNA and the Prox 1 protein, respectively. RESULTS: Prox 1 mRNA was present in the retina and in the lens (in the epithelium and bow region) of the intact eye. Prox 1 protein was found to be predominantly present in the lens and dorsal iris of the intact eye, although some trace levels of Prox 1 protein were detected in the ventral iris as well. After lentectomy, expression of the mRNA was also pronounced in the dorsal dedifferentiating iris and the regenerating lens. The ventral iris also expressed Prox 1 but seemingly at lower levels. Although Prox 1 protein showed upregulation in the dorsal iris during the process of lens regeneration, trace levels were also detected in the ventral iris. In the retina, Prox 1 protein was distributed in horizontal cells of the inner nuclear layer, whereas the mRNA was expressed in all layers of the retina. CONCLUSIONS: Prox 1 was unevenly distributed in the intact cells of the newt iris, with significantly higher levels of Prox 1 protein present in the dorsal versus the ventral margin. This protein was differentially regulated during the process of lens regeneration, with obvious upregulation in the dorsal iris. Prox 1 is the first transcriptional factor to be shown to be regulated in the dorsal versus ventral iris during the process of lens regeneration.

Expression of the third component of complement, C3, in regenerating limb blastema cells of urodeles.

In this study we have shown that complement component C3 is expressed in the regenerating tissue during urodele limb regeneration. C3 was expressed in the dedifferentiated regeneration blastema and in the redifferentiated limb tissues in the axolotl, Amblystoma mexicanum, and in Notophthalmus viridescens. This expression was verified by immunofluorescent staining using an Ab against axolotl C3 and by in situ hybridization with an axolotl C3 cDNA probe. In the early stages of regeneration C3 appeared to be equally present in all mesenchymal cells and in the wound epithelium, whereas in the later stages it was mainly expressed in the differentiating muscle cells. Since no expression was seen in the developing limb, it appears that the C3 expression was specific to the regeneration process. We then demonstrated by hybridization experiments that a blastema cell line of myogenic origin expresses C3. All these findings implicate C3 in the dedifferentiation process and may indicate a new role for this molecule in muscle differentiation.

Fibroblast growth factor receptors regulate the ability for hindlimb regeneration in Xenopus laevis.

During outgrowth of the developing limb, signals from the apical ectodermal ridge, such as fibroblast growth factors, are paramount for limb patterning. Similarly, fibroblast growth factor molecules and their receptors are synthesized in the wound epithelium of the regenerating limb blastema, implicating an analogous function to limb development. To address this issue further and to understand the role of fibroblast growth factor receptor signaling in limb regeneration, we have examined the expression patterns of x-fibroblast growth factor receptors-1, -2, -3, -4a, and -4b in Xenopus laevis. This amphibian model provides a system in which both regenerating (premetamorphic; tadpole or larva stage) and nonregenerating (postmetamorphic; froglet stage) hindlimbs can be studied. In premetamorphic hindlimbs (stage 53), all of the receptors were expressed in the wound epithelium and the underlying mesenchyme. In postmetamorphic limbs (stage 61), however, transcripts for x-fibroblast growth factor receptors-1 and -2 were absent from the wound epithelium. The expression results for x-fibroblast growth factor receptors-1 and -2 were corroborated at the protein level by employing specific antibodies. Thus, it appears that expression of both fibroblast growth factor receptors-1 and -2 is associated with the ability for limb regeneration. The role of these receptors in regeneration was further investigated by using specific inhibitors to fibroblast growth factor receptors during premetamorphic hindlimb regeneration. These compounds inhibited the normal limb outgrowth and resulted, in the majority of the cases, in outgrowths of cones or spikes reminiscent of growth that is seen in amputated postmetamorphic limbs. Thus, fibroblast growth factor receptors-1 and -2 expression and function should be regarded as paramount for the ability of limb regeneration in Xenopus.

Regulation of lens regeneration by fibroblast growth factor receptor 1.

Lens regeneration in vivo is restricted to some urodeles only. After removal of the lens, this remarkable event is initiated from the dorsal iris. The pigmented epithelial cells from the dorsal iris dedifferentiate and subsequently transdifferentiate to form the regenerating lens. This property of the dorsal iris implies specific regulation along the dorsal-ventral axis. To date, no known genes are known to be specifically expressed in the dedifferentiating cells and to be involved in lens regeneration. In this paper, we show that FGFR-1 expression and function is correlated with the process of lens regeneration from the dorsal iris. Following lentectomy, FGFR-1 protein is specifically present in the dedifferentiating pigment epithelial cells in the dorsal iris, but is absent from the ventral iris. Subsequently, FGFR-1 protein is present throughout the process of lens regeneration and fiber differentiation. Furthermore, we show that an FGFR-1-specific inhibitor is able to inhibit the process of transdifferentiation and lens regeneration. In this sense, FGFR-1 can be regarded as the first known lens regeneration-associated factor.

