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Role of the LPM in zebrafish pancreas development
Recent work has been aimed at directing in vitro differentiation of embryonic stem cells into insulinproducing pancreatic beta cells for use in treatment of Type I Diabetes. The intrinsic molecular mechanisms that
specify pancreatic beta cells have been the primary focus of such research. Extrinsic factors, however, are also
necessary for establishing pancreas specification in vivo. A better understanding of how extrinsic factors affect in
vivo differentiation of endoderm cells into pancreatic beta cells would advance in vitro differentiation studies.
In addition to signals from the notochord and blood vessel endothelium, signals from the lateral plate
mesoderm (LPM) have been shown to be important for directing a pancreas-specific fate in chick (Kumar). Signals
from the LPM may direct endodermal cells toward a pancreas fate or signals may be required for maintaining
specification or directing proper timing of differentiation, proliferation, or migration. By creating transgenic
zebrafish with LPM-specific expression of diphtheria toxin A-chain (DTA), the influence of LPM on pancreatic
organogenesis in vivo will be analyzed. Early and late genetic ablation of the LPM will determine effects of early
and late acting signals on pancreatic development in vivo.
Recent work suggests that development of the zebrafish, mouse, and chick pancreas are similar making
zebrafish a model organism for pancreas studies (Beimar, Field). Also, zebrafish can survive for several days
without circulation, which would result from loss of the LPM. This uncoupling of circulation and LPM defects may
help segregate the effects of blood vessel epithelium and LPM signaling on pancreas development. The combination
of LPM genetic ablation studies and molecular and forward genetic analysis will provide a well rounded approach
for understanding how signals from tissues surrounding the endoderm contribute to pancreas development during
zebrafish embryogenesis
Specific Aim 1
To determine whether signals that originate in the LPM direct development of the pancreatic domain in zebrafish and
when these signals act.
Rationale: Pdx-1 is the earliest known marker for pancreas specification. It is not known what initiates pdx-1
expression. Signals from the notochord and blood vessel epithelium seem to be permissive toward pancreas
specification, while signals from the LPM may be instructive. The question remains as to the nature of
mesoderm/endoderm interaction prior to establishment of the pancreatic domain. The instructive LPM signals may
initiate pdx-1 expression and direct endoderm toward the pancreatic fate. Alternatively, pancreatic fate may be the
default state of patterned endoderm and LPM may be necessary for anterior-posterior patterning. Ablation of the
LPM at a stage prior to pdx-1 expression is key to asking if the LPM provides the endoderm with a pancreas
specification signal.
After initiation of pdx-1 expression, cells directed toward a pancreatic fate continue to differentiate into
cells that produce endocrine hormones such as insulin and glucagon and cells that produce exocrine enzymes such as
trypsin and amylase. Mouse explant cultures of pdx-1 positive endoderm cells have been shown to differentiate in
the absence of surrounding tissues into insulin and glucagon producing cells as well as cells positive for p48, which
marks the exocrine lineage (Deutsch). This suggests that the pancreatic endoderm does not require signals from
LPM after initiation of pdx-1 expression to continue toward pancreatic differentiation. The pdx-1 positive tissue
explant data, however, does not address the possible role of LPM in proper temporal execution of the pancreas
differentiation and morphogenesis process. Ablating LPM cells at various time points during the differentiation and
morphogenesis of the pancreas is key to establish if the LPM directs the specified pancreatic domain to further
differentiate into a functional pancreas.
Experimental Design: In order to ablate the LPM in development prior to initiation of pdx-1 expression and at time
points thereafter, I will utilize a modified Gal4-VP16 expression system to create DNA constructs that direct
expression of DTA under the spatial control of the dHand promoter and the temporal control of an inducible Gal4VP16 that is restricted to the cytoplasm until the embryos are treated with RU486 (Chae). The basic Gal4-VP16
expression system has been successfully utilized in the Stainier lab; the inducible expression system has been
successfully utilized in Xenopus. dHAND, a bHLH transcription factor, is initially expressed in the LPM of zebrafish
around 8 hours post fertilization (hpf) (Angelo), a time point prior to the appearance of pdx-1 expressing cells at 10somites. The dHand promoter will direct expression of the Gal4-VP16 activator fused to a mutated form of the
human progesterone receptor ligand binding domain in the LPM at about 8hpf. Following treatment with RU486,
expression of the DTA will be activated by nuclear translocation of the Gal4-VP16 activation domain (Figure 1).
This approach allows development of a zebrafish line that carries an inducible diphtheria toxin A for ablation of the
LPM at various time points.
