A role of the LIM-homeobox gene Lhx2 in the regulation of pituitary development

Abstract

The mammalian pituitary gland originates from two separate germinal tissues during embryonic development. The anterior and intermediate lobes of the pituitary are derived from Rathke's pouch, a pocket formed by an invagination of the oral ectoderm. The posterior lobe is derived from the infundibulum, which is formed by evagination of the neuroectoderm in the ventral diencephalon. Previous studies have shown that development of Rathke's pouch and the generation of distinct populations of hormone-producing endocrine cell lineages in the anterior/intermediate pituitary lobes is regulated by a number of transcription factors expressed in the pouch and by inductive signals from the ventral diencephalon/infundibulum. However, little is known about factors that regulate the development of the posterior pituitary lobe. In this study, we show that the LIM-homeobox gene Lhx2 is extensively expressed in the developing ventral diencephalon, including the infundibulum and the posterior lobe of the pituitary. Deletion of Lhx2 gene results in persistent cell proliferation, a complete failure of evagination of the neuroectoderm in the ventral diencephalon, and defects in the formation of the distinct morphological features of the infundibulum and the posterior pituitary lobe. Rathke's pouch is formed and endocrine cell lineages are generated in the anterior/intermediate pituitary lobes of the Lhx2 mutant. However, the shape and organization of the pouch and the anterior/intermediate pituitary lobes are severely altered due to the defects in development of the infundibulum and the posterior lobe. Our study thus reveals an essential role for Lhx2 in the regulation of posterior pituitary development and suggests a mechanism whereby development of the posterior lobe may affect the development of the anterior and intermediate lobes of the pituitary gland.

Fig. 1

Expression of Lhx2 in the developing ventral diencephalon and posterior lobe of the pituitary gland. A-D. Lhx2 mRNA was detected by in situ hybridization on sagittal sections through the developing pituitary of E9.5 (A), E10.5 (B), E11.5 (C), and E12.5 (D) mouse embryos. Rostral is to the left. Insets in B-C show part of a coronal section through the pituitary. Arrowheads in A-B point at an area that directly abuts Rathke's pouch (R). Arrowheads in C and D point at the evaginating neuroectoderm of the ventral diencephalon and the posterior lobe of the pituitary, respectively. Bar in D represents 200 μm for all panels.

Fig. 2

Defects in morphogenesis of the pituitary gland in Lhx2^−/− mutants. A-H. Sagittal sections through the developing pituitary of E10.5 (A, E), E11.5 (B, F), E12.5 (C, G), and E13.5 (D, H) control (A-D) and Lhx2^−/− mutant (E-H) embryos were stained by hematoxylin and eosin. Rostral is to the left. In contrast to control embryos (A-D), the neuroectoderm of the ventral diencephalon in Lhx2^−/− mutants (E-H) fails to evaginate to form the posterior lobe (PL) of the pituitary. Arrowheads in A-D point at the part of the neuroectoderm bending dorsally directly above the dorsal pole of Rathke's pouch in the control embryos. Arrowheads in E-H points at a comparable region in the mutant embryos that fails to evaginate. Rathke's pouch (R) in Lhx2^−/− mutants (E-H) initially forms, but fails to extend dorsally. The dorsal part of the pouch extends instead along the rostral-caudal axis and then grows ventrally into the lumen of the pouch, resulting in misshapen anterior (AL) and intermediate lobes (IL) of the pituitary gland (arrows). Bar in A represents 200 μm for all panels.

Fig. 3

Histological defects of the pituitary gland in Lhx2^−/− mutants at E15.5. Coronal (A, D) and sagittal (B, C, E, F) sections through the pituitary of control (A-C) and Lhx2^−/− mutant (D-F) embryos were stained with hematoxylin and eosin. C and F are enlargements of the anterior/intermediated lobes of the pituitary shown in B and D, respectively. In Lhx2^−/− mutants, a dense group of cells (indicated by white asterisks in D, E) occupies the area between the third ventricle and the anterior/intermediate lobes. The median eminence (pointed out by black arrows in B, C) and the posterior lobe (PL) of the pituitary are missing. The anterior (AL) and intermediate (IL) lobes of the pituitary in Lhx2^−/− mutants are severely disorganized. The rostral tip of the anterior lobe is missing (as indicated by black asterisks in E, F), and there is an increased presence of blood vessels containing immature red blood cells (green arrow in F) and mononuclear cells (green arrowhead in F). Bar in F represents 200 μm for A, B, D, E and 70 μm for C, F.

