| 1.) Telencephalic Regionalization and Axon Tract Formation |
| The vertebrate central nervous system is characterized by region-specific
differences in the organization, morphology, and functions of its neurons. An outstanding
quest of developmental neurobiology is to understand how these regional differences arise
and how genes regulate their development. |
| The telencephalon is divided into dorsal, intermediate and ventral regions that
give rise to a variety of cell types and stereotyped axonal projections. These forebrain
subdivisions are demarcated by expression of developmental regulatory genes (transcription
factors such as emx1 (blue, dorsal) and dlx2 (red, ventral)). |
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| In 24h wild types, the axons of the anterior commissure (AC) are restricted to
ventral telencephalon (T) and do not extend into diencephalon (D). Dorsal telencephalic
neurons extend axons that form the supraoptic tract (SOT) and connect telencephalon
and diencephalon (Chitnis and Kuwada, 1990; Wilson et al., 1990). Arrow in upper panel
indicates approximate location of the SOT |
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| Goals |
We want to understand how the neurons that contribute axons to the SOT are
specified. Several lines of evidence suggest that emx genes play a key role in this event.
A fate map at the end of gastrulation suggests that most of these neurons arise from the
anterior neural plate and express emx1. In slow muscle omitted (smu) mutant embryos, Hh
signaling is blocked, because the mutation affects Smoothened, a seven-pass transmembrane
mediator of Hh signaling. In smu mutants emx expression is expanded ventrally and ventral
neurons extend axons into the diencephalons (Varga et al., 2001). Because this resembles
axon growth patterns of dorsal telencephalic cells, we investigate whether emx genes are
sufficient and/or required to instruct telencephalic cells to adopt a dorsal identity and
form SOT-like projections.
Using various genes that code for transcription factors (such as emx1/2) we tested by mRNA
overexpression, or Morpholino “knock-down” analysis how the fate of these cells
is specified and whether they extend appropriate axonal projections. |
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| Anterior views of axon tracts in the 48h zebrafish forebrain.
The embryos were labeled using anti-acetylated tubulin primary antibodies and fluorescence
labeled secondary antibodies. A stack of 100 confocal images was recorded in a Zeiss
510 LSM and merged into 2D. Left Panel; SOT, Supraoptic tract; AC, anterior commissure;
POC, post-optic commissure; Och optic commissure. The right panel was color coded
for depth (blue is closest and red is farthest from the viewer, green, yellow: intermediate
slices). |
TOP |
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| 2.) Development of the Hypothalamo / Pituitary System
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| The hypothalamus and pituitary gland are key regulators of the hormone
system. Sensory and endocrine information is processed and integrated in the brain
and hormone release is controlled by neuroendocrine secretion in the posterior pituitary
lobe. In addition, other hypothalamic neurons secrete releasing (RH) or release-inhibiting
hormones (RIH) into the portal blood system that control hormone release from specific
endocrine cells in the anterior pituitary lobe. |
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| Goals |
| Thus, the hypothalamic/pituitary system is a key link between the brain and
the endocrine system. Although these two tissues are in close proximity in the adult they
arise from distant locations in the embryo (Varga et al., 1999). Our goal is to learn |
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when and where the progenitors of these two cell populations come into contact during
embryogenesis, and how they interact |
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how pituitary and hypothalamic cell types are specified, and |
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what genes regulate their development |
To analyze pituitary cell specification we study smu mutant embryos. In these
embryos, an ectopic, distorted, or fused lens forms in place of the pituitary. This finding
suggests, that Hh signaling plays a key role in pituitary cell specification. We are investigating
how cell fate, position, gene expression, cell specification and Hh signaling interact in
pituitary development.
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| In smu mutant embryos an ectopic lens is formed at the expense of pituitary
gland |
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| A whole mount in situ hybridization with RNA probes against
a geneis expressed in lens and pituitary cells |
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We recently showed that a median anterior protrusion of the foxb1.2 (previously
mariposa, Moens et al., 1999, and forkhead-3, Odenthal and Nüsslein-Volhard, 1998)
gene expression domain demarcates the location of hypothalamic precursors in neural plate
(Varga et al., 1999). These cells are initially located posterior to retinal precursors
that express the odd-paired-like (opl) gene (Grinblat et al., 1998), and they later shift
anteriorly along the ventral midline to separate the eye primordia (Varga et al., 1999).
In cyclops (nodal) mutant embryos the movement does not occur and thus the eyes remain fused
across the midline. |
| A Cyclops mutant embryos: retina is fused at ventral midline |
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| Wild-Type cyclops Gene Function is Required for Anterior Movement of Ventral
Forebrain Precursors (Varga et al., 1999) |
 (A,
B) Single cells in corresponding regions of the neural plate of wild-type (A, arrowhead)
and cyclops mutant embryos (B, arrowhead) were injected with rhodamine dextran. The first
group of embryos (n = 9 wild type; n = 3 cyclops) was immediately fixed and labeled for
mar expression by mRNA in situ hybridization.
(C, D) The second group of embryos (n = 7 wild type; 2 cyclops) was fixed 2 hours later
and labeled for mar expression. (C) By this time the injected cell (arrowhead) had moved
anterior, and had stopped expressing mar in wild-type embryos. (D) In cyclops mutant embryos,
the injected cell failed to move anteriorly and remained near the anterior border of the
mar expression domain.
(E, F) The third group (n = 6 wild type; n = 3 cyclops) was fixed 4 hours after injection.
These embryos were labeled (black) to detect the expression of pax2 in the prospective optic
stalk region, emx1 in the prospective telencephalon, eng3 in the prospective midbrain hindbrain
border and krx20 in the prospective hindbrain. In wild-type embryos (E), the injected cell
(red, arrowhead) was found anterior and ventral to the eye primordia. A rudimentary ventral
forebrain had formed. (F) In cyclops mutants, the injected cell failed to move forward and
remained posterior to the eye (arrowhead). cyclops mutants lacked ventral forebrain.
(A – D) dorsal views, anterior to the top. (E, F) side views, anterior to the left,
dorsal to the top. Scale bar, 200 µm.
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| Proposed Model for the Separation of a Single Retinal Field by Median Posterior
Cells (Varga et al., 1999) |
 Ventral
diencephalic precursors (blue) move anterior (blue arrow) and ventral to the retinal field
(red) and form the primordium of the ventral forebrain. In late neural plate stages, they
occupy a location ventral to the retinal field and dorsal to the axial mesendoderm (green).
Retinal precursors move laterally (red arrows) to form the bilateral eyes. Telencephalic
cell fates at the anterolateral periphery of the neural plate are not shown here. Anterior
to the lower left, posterior to upper right, dorsal to the top. |
Some of the ventral diencephalic precursors give rise to cells in the
preoptic and paraventricular areas of the hypothalamus, including neuroendocrine and
RH/RIH secreting neurons. We analyze developmental regulation of these cell types
using barred b700 mutant embryos that lack most but not all of these anterior hypothalamic
regions. We have cloned several genes that characterize hypothalamic cell types in
this region and we will analyze
the
role of the gene that is affected in b700 for hypothalamic development
We
will map the mutation and clone the identified gene.
We
will prepare a substraction cDNA library between hypothalamic and pituitary tissue. |
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