Noggin regulates Bmp4 activity during pituitary induction

Bone morphogenetic protein (Bmp) signaling is critical for the development and patterning of the mouse pituitary from the initial induction of Rathke's pouch to cell specification in the anterior lobe. We examined the regulation of Bmp signaling during pituitary development by analyzing null embryos for noggin, a Bmp 2 and 4 antagonist. Noggin is expressed in the ventral diencephalon during Rathke's pouch induction, in the underlying cartilage plate during cell specification and in the adult anterior pituitary gland. Noggin null embryos have a variable pituitary phenotype, which ranges from a rostrally displaced Rathke's pouch to induction of secondary pituitary tissue. While cell specification in the anterior pituitary appears normal, patterning in the ventral diencephalon is disrupted; Bmp4 activity is expanded resulting in Fibroblast growth factor 10 repression and in a rostral shift in the boundary between the Bmp4 and Sonic hedgehog expression domains. The expanded domain of Bmp4 activity also results in additional invaginations of oral ectoderm and can shift the position of Rathke's pouch or create secondary pituitary tissue. This work demonstrates the importance of attenuating the activity of Bmp signaling during pituitary induction in order to maintain the proper balance of signaling factors necessary for pituitary organogenesis.     

Premature differentiation and aberrant movement of pituitary cells lacking both Hes1 and Prop1

In the pituitary, the transition from proliferating progenitor cell into differentiated hormone producing cell is carefully regulated in a time-dependent and spatially-restricted manner. We report that two targets of Notch signaling, Hes1 and Prop1, are needed to maintain progenitors within Rathke's pouch and for the restriction of differentiated cells to the ventral pituitary. We observed ACTH and αGSU producing cells that had prematurely differentiated within Rathke's pouch along with correlated ectopic expression of Mash1 only when both Prop1 and Hes1 were lost. We also discovered that downregulation of N-cadherin expression in cells as they transition from Rathke's pouch to the anterior lobe appears to be essential for their movement. In the Prop1 mutant, cells are trapped in Rathke's pouch and N-cadherin expression remains high. Also, Slug, a marker of epithelial-to-mesenchymal transition, is absent in the dorsal anterior lobe. When Hes1 is lost in the Prop1 mutant, N-cadherin is downregulated and cells are able to exit Rathke's pouch but have lost their migrational cues and form ectopic foci surrounding Rathke's pouch. Our data reveal important overlapping functions of Hes1 and Prop1 in cell differentiation and movement that are critical for pituitary organogenesis.     

Granulated 'marginal cell layer' in the rat anterior pituitary gland

A granulated 'marginal layer cell' was observed in the lining of Rathke's residual pouch of 5 and 10 day-old rat anterior pituitary glands. Immunohistochemistry was not employed to identify the precise function of these cells. However, the cytological characteristics of nearly all of the cells indicated that they resembled GH-secreting cells, with a few displaying morphological features of corticotrophs. In pituitary glands of 5-20 day-old rats, both ends of Rathke's residual pouch extended into the pars distalis at the site of transitional zone of this lobe and of the pars intermedia. The cells within the 'invading' residual pouch contained numerous microvilli. In the middle portion of the residual pouch, cavities lined by 'marginal layer cells' had numerous microvilli and were adjoined by junctional complexes. In the adult rat pituitary gland, there were no granulated cells in the 'marginal cell layer' and no invasion of the residual pouch into the anterior lobe. From these data the possible source of the follicle and of the folliculo-stellate cells in the anterior pituitary of the rat is proposed.     

Hes1 is required for pituitary growth and melanotrope specification

Rathke's pouch contains progenitor cells that differentiate into the endocrine cells of the pituitary gland. It gives rise to gonadotrope, thyrotrope, somatotrope, corticotrope and lactotrope cells in the anterior lobe and the intermediate lobe melanotropes. Pituitary precursor cells express many members of the Notch signaling pathway including the downstream effector gene Hes1. We hypothesized that Hes1 regulates the timing of precursor differentiation and cell fate determination. To test this idea, we expressed Hes1 in differentiating pituitary cells and found that it can inhibit gonadotrope and thyrotrope differentiation. Pituitaries of Hes1 deficient mice have anterior lobe hypoplasia. All cells in the anterior lobe are specified and differentiate, but an early period of increased cell death and reduced proliferation causes reduced growth, evident as early as e14.5. In addition, cells within the intermediate lobe differentiate into somatotropes instead of melanotropes. Thus, the Hes1 repressor is essential for melanotrope specification. These results demonstrate that Notch signaling plays multiple roles in pituitary development, influencing precursor number, organ size, cell differentiation and ultimately cell fate.     

