Multistep Control of Pituitary Organogenesis

See allHide authors and affiliations

Science  05 Dec 1997:
Vol. 278, Issue 5344, pp. 1809-1812
DOI: 10.1126/science.278.5344.1809


Lhx3 and Lhx4 (Gsh4), two closely related LIM homeobox genes, determine formation of the pituitary gland in mice. Rathke's pouch is formed in two steps—first as a rudiment and later as a definitive pouch. Lhx3 and Lhx4have redundant control over formation of the definitive pouch.Lhx3 controls a subsequent step of pituitary fate commitment. Thereafter, Lhx3 and Lhx4 together regulate proliferation and differentiation of pituitary-specific cell lineages. Thus, Lhx3 and Lhx4 dictate pituitary organ identity by controlling developmental decisions at multiple stages of organogenesis.

Pituitary organogenesis is driven by a series of developmental decisions controlled by transcription regulators.Pit-1/GHF-1 (1-4) and prophet-1 (5) direct establishment of certain pituitary cell lineages [for review, see (6)]. Targeted mutation of the Lhx3 gene revealed its role in the specification of most pituitary lineages (7). This study focuses on earlier steps in pituitary organ formation. We analyze the effects of null mutations in Lhx3and Lhx4 (8), a gene closely related toLhx3 (8-11), and show that both genes direct formation of the pituitary gland in mice.

The anterior and intermediate lobes of the pituitary are derived from the oral ectoderm that invaginates to form Rathke's pouch (12). Rathke's pouch gives rise to at least six pituitary-specific cell lineages (6, 12).

Lhx3 and Lhx4 are expressed throughout the invaginating pouch at day 9.5 of gestation (E9.5) (Fig.1, a and e). However, at E12.5,Lhx4 expression becomes restricted to the future anterior lobe of the pituitary gland, whereas Lhx3 remains expressed in the whole pouch (Fig. 1, b and f). At E15.5, Lhx4expression diminishes; Lhx3 expression is maintained (Fig.1, c and g). In the adult pituitary, Lhx3 is expressed at a higher level than Lhx4 in the anterior and intermediate lobes (Fig. 1, d and h).

Figure 1

Expression of Lhx3 and Lhx4in developing and adult pituitary. Sagittal sections of Rathke's pouch or coronal sections of adult pituitary were hybridized toLhx3-specific (7) orLhx4-specific (8) riboprobes. Antisense riboprobes were transcribed from Lhx3 or Lhx4cDNA with RNA polymerase (Ambion) in the presence of uridine 5′-[33P]triphosphate (NEN). In situ hybridization was performed on tissue sections essentially as described (16). For photography, sections were stained with bisbenzimide (10 μg/ml) and simultaneously viewed in dark-field and ultraviolet illumination. R, Rathke's pouch; PL, posterior lobe; IL, intermediate lobe; AL, anterior lobe. Bars, 100 μm.

Null mutations of either Lhx3 (7) orLhx4 (see below) do not prevent formation of Rathke's pouch. We generatedLhx3−/−/Lhx4 −/− mice carrying null mutations at both loci (3−/−4−/− for short). In these mutants the oral ectoderm invaginates normally to form a pouch rudiment (Fig. 2d). The pouch rudiment grows no further after E12.5 (Fig. 2e), and by E15.5, as the cartilage of the sphenoid bone grows across the floor of the brain, the entire rudiment is observed pressed back toward the oral cavity (Fig. 2f).

Figure 2

Ontogeny of anterior and intermediate lobes of pituitary development in WT mice (a toc), in 3−/−4−/− double mutants (d to f), and in mutants for one gene that are heterozygous at the other locus (g tol). A pouch rudiment is formed in the absence of bothLhx3 and Lhx4 genes. However, this rudiment fails to grow into a definitive pouch in the double mutant. Downgrowth of the infundibulum occurs in the absence of a definitive pouch. I, infundibulum; R, Rathke's pouch; pr, pouch rudiment. Sagittal sections of embryos were stained with hematoxylin and eosin. Lhx3and Lhx4 genotyping was done by Southern blotting and polymerase chain reaction, respectively, as described (7,8). Bar, 100 μm.

After E9.5, normal pouch development proceeds from a rudiment to a definitive pouch that is characterized by an extension of the pouch into the brain cavity, where it abuts the infundibulum. InLhx3 −/− or Lhx4 −/−mutants a definitive, albeit defective, pouch forms (7, 13). Analysis of mutants for one gene that are heterozygous at the other locus revealed that one wild-type (WT) allele of either Lhx3or Lhx4 is sufficient for formation of a definitive pouch (Fig. 2, g to l).

The next developmental step is commitment to the fate of the pituitary organ, which leads to formation of a proper pituitary structure and specification of pituitary lineages. This step is absolutely dependent on Lhx3 and is not realized in mutants that lackLhx3 (Fig. 2, j to l) [see also (7)]. Histological analysis of pituitary development in mutants with intermediate genotypes showed that Rathke's pouch gave rise to a pituitary structure in the presence of at least one copy ofLhx3 (Fig. 2, g to i) (13), but not in the absence of Lhx3 (7). Commitment to an organ fate also implies that the primordium will eventually give rise to organ-specific cell lineages. Therefore, we assessed the ability of mutant primordia to differentiate into organ-specific cell lineages. Marker gene and protein expression was assessed by in situ hybridization and immunocytochemistry. Transcripts for the α-glycoprotein subunit (GSU), Pit-1, growth hormone (GH), and thyroid-stimulating hormone β subunit (TSHβ) are present in theLhx4 −/− mutant but not in theLhx3 −/− mutant (7, 13). In the 3+/−- 4−/− pituitary, cells containingPit-1, GSU, GH, and TSHβ transcripts are present, but there are fewer than in the 3+/+4−/−pituitary (13). Therefore, the presence of one copy of theLhx3 gene is sufficient for specification of pituitary cell lineages.

