THE
MESD MUTATION
UNCOUPLES THE HEAD AND
TRUNK ORGANIZER
Mary E. Wines, Kristen Brown, Stephen Wefer, Lance Lee,
Thomas Rosenquist, and Bernadette C. Holdener
In mouse, the formation of the primitive streak begins by embryonic day (E)
6.5 at the posterior end of the embryo, and represents the first morphological
evidence of anteroposterior axis specification. The node or mouse equivalent
of the trunk organizer is localized at the anterior end of the primitive streak.
Cells ingressing through the primitive streak and node are the source of the
definitive endoderm and mesoderm of the mouse embryo. These tissues play an
important role in subsequent anteroposterior patterning of the epiblast and
are essential for formation of the blood and major organ differentiation. Recently,
reports of regionally restricted gene expression prior to primitive streak formation
provided evidence that anteroposterior polarity was established well before
the primitive streak is distinct. These dynamic expression patterns, combined
with lineage analysis of visceral endoderm and epiblast fate maps led to the
prediction that anteroposterior axis specification evolves from an earlier proximal/distal
patterning of the early egg-cylinder.
Embryos homozygous for deletions that remove the mesoderm
development (mesd) gene fail to generate mesoderm. In an effort to understand
why mesd mutant embryos fail to undergo gastrulation and form the primary
germ layers, we have characterized the mesd mutant phenotype at the molecular.Using whole mount in situ hybridization we
demonstrate that the mesd mutation not only prevents expression of genes
in the early primitive streak, but also blocks differentiation of the embryonic
ectoderm. Remarkably, in mesd mutants localization of the head organizer
and expression of an early anterior ectoderm marker occurs independent of primitive
streak function and mesoderm induction. We have identified a novel mesd candidate
gene and are currently determining the subcellular localization of the mesd
protein, identifying interacting proteins using two-hybrid analysis, and determining
the role of mesd in cell proliferation.
FIG 1:
Anterior/Posterior Axis Determination and Primitive
Streak
Establishment
FIG. 2: Mesd
Mutants Fail to Establish a Primitive streak
FIG. 3: Defects
in the Mesd Proximal Epiblast Underlie the Failure to
form a Primitive Streak
FIG. 4: Mesd
Mutants Correctly Localize the Head Organizer
FIG. 5: The
Mesd Epiblast Adopts an Anterior Fate
FIG. 6: Mesd
is Required for Differentiation of the Epiblast
FIG. 7: Mesd
is Required in Extraembryonic Tissue for Epiblast Differentiation
FIG. 8: Two
candidate genes map within the mesd interval
FIG. 9: Mesd-1
and Mesd-2 are novel genes expressed
in the early
embryo and the adult
FIG. 10: The
mesd phenotype is rescued by a BAC containing Mesd-2
Conclusions:
Mesoderm Development
Fig. 1: Mesoderm
Induction AND Anterior/Posterior Polarity
- Figure adapted from Ding et al. (1998) Nature 395: 702-707
- Proximal/distal patterning of the early egg-cylinder, visualized at E 5.5
- E 6.0, precedes anterior/posterior axis specification.
- Anteroposterior patterning occurs when directional cell movements localize
the head organizer to the anterior visceral endoderm (AVE) at E6.0 and the
trunk organizer to the node at the anterior end of the primitive streak at
E 6.5.
- Cell movements during conversion of proximal/distal patterning to anterior/posterior
patterning are visualized by changes in Nodal, Cripto, Hex, Brachyury,
and BMP4 gene expression in the developing embryo.
FIG.
2: Mesd Mutants Fail to Establish a
Primitive streak
- In Xenopus both Fgfs and Wnts are strong posteriorizing
agents; ectopic expression of these signaling molecules results in the expansion
of posterior tissues while inhibition of signals leads to expanded anterior
domains.
- Fgf8 is expressed in wild-type mouse embryos in the posterior epiblast
in those cells that will first pass through the streak. Fgf8 is not
expressed in mesd mutants.
- Wnt3 transcripts mark a broader area of the posterior epiblast and
emerging mesoderm of wild type embryos. In contrast, wnt3 transcripts
are limited the visceral endoderm of 3/6 mesd mutant embryos and not
expressed in remaining mutants. Expression was never detected in the primitive
streak of mesd mutants.
- The lack of expression of these markers (as well as Fgf4, Goosecoid,
and Evx) suggest that the failure to form a functional primitive
streak likely underlies mesoderm deficiency in mesd embryos.
FIG. 3: Defects
in the Mesd Proximal Epiblast Underlie Primitive Streak Defects
- Fate mapping of the early epiblast demonstrates that the precursors of the
primitive streak reside in the proximal epiblast. To determine if the failure
to form a primitive streak in mesd mutants is secondary to defects
in the proximal epiblast we examined expression of Brachyury, BMP4, Nodal,
and Cripto.
- Brachyury (T) expression is delayed in mesd mutants; transcripts
are detected in a ring of proximal epiblast and fail to become localized to
the posterior.
- BMP4 expression is delayed in mesd mutants; transcripts are
detected in a ring of extraembryonic ectoderm and are not localized to the
posterior.
- Nodal expression is dramatically altered in mesd mutants;
transcripts are detected in the distal epiblast and visceral endoderm and
are excluded from the proximal epiblast.
