GENES THAT PATTERN THE MOUSE EMBRYO
|
Heiner
Westphal, MD, Head,
Section on Mammalian Molecular Genetics Yangu
Zhao, PhD, Staff Scientist Lan
Chen, PhD, Postdoctoral Fellow Ipsita
Dey-Guha, PhD, Postdoctoral Fellow Dongho
Geum, PhD, Postdoctoral Fellow Marat
Gorivodsky, PhD, Postdoctoral Fellow Woon
Kyu Lee, PhD, Postdoctoral Fellow Nasir
Malik, PhD, Postdoctoral Fellow Mahua
Mukhopadhyay, PhD, Postdoctoral Fellow Edit
Hermesz, PhD, Guest Researcher Alexander
Grinberg, DVM, Senior Research Assistant Sing-Ping
Huang, MS, Senior Research Assistant Eric
Lee, DVM, Senior Research Assistant Lisa
Williams-Simons, BS, Senior Research Assistant Christina
Mailloux, BS, Predoctoral Fellow Donna Morales, BS, Predoctoral Fellow |
|
|
We
focus on the molecular genetics of embryonic development. Transcriptional control
is a key element of developmental regulation; it involves an elaborate
repertoire of cis-regulatory target gene sequences as well as the
cooperation and physical interaction of several factors that regulate gene
expression. Core elements, present in many cell contexts, are thought to form
complexes with cell- or tissue-specific factors to establish positive and
negative control of gene expression and to bring about cell specification and
tissue identity in the developing embryo. We study the molecular genetics of
this process. Much of our work focuses on the functional evaluation of
members of the LIM class of homeobox genes (termed Lhx genes) during
mouse development. During the course of our experiments, we identified two
novel classes of proteins encoded by the Ldb and Ssdp gene
families, respectively. They are co-factors whose interaction with the Lhx-encoded
LIM-homeodomain factors and other transcriptional regulators is essential for
embryonic development. We also study members of the Dkk family of Wnt
inhibitors that form a link between the action of Lhx genes and
pattern formation in the developing central nervous system. Developmental
controls exerted by Lhx genes Grinberg, Hermesz,
Huang, Lee E, Lee WK, Mailloux, Morales, Westphal, Williams-Simons,
Yamashita,a Zhao; in collaboration with members of the Palkovits
and Rubenstein research groups Over
the years, we have established that LIM-homeodomain proteins are involved in
early patterning events, in the development of the nervous system, and in the
assembly of organs, including the pituitary gland and the gonad. We have paid
special attention to the development of the mammalian central nervous system,
where the concerted action of a number of distinct Lhx gene products,
in conjunction with their co-factors, conveys regional identity. Conditional
ablation of these functions, one or more at a time, was required to probe the
intricate spatial and temporal regulation of forebrain assembly. Cre-mediated
excision of DNA sequences in the embryo, a method originally introduced by
our laboratory more than a decade ago and now widely used, is an important
research tool in this context. Several
of our recent studies have dealt with specific regional aspects of brain
development controlled by Lhx genes and their co-factors. Thus, we
found that Lhx8 mutants lack the nucleus basalis, a major source of
cholinergic input to the cerebral cortex. The number of cholinergic neurons
is also markedly reduced in several other areas of the subcortical forebrain.
However, initial steps of cholinergic neurogenesis are preserved. These
results indicate that Lhx8 plays a major role in the specification of
cholinergic lineages in the forebrain. The
closely related Lhx1 and Lhx5 genes are also topics of our
ongoing work. The two genes are prominently expressed in many regions of the
developing central nervous system, including the spinal cord and the
cerebellum. Earlier work showed that Lhx1 null embryos lack anterior
head structures and that Lhx5 null mutants are impaired in hippocampal
development. Lhx1 null embryos die at an early stage of development,
precluding a thorough analysis of possible brain defects. Our work also
addressed the possibility that functional redundancies may exist between Lhx1
and Lhx5. We therefore generated conditional Lhx1 mutants as
well as Lhx1/5 double mutants to be able to examine more closely the
individual or combined roles of both genes in brain development. The analysis
focused on the mutants’ developing cerebellum. The cerebellar phenotype
of embryos that lack either Lhx1 or Lhx5 is not remarkable.