In the Lhx4 −/− mutant, immunohistochemistry revealed that only one of three mutant pouches contained a few luteinizing hormone–positive (LH+) cells (Fig. 3A). In situ hybridization with probes specific for LH and for the receptor for gonadotropin-releasing hormone (GnRHR) showed similar results at both E15.5 and E18.5 (13). Thus, Lhx4may support, but is not required for, specification of gonadotroph cells.

Figure 3

Immunohistological (A), morphological (B), and in situ hybridization (C) analysis of pituitary development in the Lhx4 −/−mutant. (A) Immunohistological analysis (17) of pituitary-specific lineage development in the Lhx4 −/− mutant. Paraffin sections derived from E18.5 normal and mutant mice were stained with antibodies specific to adrenocorticotropic hormone (ACTH) (13), PRL (13), melanocyte-stimulating hormone (MSH), GSU, GH, TSH, and LH. All five anterior pituitary–specific cell lineages are present in the Lhx4 −/− pituitary but in dramatically reduced numbers. There are about the same number of melanotrophs in the intermediate lobe of theLhx4 −/− pituitary and in the control. Bar, 100 μm. (B) (a and c) Sagittal sections derived from E15.5 embryos and stained with hematoxylin and eosin, showing that the ventral wall (the perspective anterior lobe) of theLhx4 −/− pouch is hypoplastic. The lumen is enlarged as a result of reduced proliferation of the precursors. (b and d) Pituitary glands were dissected from newborn mice. The anterior lobe of the Lhx4 −/−pituitary is clearly reduced in size. Bars, 100 μm. (C) Development of lineage precursors in the Lhx4 −/− pituitary. Sagittal sections of Lhx4 −/− or control mouse at E15.5 were hybridized to GnRHR-specific and Pit-1–specific riboprobes. Both Pit-1 + and GnRHR+precursors are present. Arrow highlights a few LH+ cells in the anterior lobe. Bar, 100 μm.

We conclude that elaboration of a definitive pouch is directed by either Lhx3 or Lhx4, and subsequent organ fate commitment is regulated solely by Lhx3. Formation of Rathke's pouch and commitment of cells to the fate of the pituitary organ are distinct developmental events. The function ofLhx3 in the latter event is not replaceable by Lhx4.

All defective pouches display defects in cell proliferation (Figs. 2 and 3), with no detectable increase in programmed cell death (13). These defects in cell proliferation depend on Lhx3 andLhx4 gene dosage. The severity of cell proliferation defects aligned as follows: 3−/−4−/− > 3−/−4+/− > 3−/−4+/+ > 3+/−4−/− > 3+/+4−/− > 3+/−4+/− (Figs. 2 and 3) (13). InLhx3 −/− mutants, pituitary precursor cells cease proliferation before most lineage markers are expressed (7). In Lhx4 −/− mutants, cell proliferation in the intermediate lobe is less affected, but the anterior lobe is distinctly hypoplastic (Fig. 3, A and B) and all five cell lineages in the anterior lobe show reduced numbers (Fig. 3A) (13). The GnRHR+ gonadotroph precursors and the Pit-1+ somatotroph, lactotroph, and thyrotroph precursors are present at E15.5 (Fig. 3C, b and d). This suggests that the reduction in terminally differentiated pituitary cell lineages in the Lhx4 −/− mouse is caused by a cell proliferation defect at the precursor level and that proliferation of these precursors requires the function of the Lhx4 gene.

Thus, pituitary organogenesis is a multistep process, and each step is controlled by a distinct genetic program (Fig.4). Formation of Rathke's pouch involves at least two independent developmental decisions. At the beginning, a portion of the oral ectoderm apposing the neural ectoderm of the diencephalon diverges from its original ectodermal fate to become the pituitary anlage. Tissue recombination experiments have provided evidence that surrounding neural tissues are a source of inductive signals for determination of the primordium (14). Formation of the pouch rudiment does not require the function of eitherLhx3 or Lhx4 (Fig. 4, arrow a). Lhx3or Lhx4 controls development of the pouch rudiment into a definitive pouch (Fig. 4, arrow b). Commitment of precursor cells in Rathke's pouch to a pituitary organ fate is controlled byLhx3 (Fig. 4, arrow c). From E12.5 onward, cells begin to express lineage-specific molecules. Lhx4 is required for proliferation of lineage precursors. In theLhx3 −/− mutant, pouch development is arrested before the appearance of most pituitary cell lineages (7), precluding an exhaustive evaluation of Lhx3 function in lineage development. However, Lhx3 regulates the expression of Pit-1, GSU, and, in synergy with Pit-1, GH and prolactin (PRL) in vitro (7, 15), suggesting thatLhx3 also regulates cellular differentiation (Fig. 4, arrow d). Because Lhx3 is expressed in almost all pituitary precursor cells, it is unlikely that this gene determines cell type identity. Rather, it may act in concert with genes that are lineage restricted, such as Pit-1/GHF-1 (1-4) and prophet-1 (5).

Figure 4

Schematic illustration of ontogenetic events leading to pituitary formation.

  • * To whom correspondence should be addressed. E-mail: hw{at}


View Abstract

Stay Connected to Science

Navigate This Article