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di encephalon Bmp4 stomadeal *Shh ectoderm B ventral di encephalon Rathke's pouch

cell lineage

melanotropes corti cotropes Pit-1 lineage gonadotropes dorsal factors

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ventral factors

The Six3 gene (Oliver et al., 1995) is similarly associated with pituitary abnormalities in human and chick, (Gallardo et a l, 1999), although either the entire midline o f the forebrain is effected (holoprosencephaly) or an abnormal pituitary develops. A specific lack o f pituitary induction is not observed in Six3 mutants. Targeted disruption of Six3 in the hypothalamus reveals a loss of three specific nuclei (Wang & Lufkin, 2000), thus it is possible that conditional and site-specific mutants may provide useful data as to invagination events of the early pouch.

The Isll and P txl genes are both expressed in the stomadeum and Rathke’s pouch. Isll is expressed from around E9, and is down regulated by E l 1.5, corresponding closely with the dynamic expression of Bmp4 in the ventral diencephalon, suggesting a possible regulatory relationship (Ericson et al, 1998), while P txl is maintained in Rathke’s pouch throughout development into adulthood (Bach et al., 1997; Lanctot et al., 1997). Ptxl is expressed throughout the stomadeum prior to formation of Rathke’s pouch, and this broad expression may be dependent upon earlier embryonic patterning events not specific to the pituitary.

Bmp4 function is difficult to assay in the mouse as most Bmp4'^' mutant embryos die within the first 8 days o f development. A small proportion however survive to ElO,

and histological examination reveals no thickening or invagination of the oral ectoderm (Takuma et a l, 1998). Thus Bmp4 may have a role in the early induction event in pituitary organogenesis. Despite Bmp4’s possible role in controlling Isll expression in Rathke’s pouch tissue (Ericson et al., 1998), Isll itself does not appear to be involved in the invagination event.

Analysis of Rathke’s pouch development in Isll'^' mice reveals that a degree of invagination still occurs, however the pouch epithelium is unable to differentiate (Takuma et al., 1998). This indicates that Isll may be more involved in differentiation o f the tissue as opposed to controlling the invagination event itself.

Similarly a P txl knockout mouse also shows normal early development o f Rathke’s pouch (Szeto et ah, 1999).

Additionally, Sonic hedgehog {Shh) is expressed throughout the stomadeal ectoderm (and the ventral midline o f the diencephalon). B m p 4 expression from the diencephalon may be involved in suppression of Shh expression in the presumptive Rathke’s pouch tissue, creating a molecular boundary within the ectoderm (Trier et al., 1998). This event is required for early patterning of the pouch, but how or if this results in invagination is unclear. Evidence from chick suggests a role for the most rostral notochord tissue in invagination o f the non-neural ectoderm to form Rathke’s pouch, possibly implicating Shh activity (Gleiberman et al., 1999), although a molecular pathway has not yet been elucidated.

The second step o f induction is the commitment o f the early pouch to developmentally proceed (panel 3.3B). Bmp4, and then FgfS are expressed in the diencephalon during the development of Rathke’s pouch, (specifically in the presumptive infundibula process) both of which are shown to be vital for the development of the anterior pituitary (Takuma et al., 1998; Ericson et a l, 1998; Treier et al, 1998). A mouse mutant that lacks the ability to produce functional Nkx2.1 subsequently lacks FgfS expression in the ventral diencephalon. The resulting phenotype is the development of an abnormal pouch that is subsequently eliminated by apoptosis by ElO.5. Thus the anterior pituitary fails to develop.

Analysis of the abnormal pouch in Nkx2.F^' mutants reveals that the tissue expresses Isll and P txl, although both at lower levels than in wild type. More significantly, expression o f both Lim3 and Lim4 expression is absent (Takuma et al., 1998). Morphologically, the arrested phenotype o f Rathke’s pouch development in Nkx2.F^' mouse is similar to that observed in the Lim3'^';Lim4'^' double mutant mouse (Sheng et a l, 1997).