- Cripto, unexpectedly, is localized to the posterior in 2/6 mesd
mutants. The remaining mutants express Cripto throughout the epiblast
or in a proximal/distal gradient of expression.
- Failure to activate Nodal in the proximal epiblast suggest that a
defect in the proximal epiblast results in the failure to establish a primitive
streak in mesd mutants. Correct expression of T provides limited
evidence to suggest that patterning of the proximal epiblast is normal, suggesting
that the defect may lie in the control of cell proliferation.
FIG. 4: Mesd
Mutants Correctly Localize the Head Organizer
- Hex transcripts are properly localized to the AVE in mesd
mutants; expression of Hex is expanded laterally
- Cerberus-1 transcripts are properly localized to the AVE in mesd
mutants
- LIM1 transcripts are properly localized to the AVE in mesd
mutants; a patch of Lim1 expressing cells are also detected on the
posterior of this embryo
- Mrg1 is correctly localized to the AVE and the extraembryonic tissue
in mesd mutants
- These results demonstrate that localization of the head organizer can occur
independent of primitive streak function or mesoderm induction.
FIG. 5: The Mesd
Epiblast Adopts an Anterior Fate
- In mouse, the restriction of Otx2 transcripts to the anterior region
of the developing brain is dependent upon posteriorizing signals. Consistent
with the absence of Fgf8 and Wnt3, or a functional primitive
streak, Otx2 transcripts remain expressed throughout the epiblast of
mesd embryos.
- Hesx1 transcripts are localized to the AVE and the overlying ectoderm
in mesd mutants; importantly, transcripts were detected throughout
the epiblast of four embryos
FIG. 6: Mesd
is Required for Differentiation of the Epiblast
- T, Nodal, Hex, Cerberus, Mrg1, and Hesx1 are down-regulated
after E 8.5 in mesd mutants
- Oct4, normally repressed in differentiating cells, is highly expressed
in the mesd epiblast
FIG. 7: Mesd
is Required in Extraembryonic Tissue for Epiblast Differentiation
- Wild-type Rosa26 cells (BLUE) injected into blastocysts from and
Ai3/ch intercross populate the epiblast and colonize embryonic tissues but
are excluded from the host extraembryonic tissue. Rosa26 fail to rescue
the mesd mutant phenotype, demonstrating that Mesd is required
in the extraembryonic tissue.
- Previous studies demonstrate an additional requirement for Mesd in
the epiblast for growth and differentiation.
FIG. 8:
Two candidate genes map within the mesd interval
- The 1.3 Mb BAC contig spans the mesd and neur functional
regions and links D7Mit350 to D7Mit62, which map 0.5 cM apart.
- The mesd functional region (320-360 kb) contains two genes, Mesd-1
and Mesd-2.
- The neur functional region (320-350 kb) contains the Arnt2 gene.
- A new 330-430 kb predicted functional region maps between the c23DVT
and c3YPSd proximal breakpoints; it contains
the 3.5 Blast, Il-16, and Pgdh-r genes.
- The human homologues FAH, ARNT2, MESD-1, and MESD-2 are localized
to chromosome 15 in the same linkage group (bin 60 of the Stanford G3 RH panel),
indicating a conserved region of synteny.
- MESD-1, FAH, ARNT2, IL-16, 3.5 BLAST, D15S1041, and D15S211
map within overlapping human BAC clones; MESD-2 does not map
to the contig, but maps to the same RH bin as D15S211.
- D15S211 cosegregates with two human disease loci causing autosomal
dominant nocturnal frontal lobe epilepsy (ADNFLE) and tapetoretinal degeneration,
mental retardation, and spasticity (TD/MR/S).
FIG. 9:
Mesd-1 and Mesd-2 are novel genes expressed in the early embryo
and the adult
- Both Mesd1 and Mesd2 are expressed in the developing embryo.
Mesd1 transcripts (~9.0 kb) are also detected in adult Brain and Testis.
Mesd2 transcripts (3.2 kb) are detected at low levels in all tissues.
- Partial sequence of the Mesd1 cDNA identified a region of the predicted
Mesd-1 protein that shares 41% similarity (132/517 aa) to the
armadillo repeat region of the Adenomatous Polyposis Coli (APC) signaling
factor.
- Mesd-2 encodes a predicted protein of 362 aa; the sequence
is 44% similar across its entire length to the central region of the mouse
Talin protein.
FIG. 10:
The mesd phenotype is rescued by a BAC containing Mesd-2
Conclusions:
Mesoderm Development
- Failure to form a primitive streak in mesd mutants results from proximal/distal
patterning defects in the pregastrula egg cylinder
- Analysis of Rosa26 ES cell / mesd blastocyst chimeras demonstrates
that mesd is required in extraembryonic tissue for epiblast patterning
and differentiation
- Localization of the head organizer occurs independent of primitive streak
/ trunk organizer localization
- Expansion of Hex, Cerberus, Lim1, and Hesx1 expression in
mesd mutants suggests that antagonizing signals from the posterior
normally restrict anterior fates
- Posterior localization of Cripto may result from repression by anterior
signals
- Two candidate genes are located in the 350 kb mesd functional region
- A BAC containing Mesd2 rescues the mesd phenotype
- Mesd2 encodes a novel 362 amino acid protein with similarity to
an internal domain in talin.
- Future studies will focus on determining subcellular localization of mesd
protein, identification of interacting factors, and characterization of cell
proliferation in mesd mutant embryos.