The dHand promoter has not been identified in zebrafish to date. Therefore, I would first identify the
elements of the dHand promoter directing LPM expression by using information from the available Fugu and
zebrafish genomes and testing promoter constructs driving green fluorescent protein (GFP) for LPM-specific
expression. This technique has been used to successfully identify other promoter sequences in this lab. After
injecting the constructs and identifying stable expression lines, I will analyze the effects of LPM genetic ablation on
pancreas development by collecting staged embryos with induced DTA expression in the LPM.
In order to determine if absence of the LPM prevents pancreatic specification in these embryos, I will use
immunohistochemistry, in situ hybridization, and confocal microscopy to examine expression of pdx-1, the earliest
known pancreas specification marker. In order to determine if absence of LPM signaling affects later stages of
development, I will use immunohistochemistry, in situ hybridization, and confocal microscopy to examine late
differentiation markers such as islet-1, insulin, glucagon, trypsin, and amylase in transverse sections and whole
mount stainings of zebrafish embryos. This analysis will allow me to assay differentiation molecularly and
morphologically.
Outcomes: Without the early presence of the LPM and its signals, the pancreatic domain may or may not be
specified. This will be addressed by inducing DTA expression early at gastrulation and looking for pdx-1 expression
starting at 10-somites. If pdx-1 expression occurs as early as 10-somites, early signals from the LPM are not
required for directing endoderm cells toward the pancreatic lineage or these signals occur prior to the induction time
point. If pdx-1 expression occurs later, there may be redundant pancreas-specifying signals, early signals originating
from the LPM and later signals originating from another surrounding tissue or simply acting intrinsic to the
endoderm. If pdx-1 expression does not occur, genetic ablation of the LPM has also resulted in the ablation of
signals directing endoderm toward a pancreatic fate. The lack of pdx-1 expression could result from ablation of a
direct signal arising from the LPM or ablation of an indirect signal arising from the LPM but acting on other tissues
that, in turn, direct the establishment of the pancreatic domain.
If expression of later pancreatic markers such as glucagon and insulin are similar to wild type embryos,
LPM signals are not required for proper timing of the differentiation of pre-pancreatic cells into endocrine and
exocrine cell-types. Alternatively, late pancreatic markers may be delayed in expression as compared to wildtype
embryos. This result suggests that LPM signaling assists in proper timing of differentiation. Despite the data
showing that pdx-1 positive endoderm explants can differentiate into endocrine and exocrine lineages without
surrounding tissues, it is possible that this could not occur in the context of the whole embryo with LPM ablation
after 10-somites. This observation would suggest that the LPM also signals for later differentiation and
morphogenesis and that the explant cultures from previous studies may have already received the signal(s) or
contained contaminant tissue.
Specific Aim 2
To characterize and positionally clone a gene causing a zebrafish mutant with both LPM and pancreatic defects, and,
therefore to identify signaling molecules expressed in the LPM that directly or indirectly instruct endoderm to
become pancreas.
Rationale: Several mutants in pancreas development have recently been identified by a forward genetic screen
using the gut-GFP transgenic line, which expresses GFP in endodermal cells. One of these mutants, CF-5.4,shows
defects in fin, liver, and pancreas development (Figure 2). These phenotypes suggest the locus affected by the lesion
acts in the LPM. Further, by identifying the locus mutated in CF-5.4, I will most likely identify one of the extrinsic
molecules in the LPM that act to direct pancreatic development in vivo.
Experimental Design: Using positional cloning techniques and genetic mapping techniques, I will identify the gene
containing the lesion causing the CF-5.4 phenotype.
Outcomes: Once the lesion is cloned, the gene’s identity and expression profile will help determine the primary
effect of the mutation. Regardless of whether it is acting autonomously in LPM or non-autonomously on the
pancreas, cloning this gene will identify a molecule important for proper pancreas development.
Conclusion
By developing technology for directing differentiation of stem cells into pancreatic beta cells, islet transplantation
procedures could provide a cure for diabetes. Much of the information for developing such technology focuses on
intrinsic transcription factors. This approach is missing the influence of extrinsic signaling molecules that originate
from tissues surrounding the developing pancreas in vivo. LPM directed expression of DTA will ablate one of these
tissues surrounding the endoderm, and, therefore, any instructive signals arising from the LPM and acting on the
endoderm. By observing the effects of LPM ablation and cloning genes that encode LPM-specific signals, we will
identify and better understand the role of extrinsic signaling molecules in directing endoderm toward a pancreatic fate.
Figure 2. Confocal images of wt and mutant day 2 guts
Figure 1
pdHan
d
+RU 486
VP
Gal
GALPR is inactive in the
absence of RU 486
References
P
R
UA
S
DTA
DTA expression is induced
only in the presence of RU 486
WT
CF-5.4
= gut-GFP
= islet-1,2,3
= insulin
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