In normal pouch development, Bmp4 is expressed from E8.5 to E l 1.5 (Jones et a l, 1991). FgfS is expressed from E9.25 to E14.5 (Ericson et a l, 1998). The transition o f Bmp4 expression to FgfS expression in the ventral diencephalon roughly coincides

with a transition from Isll to Lim3 expression in Rathke’s pouch. In-vitro experiments with Fgf-soaked beads and ElO Rathke’s pouch expiants indicate that either Fgf8 is responsible for the down regulation of Isll and the maintenance of Lim3

expression, or a source of Bmp4 is required for continued Isll expression (Ericson et al., 1998). (Transplants were done on tissue that was already expressing Lim3 so the inductive ability of FgfS could not be assessed).

Activity of the Lim3 and Lim4 genes appears to be required for the survival and developmental progression of the early pouch tissue (Lin et al., 1994; Sheng et al.,

1997). These genes act with a degree of redundancy in the mouse, with one wild type allele of either Lim3 or Lim4 being sufficient to enable progression of the early invaginated tissue forward to a definitive pouch (Sheng et al., 1997). The following step however, a progression from Rathke’s pouch to anterior pituitary, is reliant on Lim3. At a genetic level, the function of Lim3 is to initiate the expression of lineage specific markers in synergy with other transcription factors, thus patterning the gland.

The Ptx2 gene appears to have an effect on development between the induction and patterning phases of development. Ptx2'^' knockout mice show arrested development of the anterior pituitary tissue following successful Lim3 induction. The ventral cells of Rathke’s pouch fail to proliferate (Lin et al., 1999). Recent work has determined a requirement for Ptx2 in maintaining Rpx and Prop-1 expression within the embryonic gland. At later stages, Ptx functions in synergy with a number of transcription factors (including Pit-1, GATA2 and SF-1) in terminal differentiation steps and hormone production (Suh et a l, 2002). Mutations in the Ptx2 gene are responsible for the human condition Rieger syndrome (Semina et a l , 1996) the symptoms of which include abnormal craniofacial development as well as asymmetry defects (Lu et a l,

1999). The Ptx2 gene has also been suggested to act downstream o f the nodal pathway in asymmetrical aspects of zebrafish development (Concha et a l, 2000; Bisgrove & Yost, 2001).

3.1.4 Patterning the anterior pituitary gland.

The murine anterior pituitary is composed of six distinct cell types, all o f which are derived from a single unit o f ectodermal tissue as described in the previous section. Patterning the gland again relies on external signalling from the diencephalon, a possible role of the mesenchyme tissue and a large number o f transcription factors expressed within the developing gland itself (reviews include Scully & Rosenfeld, 2002; Sheng & Westpal, 1999; Dansen & Rosenfeld, 1999). A summary of cell lineage development at a molecular level is given in figure 3.4.

As described earlier, Shh expression is down regulated in the developing pouch. The boundary of non-Shh expressing cells in the ectoderm is marked by Bmp2 expression from the boundary site across the pouch in a decreasing ventral to dorsal gradient (Treier et al., 1998). A DV polarity is established in the pouch by the ventral Bmp2 expression along with Fgf8 expression from the ventral diencephalon which creates an opposing but overlapping gradient from the dorsal edge of the pouch (Ericson et al., 1998). Other proteins involved in the process may be WntSa and Chordin, both of which are expressed in the mesenchyme immediately caudal to Rathke’s pouch (Treier et al., 1998). The ventral pouch will give rise to the pars distalis and the dorsal portion to the medial eminence. At these initial stages, it is proposed that FgfS promotes proliferation, where as Bmp2 promotes differentiation (Sheng & Westphal,

1999).

FgfS secreted from the diencephalon may induce Lim3 and Lim4 expression within Rathke’s pouch. Lim3 serves not only to stabilise the developing pouch, but also to specify populations o f cell types (Bach et al., 1995). The aforementioned experiments mimicking the activity of FgfS expression in the diencephalon, initiating Lim3 expression have also revealed the importance o f later FgfS activity in patterning the anterior pituitary (Ericson et al., 1998). The first differentiating cell type in the dorsal pituitary is a population of corticotropes, followed ventrally by a population of thyrotropes which lie medially in the anterior pituitary. FgfS activity is required for thyrotrope development, but not for corticotrope development. Thus isolation of the

Figure 3.4 Cell lineage specification within the developing anterior