Appearence of LH-Immunoreactive Cells in the Rathke's Pouch of the Chicken Embryo

The differentiation of the pituitary of the chicken embryo was studied by means of an immunohistochemical technique using antisera to turkey and chicken pituitary hormones. Immunoreactive LH-cells are detected in 4-day embryos (stage 23 of Hamburger and Hamilton) when the primordium of the anterior pituitary, the Rathke's pouch is only composed of a single-layer epithelium lined with an undifferentiated mesenchyme. A few immunoreactive cells are observed grouped on the posterior aspect of the pouch. As development proceeds, a strip of positive cells is detected encircling the Rathke's pouch. Prolactin-, growth hormone-, and ACTH-immunoreactive cells are detected in 6- and 7-day embryos, only after the pituitary has acquired its characteristic structure with cords in which different cell types become progressively recognizable. The early appearance of immunoreactive LH-cells following a precise distribution shows that secretory properties and differentiation capacities are acquired simultaneously in the epithelium of the Rathke's pouch and may be induced by the same stimulus.     

Appearance of LH-immunoreactive cells in the Rathke's pouch of the chicken embryo

The differentiation of the pituitary of the chicken embryo was studied by means of an immunohistochemical technique using antisera to turkey and chicken pituitary hormones. Immunoreactive LH-cells are detected in 4-day embryos (stage 23 of Hamburger and Hamilton) when the primordium of the anterior pituitary, the Rathke's pouch is only composed of a single-layer epithelium lined with an undifferentiated mesenchyme. A few immunoreactive cells are observed grouped on the posterior aspect of the pouch. As development proceeds, a strip of positive cells is detected encircling the Rathke's pouch. Prolactin-, growth hormone-, and ACTH-immunoreactive cells are detected in 6- and 7-day embryos, only after the pituitary has acquired its characteristic structure with cords in which different cell types become progressively recognizable. The early appearance of immunoreactive LH-cells following a precise distribution shows that secretory properties and differentiation capacities are acquired simultaneously in the epithelium of the Rathke's pouch and may be induced by the same stimulus.     

Granulated ‘marginal cell layer’ in the rat anterior pituitary gland

A granulated ‘marginal layer cell’ was observed in the lining of Rathke's residual pouch of 5 and 10 day-old rat anterior pituitary glands. Immunohistochemistry was not employed to identify the precise function of these cells. However, the cytological characteristics of nearly all of the cells indicated that they resembled GH-secreting cells, with a few displaying morphological features of corticotrophs. In pituitary glands of 5–20 day-old rats, both ends of Rathke's residual pouch extended into the pars distalis at the site of transitional zone of this lobe and of the pars intermedia. The cells within the ‘invading’ residual pouch contained numerous microvilli. In the middle portion of the residual pouch, cavities lined by ‘marginal layer cells’ had numerous microvilli and were adjoined by junctional complexes. In the adult rat pituitary gland, there were no granulated cells in the ‘marginal cell layer’ and no invasion of the residual pouch into the anterior lobe. From these data the possible source of the follicle and of the folliculo-stellate cells in the anterior pituitary of the rat is proposed.     

Birthdating studies reshape models for pituitary gland cell specification

The intermediate and anterior lobes of the pituitary gland are derived from an invagination of oral ectoderm that forms Rathke's pouch. During gestation proliferating cells are enriched around the pouch lumen, and they appear to delaminate as they exit the cell cycle and differentiate. During late mouse gestation and the postnatal period, anterior lobe progenitors re-enter the cell cycle and expand the populations of specialized, hormone-producing cells. At birth, all cell types are present, and their localization appears stratified based on cell type. We conducted a birth dating study of Rathke's pouch derivatives to determine whether the location of specialized cells at birth is correlated with the timing of cell cycle exit. We find that all of the anterior lobe cell types initiate differentiation concurrently with a peak between e11.5 and e13.5. Differentiation of intermediate lobe melanotropes is delayed relative to anterior lobe cell types. We discovered that specialized cell types are not grouped together based on birth date and are dispersed throughout the anterior lobe. Thus, the apparent stratification of specialized cells at birth is not correlated with cell cycle exit. Thus, the currently popular model of cell specification, dependent upon timing of extrinsic, directional gradients of signaling molecules, needs revision. We propose that signals intrinsic to Rathke's pouch are necessary for cell specification between e11.5 and e13.5 and that cell–cell communication likely plays an important role in regulating this process.     

Involvement of pro-apoptotic and anti-apoptotic factors in the early development of the human pituitary gland

