A dynamic Gli code interprets Hh signals to regulate induction, patterning, and endocrine cell specification in the zebrafish pituitary.

Hedgehog (Hh) signaling is necessary for the induction and functional patterning of the pituitary placode, however the mechanisms by which Hh signals are interpreted by placodal cells are unknown. Here we show distinct temporal requirements for Hh signaling in endocrine cell differentiation and describe a dynamic Gli transcriptional response code that interprets these Hh signals within the developing adenohypophysis. Gli1 is required for the differentiation of selected endocrine cell types and acts as the major activator of Hh-mediated pituitary induction, while Gli2a and Gli2b contribute more minor activator functions. Intriguingly, this Gli response code changes as development proceeds. Gli1 continues to be required for the activation of the Hh response anteriorly in the pars distalis. In contrast, Gli2b is required to repress Hh target gene expression posteriorly in the pars intermedia. Consistent with these changing roles, gli1, gli2a, and gli2b, but not gli3, are expressed in pituitary precursor cells at the anterior neural ridge. Later in development, gli1 expression is maintained throughout the adenohypophysis while gli2a and gli2b expression are restricted to the pars intermedia. Given the link between Hh signaling and pituitary adenomas in humans, our data suggest misregulation of Gli function may contribute to these common pituitary tumors.

Multiple roles for Hedgehog signaling in zebrafish pituitary development.

The endocrine-secreting lobe of the pituitary gland, or adenohypophysis, forms from cells at the anterior margin of the neural plate through inductive interactions involving secreted morphogens of the Hedgehog (Hh), fibroblast growth factor (FGF), and bone morphogenetic protein (BMP) families. To better understand when and where Hh signaling influences pituitary development, we have analyzed the effects of blocking Hh signaling both pharmacologically (cyclopamine treatments) and genetically (zebrafish Hh pathway mutants). While current models state that Shh signaling from the oral ectoderm patterns the pituitary after placode induction, our data suggest that Shh plays a direct early role in both pituitary induction and patterning, and that early Hh signals comes from adjacent neural ectoderm. We report that Hh signaling is necessary between 10 and 15 h of development for induction of the zebrafish adenohypophysis, a time when shh is expressed only in neural tissue. We show that the Hh responsive genes ptc1 and nk2.2 are expressed in preplacodal cells at the anterior margin of the neural tube at this time, indicating that these cells are directly receiving Hh signals. Later (15-20 h) cyclopamine treatments disrupt anterior expression of nk2.2 and Prolactin, showing that early functional patterning requires Hh signals. Consistent with a direct role for Hh signaling in pituitary induction and patterning, overexpression of Shh results in expanded adenohypophyseal expression of lim3, expansion of nk2.2 into the posterior adenohypophysis, and an increase in Prolactin- and Somatolactin-secreting cells. We also use the zebrafish Hh pathway mutants to document the range of pituitary defects that occur when different elements of the Hh signaling pathway are mutated. These defects, ranging from a complete loss of the adenohypophysis (smu/smo and yot/gli2 mutants) to more subtle patterning defects (dtr/gli1 mutants), may correlate to human Hh signaling mutant phenotypes seen in Holoprosencephaly and other congenital disorders. Our results reveal multiple and distinct roles for Hh signaling in the formation of the vertebrate pituitary gland, and suggest that Hh signaling from neural ectoderm is necessary for induction and functional patterning of the vertebrate pituitary gland.

Sonic hedgehog is required early in pancreatic islet development.

Pancreatic organogenesis relies on a complex interplay of cell-autonomous and extracellular signals. We demonstrate that the morphogen sonic hedgehog (Shh) is required for pancreatic development in zebrafish. Genetic mutants of Shh and its signaling pathway establish this dependence as specific to endocrine, but not exocrine, pancreas. Using cyclopamine to inhibit hedgehog signaling, we show that transient Shh signaling is necessary during gastrulation for subsequent differentiation of endoderm into islet tissue. A second hedgehog-dependent activity occurring later in development was also identified and may be analogous to the known action of Shh in gut endoderm to direct localization of pancreatic development. The early action of Shh may be part of a more general process allowing neuroendocrine cells to originate in nonneuroectodermally derived tissues.