Molecular mechanisms of pituitary organogenesis: In search of novel regulatory genes.

Defects in pituitary gland organogenesis are sometimes associated with congenital anomalies that affect head development. Lesions in transcription factors and signaling pathways explain some of these developmental syndromes. Basic research studies, including the characterization of genetically engineered mice, provide a mechanistic framework for understanding how mutations create the clinical characteristics observed in patients. Defects in BMP, WNT, Notch, and FGF signaling pathways affect induction and growth of the pituitary primordium and other organ systems partly by altering the balance between signaling pathways. The PITX and LHX transcription factor families influence pituitary and head development and are clinically relevant. A few later-acting transcription factors have pituitary-specific effects, including PROP1, POU1F1 (PIT1), and TPIT (TBX19), while others, such as NeuroD1 and NR5A1 (SF1), are syndromic, influencing development of other endocrine organs. We conducted a survey of genes transcribed in developing mouse pituitary to find candidates for cases of pituitary hormone deficiency of unknown etiology. We identified numerous transcription factors that are members of gene families with roles in syndromic or non-syndromic pituitary hormone deficiency. This collection is a rich source for future basic and clinical studies.

Developmental regulation of Notch signaling genes in the embryonic pituitary: Prop1 deficiency affects Notch2 expression.

Normal development of the pituitary gland requires coordination between the maintenance of a progenitor cell pool and the selection of progenitor cells for differentiation. As Notch signaling controls progenitor cell differentiation in many embryonic tissues, we investigated the involvement of this important developmental pathway in the embryonic pituitary. We report that expression of Notch signaling genes is spatially and temporally regulated in pituitary embryogenesis and implicate Notch2 in the differentiation of several cell lineages. Notch2, Notch3, and Dll1 are initially expressed by most cells within the pituitary primordium and become restricted to a subset of the progenitor cell pool as differentiated pituitary cells begin to appear. Mutations in the transcription factor Prop1 interfere with pituitary growth and cell specification, although the mechanism is unknown. Notch2 expression is nearly absent in the developing pituitaries of Prop1 mutant mice, but unaltered in some other panhypopituitary mutants, revealing that Prop1 is directly or indirectly required for normal Notch2 expression. Transgenic overexpression of Prop1 is not sufficient for enhancement of endogenous Notch2 expression, indicating that there are multiple inputs into this pathway. Dll3 is expressed only in the presumptive corticotrope and melanotrope cells. Analysis of Dll3 null mutants indicates that Dll3 is not required for specification of these two cell types, although there may be functional overlap with Dll1. The spatial and temporal expression patterns of Notch signaling genes in the pituitary suggest overlapping roles in pituitary growth and cell specification.

Persistent Prop1 expression delays gonadotrope differentiation and enhances pituitary tumor susceptibility.

The 'paired'-like homeodomain transcription factor Prop1 is essential for the expansion of the pituitary primordia and for the differentiation and/or function of the hormone-producing cells of the anterior pituitary gland. Prop1 expression is normally extinguished before transcription of most differentiation markers is initiated. We report that constitutive expression of Prop1 interferes with anterior pituitary cell differentiation and increases the susceptibility for pituitary tumors. The terminal differentiation of pituitary gonadotropes is delayed, resulting in transient hypogonadism and a delay in the onset of puberty. Thyrotrope differentiation occurs normally, but thyrotrope function is impaired resulting in mild hypothyroidism. Aged mice exhibit defects consistent with misregulation of pituitary cell proliferation, including adenomatous hyperplasia with the formation of Rathke's cleft cysts and tumors. Thus, silencing Prop1 is important for normal pituitary development and function. These data suggest that gain-of-function mutations in PROP1 could contribute to the most common human pituitary endocrinopathies and tumors.

Neuregulin induces GABA(A) receptor subunit expression and neurite outgrowth in cerebellar granule cells.