Role of 5' HoxD genes in chondrogenesis in vitro.

In the present study, we have examined by in situ hybridization, expression of five 5' HoxD cluster genes (D9, D10, D11, D12 and D13) during chondrogenesis of chick limb bud mesenchymal cells in vitro. After one day in culture, D9 and D13 gene expression was restricted to patches of mesenchymal cells, while expression of D10, Dll, and D12 gene was prominent in all mesenchymal cells. In 3-day cultures, D9 and D13 genes were expressed only in cartilage nodules, while D10, Dll, and D12 genes were expressed in both cartilage nodules and in all mesenchymal cells. These observations indicate two different patterns of expression; one for D9 and D13, and a different one for D10, Dll, and D12. These patterns of expression seem to correlate with patterns of cell proliferation and differentiation to chondrocytes. The role of these HoxD genes was further investigated by employing antisense S-oligomers. We found that oligodeoxynucleotides complementary to HoxD (D10-D 13) mRNAs were capable of inhibiting chondrogenesis. These data suggest that expression of HoxD genes is required for mesenchymal condensation, and differentiation to chondrocytes. This in turn implies that these HoxD genes aside from their role in the patterning of the developing skeletal elements might regulate down-stream factors necessary for cartilage differentiation as well.

Graded expression of Emx-2 in the adult newt limb and its corresponding regeneration blastema.

Amputation of a newt limb causes stump cells to organize the reformation of the missing structures. The phenomenon is remarkably precise in that the regeneration is perfect. During the first few days following amputation, the tissue proximal to the plane of amputation gives rise to the blastema, an area of growth composed of mesenchymal cells covered by a single epithelium. The blastema possesses a morphogenetic potential characteristic of the structures that have been amputated. Looking for control genes putatively involved in regeneration, we cloned the newt version of the mouse and human Emx-2. Its expression is restricted to the skin of the regeneration territories and is graded along the proximal-distal axis of both forelimb and hindlimb, with higher levels in distal regions. The regeneration blastema also show this proximal-distal graded level of expression with distal blastemas (mid-radius and ulna) showing higher levels of expression when compared to blastemas of more proximal origin (mid-humerus). Finally, retinoic acid proximalizes both the level of Emx-2 expression and the positional memory of the blastema suggesting Emx-2 may participate in pattern formation by specifying positional information.

Regulation of homeobox-containing genes during lens regeneration.

In this study, the expression of homeobox-containing genes was evaluated after lentectomy in the newt, which is competent for lens regeneration, and in the axolotl which is not. Such a comparison was designed to offer insights about possible regulation due to regenerative abilities. Six homeobox-containing genes were examined: NvHox A4, NvHox B1, NvHox 7, NvHox X, Nvmsx-1 and Xbr1. For all genes examined, it was found that soon after lentectomy in the newt there was a general down-regulation in the retina. This down-regulation varied among the Hox genes with NvHox 7 and NvHox B1 being virtually absent in the initial stages; their expression was re-established to the original levels after the reappearance of lens. The expression patterns, for NvHox 7 and NvHox B1 were the same when the lens was removed and then displaced. However, in axolotl, down-regulation was not observed. These data suggest that the observed regulation is related to the process of lens regeneration and provide the first molecular evidence that lens regeneration could be dependent on retina and underline the importance of this tissue in lens regeneration. Such patterns link expression of homeobox-containing genes and lens regeneration and can be now used to understand the underlying mechanisms of lens regeneration and transdifferentiation.

Expression of integrins during axolotl limb regeneration.

Limb regeneration in urodeles is achieved through the dedifferentiation of tissues at the amputation plane and through the production of the blastema. This tissue breakdown is possible by extensive alterations in molecules of the extracellular matrix. In this respect we describe the regulation of several integrins during such events. It was found that alpha 1 and beta 1 integrins were down-regulated as blastema formation proceeded. In contrast, the expression of alpha 3, alpha 6 and alpha v integrins were upregulated in the blastema. These data are consistent with the roles of integrins in developmental phenomena and are discussed in light of the mechanisms of dedifferentiation.