The spatial and temporal pattern of appearance of pro-apoptotic caspase-3 and p53 proteins, and anti-apoptotic bcl-2 protein was investigated in the developing pituitary gland of 6 human embryos 5-8-weeks old, using morphological and immunohistochemical techniques. Their dynamic appearance was analyzed in the Rathke's pouch (future adenohypophysis), mesenchyme, and in the developing neurohypophysis. In the 5th and 6th week, caspase-3 positive cells appeared in the Rathke's pouch (5%) and stalk (11%), in the mesenchyme, but not in the neurohypophysis. In the 6th and 7th week, apoptotic cells were more numerous in the caudal part of the Rathke's pouch due to its separation from the oral epithelium. Pro-apoptotic p53 protein was detected in all parts of the pituitary gland throughout the investigated period. Nuclear condensations characterized cells positive to caspase-3 and p53 proteins. Apoptotic cells displayed condensations of nuclear chromatin on an ultrastructural level as well. While caspase-3 dependent pathway of cell death participated in morphogenesis of the adenohypophysis and associated connective tissue, p53-mediated apoptosis most likely participates in morphogenesis of all parts of the gland, including neurohypophysis. The anti-apoptotic bcl-2 protein was also detected in all parts of the developing gland. With advancing development, the positivity to bcl-2 protein increased in the cells of the adenohypophysis, while it decreased in the neurohypophysis. Bcl-2 protein probably prevented cell death in all parts of the gland and enhanced cell differentiation. The described pattern of appearance of the investigated pro-apoptotic and anti-apoptotic factors might be important for normal morphogenesis and function of the pituitary gland.     

Immunohistochemical distribution of simple-epithelial-type keratins and other intermediate filament proteins in the developing human pituitary gland

Summary An immunohistochemical study of the production of the intermediate filaments [vimentin, cytokeratin, and glial filament acidic protein (GFAP)] during development of the pituitary gland was made by use of fetal and adult human pituitary tissue. Among these intermediate filament proteins in the anterior and intermediate lobes of the pituitary, cytokeratin is the first to appear, followed by GFAP and vimentin. However, only cytokeratin is seen during the period of morphogenesis of the pituitary gland, with the type-II subfamily cytokeratin 8 being the earliest to appear. Among the simple-epithelial-type cytokeratins, cytokeratins 8 and 19 were observed within the pituitary primordium during morphogenesis. Cells immunoreactive for cytokeratins 8 and 19 showed a heterogeneous three-dimensional distribution pattern in Rathke's pouch. Both cytokeratins 8 and 19 tended to be strongly positive at sites in the pituitary primordium where cells had become more loosely arranged (i.e., areas far from the diencephalon) but were only weakly positive in areas in which the epithelial cells were densely packed (i.e., areas closely associated with the diencephalon). It is concluded that, during the period of morphogenesis, Rathke's pouch has the intermediate filaments characteristic of simple epithelium and shows different immunoreactivity for simple-epithelial-type cytokeratins from place to place according to the extent of cellular differentiation.     

Cloning and characterization of porcine 4Ig-B7-H3: a potent inhibitor of porcine T-cell activation

Background

Members of the B7 superfamily costimulate the proliferation of lymphocytes during the initiation and maintenance of antigen-specific humoral and cell-mediated immune responses. B7-H3 (CD276) is a newly identified member of the B7 superfamily. It has been shown that B7-H3 plays a significant role in regulating T cell response in humans and mice, but it is not known whether a counterpart of human or murine B7-H3 exists in porcine species.

Methodology/Principal Findings

We cloned the porcine 4ig-b7-h3 gene using a blast search at the NCBI database with human b7-h3, RT-PCR and 3′-terminus RACE. Protein sequence analysis showed that the protein encoded by this gene contained 4Ig-like domains and was 90.88% identical with human 4Ig-B7-H3. Results of Dot-blot hybridization and RT-PCR showed that B7-H3 was broadly distributed in porcine tissues mainly as two isoforms, 2Ig-B7-H3 and 4Ig-B7-H3, of which 4Ig-B7-H3 was dominant. We further demonstrated that porcine 4Ig-B7-H3 was able to inhibit the proliferation and cytokine production of porcine T cells activated through the TCR pathway, similar to human B7-H3.

Conclusion

We cloned the porcine 4ig-b7-h3 gene and demonstrated that the porcine 4Ig-B7-H3 serves as a negative regulator for the T-cell immune response.     

Molecular characterization of human 4Ig-B7-H3, a member of the B7 family with four Ig-like domains

In an effort to characterize molecules with immunoregulatory potential, we raised mAbs to human dendritic cells. We selected an Ab that recognizes a molecule that is induced on monocytes differentiated in vitro toward dendritic cells. Retroviral expression cloning identified this molecule as B7-H3, a member of the B7 family described recently. In contrast to an earlier report, in which B7-H3 was described as a molecule consisting of two Ig-like domains, our cDNA encoded a type I membrane protein with four Ig-like domains, and the molecule identified by us was therefore named 4Ig-B7-H3. mRNA analysis as well as Western blotting experiments performed by us did not reveal evidence for a small B7-H3. B7-H3 is not expressed on peripheral blood lymphocytes, monocytes, or granulocytes. Upon in vitro stimulation, the expression of B7-H3 is induced on T cells, B cells, and NK cells. A number of different approaches were used to investigate the function of human B7-H3. In contrast to an earlier report, our data do not support a costimulatory role of B7-H3 in anti-CD3-mediated activation of the TCR-complex resulting in T cell proliferation and IFN-gamma production.