BIOL2010 LT8 Xenopus Mesoderm Induction

Xenopus: Mesoderm Induction

Mesoderm Induction

 Mesoderm is formed in the marginal zone of Xenopus late blastulae, after the onset
of zygotic transcription at the MBT
 Seen in the expression of mesoderm specific genes such as bra, gsc, myf5 and
vent1. All four of these genes encode transcription factors that are required for
mesodermal development.
 Brachyury (Bra) is a Tbox
transcription factor required for
notochord and tail development
that is expressed by all mesoderm
cells as they involute over the
blastopore during gastrulation,
but is then switched off in all but
the involuting notochord cells.
 Goosecoid (Gsc) is a homeobox
transcription factor required for
head development that is
expressed by mesoderm cells
above the dorsal blastopore lip of early gastrulae.
 Myogenic Factor 5 (Myf5) is a basic helix-loop-helix transcription factor required
for skeletal muscle development that is expressed in dorsallateral mesoderm of the
early gastrula
 Ventral Homeobox 1 (Vent1) is a homeobox transcription factor required for the
development of ventral mesoderm that is expressed in mesoderm of the ventral
half of the early gastrula.
- Microarray analysis showed that transcripts all four genes are present at very
low levels before MBT but increase significantly between MBT and the onset of
gastrulation.

How then is the mesoderm specified in the marginal zone?

 Pieter Nieuwkoop (1917-1996), isolated animal
cap fragments and vegetal fragments from
Ambystoma mid-blastulae and found that they
formed ectoderm and endoderm, respectively,
when incubated alone.

, - However, they also formed mesoderm (e.g. notochord, muscle, pronephros) when
grafted together and incubated until a tadpole stage (Nieuwkoop 1969).
- fragments from either pigmented (normal) or albino embryos, which allowed him
to discriminate between animal and vegetal cells.
- suggested that all the mesoderm was formed by the animal cap and concluded
that mesoderm was induced by the vegetal pole.
- Mesoderm was not induced when explants were isolated from early gastrulae,
indicating that mesoderm induction occurs during blastula stages.
 Experiments repeated on Xenopus blastulae, combining fluorescently labelled animal
caps with unlabelled vegetal poles, and vice versa, confirmed that all the mesoderm
was derived from the animal cap (Dale et al., 1985).
 Nieuwkoop also showed that the
dorsal-vegetal hemisphere induced
dorsal mesoderm while the ventral-
vegetal hemisphere induced ventral
mesoderm, suggesting different
inducing centres.
- confirmed using the 32-cell
Xenopus embryo, which is
composed of 4-tiers (A-D) of 8
cells (Dale et al., 1987)
- Individual blastomeres from the vegetal D-tier were isolated and combined with
the 8-cells of the animal A-tier, labelled with a fluorescent marker
- Only 1 pair of D-tier blastomeres induced dorsal mesoderm (notochord) in the
animal cap (D1 on dorsal side) The remaining D-tier blastomeres (D2, D3, D4)
only induced ventral mesoderm (blood)
- Blastomere D1 will also induce a second dorsal axis (conjoined twin) when
grafted to the ventral side of a 32-cell embryo, replacing blastomere D4
(Gimlich & Gerhart, 1984)

This dorsalvegetal fragment is now known as the Nieuwkoop centre

 location of both sia1 and sia2
expression in Xenopus late-blastulae.
 results suggest that the vegetal
hemisphere possesses two inducing
centres; a small dorsal centre (the
Nieuwkoop centre) that induces dorsal mesoderm and a large ventral centre that
induces ventral mesoderm (Smith et al., 1985).

,  Only explants of the dorsal marginal
zone differentiated notochord and
muscle, while ventral and lateral
explants differentiated blood,
mesothelium and mesenchyme (Dale &
Slack, 1987)

What gives the Nieuwkoop centre its
properties?

1) Beta-catenin
 Major candidate for what gives the
centre its properties
 Multifunctional protein that can act as a centre
for cell membrane cadherins or as a nuclear
transcription factor
 Accumulates at the dorsal region of the egg
during cytoplasmic movements of fertilisation and
continues to accumulate
 Accumulation seen in the nuclei of dorsal cells and
appears to cover the centre and organiser regions
– refer to how DV polarity is established  how is accumulation achieved
 Animal caps were isolated from midblastulae and grafted to vegetal poles
isolated from either control, vegt depleted, or ß-catenin depleted blastulae
 Whereas control vegetal poles induced dorsal mesoderm, vegt depleted vegetal
poles did not induce any mesoderm and ß-catenin depleted vegetal poles only
induced ventral mesoderm
 Thus, VegT is necessary for both dorsal and ventral mesoderm induction, while ß-
catenin is only required for dorsal mesoderm induction. Zhang et al, (1998)




2) TGF-beta like signal
 The blastula stage animal cap assay: identifies molecules with mesoderm inducing
activity, by incubating them in a simple salt solution containing the molecule to be
tested
- Isolated animal caps normally form round balls that differentiate as epidermis,
but if a mesoderm-inducing factor is present then the animal cap changes its
morphology and differentiates mesoderm (Smith, 1987)
- The changed morphology is particularly evident if dorsal mesoderm is induced,
since the cap undergoes considerable elongation