Fig. 4

Increased cell proliferation in the area of the infundibulum of Lhx2^−/− mutants. Anti-BrdU staining of sagittal (A-F) or coronal (G, H) sections through the pituitary of E11.5 (A, B), E12.5 (C, D), E13.5 (E, F), and E15.5 (G, H) control (A, C, E, G) and Lhx2^−/− mutant (B, D, F, H) embryos. Black bars in A-B and C-D, which are placed 100 μm from the most ventral-caudal point of the infundibulum (indicated by black arrows), mark the areas of the infundibulum where BrdU-positive cells were counted. Bar in H represents 100 μm for A-F and 200 μm for G-H.

Fig. 5

Molecular marker analysis of pituitary defects in Lhx2^−/− mutants. A-B. Sagittal sections through the developing pituitary of E12.5 control (A) and Lhx2^−/− mutant (B) embryos stained with an antibody to calbindin (CB). C-F. Sagittal sections through the pituitary of E15.5 control (C, E) and Lhx2^−/− mutant (D, F) embryos stained with antibodies to calbindin (CB) (C, D) and vasopressin (VP) (E, F), respectively. Arrowhead in A points at CB-positive cells in the posterior lobe of the pituitary. Arrow in E points at the VP-positive axons in the posterior lobe of the pituitary. Bar in F represents 200 μm for A-B and 140 μm for C-F.

Fig. 6

Increased cell apoptosis in the ventral diencephalon of Lhx2^−/− mutants. TUNEL staining of sections from E13.5 (A, B) and E15.5 (C, D) embryos shows an increase of cell apoptosis in the ventral diencephalic region adjacent to the anterior/intermediate pituitary lobes in Lhx2^−/− mutants (B, D, indicated by arrowheads) as compared to the controls (A, C). Bar in D represents 100 μm for all panels.

Fig. 7

Analysis of endocrine cell lineages of the pituitary gland in Lhx2^−/− mutants. Immunostaining of sagittal sections through the developing pituitary of E15.5 embryos shows the presence but abnormal distribution of the various endocrine cell lineages, including thyrotropes (A, F), lactotropes (B, G), corticotropes (C, H), somatotropes (D, I), and melantropes (E, J), in Lhx2^−/− mutants (F-J) as compared to the controls (A-E). Bar in J represents 300 μm for all panels.

Fig. 8

Dorsal-ventral polarity of Rathke's pouch is maintained in Lhx2^−/− mutants. In situ hybridization analysis of Six3 (A, B), and immunostaining of Islet1 (C, D) of sagittal sections through the developing pituitary of E12.5 embryos shows that the dorsal-ventral polarity of Rathke's pouch is preserved in Lhx2^−/− mutants (B, D) and comparable to the controls (A, C). Bar in D represents 170 μm for all panels.

Fig. 9

Analysis of expression of signaling molecules in Lhx2^−/− mutants. In situ hybridization analysis of sagittal sections through the developing ventral diencephalon and Rathke's pouch of E11.5 embryos shows that Wnt 5a (A, D) and Bmp4 (B, E) mRNAs are expressed normally in Lhx2^−/− mutants (D, E) as compared to the controls (A, B). Fgf8 (C, C’, F, F’) mRNA is detected in the ventral diencephalon of both mutant (F, F’) and control (C, C’) embryos, but the expression domain extends more rostrally in the mutants as compared to the controls (arrowheads). Rathke's pouch shown in A, B, D, E is outlined with a white dotted line. C’ and F’ are enlargements of the outlined areas shown in C and F, respectively. Bar in A represents 200 μm for A-F and 100 μm for C’ and F’.

Fig. 10

A schematic drawing showing the morphogenesis of the anterior /intermediate lobes of the pituitary in normal and Lhx2^−/− mutant embryos. Early in development (E12.5), proliferating cells (green dots) are located in the dorsal region whereas early differentiating cells positive for calbindin (purple dots) are located in the ventral regions of Rathke's pouch in both control and the Lhx2 mutant embryos. In control embryos, the cells leaving the cell cycle (yellow dots) move ventrally along the lumen of the pouch and eventually (E15.5) form the large mass of the anterior lobe, pushing the early population of calbindin-positive cells toward the rostral tip of the anterior lobe. In Lhx2 mutant embryos, as the pouch fails to extend dorsally, many of the cells leaving the cell cycle are unable to move ventrally along the side of the pouch. These cells instead push ventrally into the lumen of the pouch to form the ectopic mass. As fewer cells reach the ventral region of the pouch, the calbindin-positive cells failed to be pushed rostrally to form the rostral tip of the anterior lobe in Lhx2 mutants.