Karlstrom Lab Research Movies(Fish and Axons)

Zebrafish Forebrain Patterning
We are using a variety of genetic and experimental approaches to determine how cell-cell signaling via secreted proteins of the hedgehog family functions to pattern the zebrafish ventral forebrain.  We are analyzing several mutations that affect both hedgehog signaling and cell differentiation in the forebrain.  The you-too locus encodes zebrafish gli2, a hedgehog responsive transcription factor.  Mutations in gli2 result in forebrain and axon guidance defects.  Forebrain defects include transdifferentiation of the pituitary into an ectopic lens (Fig.1).

Fig. 1. Forebrain and axon defects in yout-too mutants.  Ventral (facial) views of wildtype (left) and you-too mutant zebrafish embryos labeled with the ZN-5 antibody to visualize retinal ganglion cells and their axons.  Retinal axons fail to cross the midline in you-too mutants, and an ectopic lens often develops at the ventral midline in place of the anterior pituitary (center of right picture, between eyes).

Pituitary Induction and Patterning
The zebrafish is a relatively untapped resource in the study of endocrine development, and has profound advantages for research into pituitary induction, patterning and cellular differentiation during embryogenesis. The zebrafish is genetically tractable, and the physical and optical accessibility of the embryo makes it possible to observe the earliest events in pituitary formation.  These events occur at embryonic stages that are extremely difficult to monitor in mammals. Importantly, pituitary structure and cell-types are remarkably conserved from fish to humans, making studies in zebrafish relevant to human development.

Based on our analysis of a series of zebrafish mutations that affect forebrain patterning and axon guidance (Karlstrom et al, 1996), the small protein Hedgehog (Hh) has emerged as a critical player in the early induction of the pituitary placode, as well as in the functional patterning of the adenohypophysis and the differentiation of a subset of endocrine cell types (Sbrogna et al, 2003).  This role for Hh is conserved across vertebrates, with defects in human Hh signaling leading to common congenital defects including holoprosencephaly.  Further, a recent report links aberrant Hh signaling to common pituitary adenomas in adults. Thus an understanding of how Hh guides pituitary development is a key issue for human health, and the zebrafish provides a window into the molecular and cellular mechanisms involved.

Fig. 2.  Conserved organization of the adult vertebrate pituitary.  Left two panels show the location of the pituitary gland in humans and fish (arrows).  The right two panels show conserved structure within the pituitary (Adapted from Liem et al).  In both species, the adenohypophysis is divided into two lobes (purple and pink) along the anterior-posterior axis.  In fish, the neurohypophysis (light blue) is positioned dorsally rather than posteriorly.  Within the adenohypophysis, endocrine cell positions are largely conserved, with PRL and AcTH cells being anterior (green), GH and TSH secreting cells being medial (yellow), and MSH secreting cells being posterior (blue).


Fig. 3.  shh and A-P patterning. (A) lim3 and (B) somatolactin expression mark the developing adenohypophysis in 36 hour embryos.  (C) nk2.2 is a Hh responsive transcription factor and is expressed near the source of Hh in the diencephalon. (D) prolactin cells differentiate in this anterior domain. (E) Model showing expression of Hh responsive transcription factors and endocrine cell types in relation to the source of Hh  (adapted from Sbrogna et al., 2003).

Axon Guidance
The mutant belladonna appears to specifically affect guidance of only a few axons.  Defects are limited to three axon pathways, the optic nerve and the post optic commissure and the anterior commissure (Fig. 2).  We are attempting to clone the belladonna gene in order to determine the molecular nature of this specific mutation.   In another project, we are beginning experiments to identify the guidance cells that appear to be missing or mis-specified in you-too.  By  transplanting wildtype cells into the ventral forebrain of you-too mutant embryos, we hope to rescue midline crossing and therefore identify cells important for axon guidance across the midline.

Fig. 4.  Axon guidance errors in belladonna mutants.  Ventral (facial) views of wildtype (left) and belladonna mutant zebrafish embryos labeled with an antibody to visualize axons. Left: Wildype axons cross the midline of the forebrain to form the postoptic (lower) and anterior (upper) commissure.  Axons in these two pathways fail to cross the midline in belladonna mutants.

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