However, examination of Lhx1/5 double mutants revealed that the
Purkinje cells of the primordial cerebellum are largely missing, as defined
by the absence of the calbindin marker. Our experiment showed that both Lhx1
and Lhx5 are essential for the generation of calbindin-positive
Purkinje cells and that there is redundancy in this function. Another
aspect of our current work concerns functions of Lhx2 in the context
of the development of the pituitary gland. Our previous studies had
established a role for this gene in brain and eye development and in
hematopoiesis. Null embryos were anophthalmic because of a developmental
arrest of the eye anlagen before formation of the optic cup. In addition,
deficient cell proliferation in the forebrain resulted in hypoplasia of the
neocortex and aplasia of the hippocampal anlagen. The Lhx2-deficient
mutants died in utero, possibly because of a cell-nonautonomous defect
of definitive erythropoiesis that caused severe anemia. Here we describe an
additional phenotype. Deletion of Lhx2 impairs formation of the
posterior lobe of the pituitary gland. We observed overproliferation and a
lack of proper differentiation of precursor cells in this tissue. Loss of Lhx2
function also leads to a disorganization of the anterior and intermediate
lobes of the pituitary gland, possibly secondary to defects in the formation
of the posterior pituitary. Our earlier work identified Lhx3 and Lhx4
as essential regulators of pituitary development. The present work shows that
Lhx2 plays an important role in the process as well. Zhao Y, Marin O, Hermesz E, Powell A, Flames N, Palkovits M,
Rubenstein JL, Westphal H. The LIM-homeobox gene Lhx8 is required for
the development of many cholinergic neurons in the mouse forebrain. Proc
Natl Acad Sci USA 2003;100:9005-9010. Mediators
of Lhx gene function Chen, Dey-Guha,
Grinberg, Geum, Gorivodsky, Huang, Malik, Mukhopadhyay, Schindler,b
Teufel,c Westphal, Zhao; in collaboration with members of the
Agulnick, Bayarsaihan, Dawid, Dorward, Downs, Kennison, Niehrs, Ogryzko,
Podtelejnikov, and Segal research groups Our
functional analysis of Ldb1 and Ssdp1, genes that we identified
as important co-factors of Lhx gene action, has contributed to an
understanding of the intricate mechanisms of transcriptional control that
govern brain patterning in the developing embryo. Ldb1 null embryos
show severe rostral defects, underscoring the essential function of the gene
in neuronal development. The Ldb1 protein, which binds to the LIM domain of Lhx-encoded
transcription factors, is an obligatory co-factor that mediates the
transcription factors’ action, as we were able to demonstrate in
vivo; embryos lacking Ldb1 function in the developing cerebellum
display a phenotype virtually identical to that of the Lhx1/5 double
mutant. Two experimental systems established in vivo the role of Ssdp
in the context of the Ldb-based complex. When we introduced mRNA encoding
mouse or Drosophila Ssdp into four-cell–stage Xenopus
embryos, the ability of co-injected Lhx1 plus Ldb1 mRNAs to
form ectopic axes was markedly enhanced, whereas Ssdp mRNA alone had
no effect. The experiment demonstrated that Ssdp interacts functionally with
the Ldb1-based complex and that the interaction has been conserved
during evolution. We were also able to show that the interaction is crucial
for embryonic development. Removal of one copy of the Drosophila Ssdp
gene markedly enhances the mildly defective wing phenotype of flies
heterozygous for a chromosomal deletion that includes Ssdp. The task
ahead is to find the genes that are regulated by Ssdp. Members
of the Dkk family of Wnt inhibitors are widely expressed in the
developing embryo. We previously reported on Dkk1, a mediator of Lhx
and other transcriptional activity during head induction. Dkk1
functional ablation results in severe rostral defects. Our recent work has
dealt with the phenotype of Dkk2 knockout mice. The mutants are
characterized by a profound defect in cornea cell turnover, indicating that Wnt
pathway regulation is essential for the maintenance of ocular surface
integrity. The cornea is a highly ordered, transparent structure whose
epithelium is replaced every three or four weeks by derivatives of stem cells
that reside in the limbus, a transitional zone between the corneal and
abutting conjunctival epithelium. The cornea of young adult Dkk2 null
mice is opaque and contains cells normally found in skin and conjunctiva,
including hair follicles, sebaceous glands, and goblet cells. Wnt signals are
upregulated in the mutant limbus region. We have therefore identified Dkk2
as a Wnt inhibitor that regulates the turnover of corneal epithelia. Chen L, Segal D, Hukriede N, Podtelejnikov A, Bayarsaihan D,
Kennison JA, Ogryzko V, Dawid IB, Westphal H. Ssdp proteins interact with the
LIM-domain binding protein Ldb1 to regulate development. Proc Natl Acad
Sci USA 2002;99:14320-14325. del Barco Barrantes I, Davidson G, Grone HJ, Westphal H, Niehrs
C. Dkk1 and noggin cooperate in mammalian head induction. Genes
Dev 2003;17:2239-2244. Mukhopadhyay M, Teufel A, Yamashita T, Agulnick AD, Chen L,
Downs KM, Schindler A, Grinberg A, Huang SP, Dorward D, Westphal H.
Functional ablation of the mouse Ldb1 gene results in severe
patterning defects during gastrulation. Development 2003;130:495-505. aTsyoshi bAlice Schindler, BS, former
Predoctoral Fellow cAndreas COLLABORATORS Alan Agulnick, PhD, CyThera, Inc., Dashzeveg Bayarsaihan, PhD, Igor Dawid, PhD, Laboratory of Molecular
Genetics, NICHD, Dave Dorward, PhD, Rocky Mountain
Laboratory, NIAID, Karen Downs, PhD, James A. Kennison, PhD, Laboratory of
Molecular Genetics, NICHD, Christof Niehrs, PhD, Deutsches
Krebsforschungszentrum, Vasily Ogryzko, PhD, Institut André Lwoff, Miklos Palkovits, MD, Alexandre Podtelejnikov, PhD, John L. Rubenstein, MD, PhD, Daniel Segal, PhD,
|
|