Is DNA a language?

DNA sequences usually involve local construction rules that affect different scales. As such their "dictionary" may not follow Zipf's law (a power law) which is followed in every natural language. Indeed, analysis of many DNA sequences suggests that no linguistics connections to DNA exist and that even though it has structure DNA is not a language. Computer simulations and a biological approach to this problem further support these results.

Conservation of fibroblast growth factor function in lens regeneration.

In urodele amphibians, lens induction during development and regeneration occurs through different pathways. During development, the lens is induced from the mutual interaction of the ectoderm and the optic vesicle, whereas after lentectomy the lens is regenerated through the transdifferentiation of the iris-pigmented epithelial cells. Given the known role of fibroblast growth factors (FGFs) during lens development, we examined whether or not the expression and the effects of exogenous FGF during urodele lens regeneration were conserved. In this paper, we describe expression of FGF-1 and its receptors, FGFR-2 (KGFR and bek variants) and FGFR-3, in newts during lens regeneration. Expression of these genes was readily observed in the dedifferentiating pigmented epithelial cells, and the levels of expression were high in the lens epithelium and the differentiating fibers and lower in the retina. These patterns of expression implied involvement of FGFs in lens regeneration. To further elucidate this function, we examined the effects of exogenous FGF-1 and FGF-4 during lens regeneration. FGF-1 or FGF-4 treatment in lentectomized eyes resulted in the induction of abnormalities reminiscent to the ones induced during lens development in transgenic mice. Effects included transformation of epithelial cells to fiber cells, double lens regeneration, and lenses with abnormal polarity. These results establish that FGF molecules are key factors in fiber differentiation, polarity, and morphogenesis of the lens during regeneration even though the regenerating lens is induced by a different mechanism than in lens development. In this sense, FGF function in lens regeneration and development should be regarded as conserved. Such conservation should help elucidate the mechanisms of lens regeneration in urodeles and its absence in higher vertebrates.

Regulation of the Wilms' tumor gene during spermatogenesis.

Spermatogenesis is the process by which male germ cells develop and mature, a pathway that includes a transition from a mitotic to a meiotic cell cycle. Throughout this pathway, the germ cells are in close contact with their nurturing cells, the Sertoli cells. Sertoli-germ cell interactions are difficult to study in mammals due to the complex cellular organization of their seminiferous tubules. The urodele amphibian testis, however, provides a unique system to study the process of germ cell maturation; it is organized in a gradient-like cystic structure, in which synchronized germ cells can be found within the same cyst. The Wilms' tumor gene (WT1) has been shown to be an essential gene for the formation of the gonads in mice, and it has been implicated in a variety of differentiation processes. The WT1 gene is thus a good candidate for the study of the differentiation processes involved in the maturation of the male germ cells. By using a probe for the urodele WT1 homologue in in situ hybridization studies, as well as an antibody against the WT1 protein in immunohistochemistry studies, we determined that WT1 gene expression in Sertoli cells depends on the stage of maturation of the associated germ cell. Thus, WT1 mRNA was detected only in Sertoli cells of cysts that contained early spermatogonia. No mRNA expression was observed in cysts containing late spermatogonia, germ cells undergoing meiosis, or germ cells going through spermiogenesis. Immunohistochemistry studies confirmed that WT1 protein was strongly expressed in Sertoli cells associated with early spermatogonia but not in late ones. The protein was also found in Sertoli cells associated with germ cells that undergo the subsequent stages of meiosis and spermiogenesis. These results suggest that WT1 could be involved in the regulation by Sertoli cells of germ cell maturation and possibly in the progression from a mitotic to a meiotic cell cycle.

9-cis retinoic acid antagonizes the stimulatory effect of 1,25 dihydroxyvitamin D3 on chondrogenesis of chick limb bud mesenchymal cells: interactions of their receptors.

Retinoids or vitamin D have been found to profoundly affect pattern formation and chondrogenesis in the developing limb. These substances mediate their actions through their nuclear receptors. In the present investigation, we present data showing that 9-cis RA, the ligand for RXR can stimulate chondrogenesis of chick limb bud mesenchymal cells, however, in combination, it antagonizes the stimulatory effect of vitamin D in the same system. The receptors for 9-cis RA (RXR) and vitamin D (VDR) were also shown to be present in the mesenchymal cells and to form heterodimers. These results implicate these receptors in cartilage differentiation during limb development.