Neuregulin (NRG), a growth and differentiation factor that signals via erbB receptor tyrosine kinases, has been shown to have biological effects in both the CNS and the peripheral nervous system. We report here that erbB4 is expressed in mature cerebellar granule cells, where it appears to be concentrated at the granule cell postsynaptic terminals. We also show that one form of NRG, Ig-NRG, plays a crucial role in aspects of cerebellar granule cell development in vitro. First, Ig-NRG treatment of granule cells in culture selectively induces the expression of the GABA(A) receptor beta2 subunit. This increase in subunit expression is paralleled by an increase in functional GABA(A) receptors. In contrast to its effects on GABA(A) receptor subunit expression, Ig-NRG does not upregulate NMDA receptor N2B and N2C subunit expression. Second, we demonstrate that Ig-NRG also enhances neurite outgrowth from cultured granule cells. Ig-NRG does not, however, act as a survival factor for the granule cells. We have compared the effect of Ig-NRG with the effects of brain-derived neurotrophic factor (BDNF), a neurotrophin that exerts specific effects on granule cells in culture, and found that BDNF does not mimic the effects of Ig-NRG on GABA(A) receptor subunit expression. Our results show that Ig-NRG has specific effects on granule cell development and maturation and may regulate these processes in vivo.

Immature granule neurons from cerebella of different ages exhibit distinct developmental potentials.

Cultured cerebellar granule neurons exhibit different developmental potentials in culture dependent on cerebellar age at plating. In cultures prepared at postnatal days (P)2-6, when all granule neurons reside in the external germinal layer (EGL) in vivo, levels of the GABA(A) receptor beta2 and gamma2 subunit mRNAs are constant. In contrast, in cultures prepared at P8-10, when neurons have begun to migrate into the internal granule cell layer (IGL), the mRNAs increase several-fold in a pattern mimicking that found in vivo. To determine the relationship between neuronal differentiation in culture and potential to express GABA(A) receptor beta2 and gamma2 subunit transcripts in the mature pattern, neuronal maturity in P6 and P10 cultures was compared. Bromodeoxyuridine labeling studies demonstrated that P10 as well as P6 cultures contained neurons only from the EGL. Moreover, the maturation of cultured P10 and P6 neurons appeared virtually identical. Cells dissociated at both ages expressed mRNAs encoding the EGL markers MATH-1 and TAG-1. The MATH-1 transcript disappeared from cultures maintained 4 days when expression of the GABA(A) receptor alpha6 subunit, a marker of mature cells, was initiated. Thus, although cultured P6 and P10 granule neurons exhibit the same maturation markers, P10 neurons presumably have been modulated by environmental cues specifying increases in GABA(A) receptor beta2 and gamma2 subunit expression. This possibility is supported by the finding that extracts of dissociated P10 cells but not P6 cells induce increases in GABA(A) receptor subunit expression in P6 cultures.

GABAA receptor subunit expression and assembly in cultured rat cerebellar granule neurons.

The assembly of multisubunit GABAA receptors in specific neuronal populations is a complex process which is poorly understood. To begin to examine receptor assembly, alpha 1, beta 2/3, and gamma 2 subunit polypeptide expression and association, as well as receptor binding, were examined in cultured rat cerebellar granule neurons. Western blots revealed two alpha 1-immunoreactive proteins. A 39 kDa species was maximal at 2 days in culture and subsequently declined. In contrast, a 51 kDa polypeptide, the anticipated size of the mature alpha 1 subunit, was first detected at 4 days and increased throughout the culture period. Additional studies demonstrated that the beta 2/3 and gamma 2 subunits were detectable at 2 days and attained maximal levels by 6 days. The level of [3H]Ro15-1788 binding, a measure of assembled receptors, rose in parallel with the increases in the 51 kDa alpha 1, beta 2/3 and gamma 2 subunits. Moreover, the 51 kDa alpha 1, beta 2/3, and gamma 2 subunits were associated in receptor complexes. However, immunohistochemical studies demonstrated the presence of substantial intracellular subunit staining. This finding suggest that only some of the subunits expressed in granule neurons contribute to functional GABAA receptors on the cell surface.