PRECIOS REFERENCIALES DE LOS EQUIPOS SEPURA

All mD2LIC mutants exhibited reduction in anterior structures., with truncations in the forebrain being the most obvious defect. In addition to these defects, Class I mutants never developed more than one branchial arch. Normal anterior patterning requires the anterior definitive endoderm (ADE) for the maintenance of an Fg/5 signalling centre in the anterior neural ridge (ANR) (Martinez-Barbera and Beddington, 2001). I therefore examined these structures to ask if they might be the cause of anterior deficiencies.

HnfSP is normally expressed in the definitive endoderm at the late neural plate/early headfold stage. However I observed a decrease in the expression domain of this gene in

mD2LIC null mutants, suggesting that the definitive endoderm is decreased in size before

mD2LIC is expressed in gut endoderm later in development (Figure 6.11).

s ta g e 1

S tage 2

S tag e 3

WT Class 1

(notochord not always distinguishable)

Class II

(notochord not always distinguishable) Class III 5 .8 % 3 0 .8 % 1 9 .3 % 1 0 .2 % / / " / / / I 3 5 .3 % 5 1 .4 % 4 3 .5 % 4 2 .4 % m \

m

Figure 6.14 Analysis o f notochord formation in wild-type and mD2LIC -/- mutants. Percentage o f sections exhibiting the different stages o f notochordal development. Class 1 mutants have the l^ t mature notochord

Fgf8

WT - /- WT

H

t

Figure 6.15 Fg/8 expression in wild-type and mD2LIC -/- mutants Normal expression in streak. At 15 somite stage expression in anterior neural ridge appears greatly reduced or absent and lateral mid/hind-brain boundary expression reduced.

Chapter 6: Functional analysis o f m P2LIC

TUNEL staining of late streak/neural plate stage mutant embryos revealed high levels of apoptosis in the ADE (Figure 6.12) whereas as no apoptosis was observed in the ADE of wild-type embryos. This provides a potential explanation for the reduced expression

domain o f HnfSP in the definitive endoderm at these stage o f development. It is important

to note that mD2LIC expression is restricted to the node at these stages, and is not

detectable in the anterior definitive endoderm. Thus the effect o f this mutation on the survival of ADE is probably indirect, perhaps a result of defective signalling from one of the node derivatives.

In addition to the ADE, anterior patterning also requires the anterior neural ridge (ANR), a

signalling centre necessary for correct forebrain patterning. Expression o f Fg/8, a molecular

marker o f the ANR, was therefore examined in 12-15 somite stage embryos. In control

littermates, Fg/8 was expressed as previously described (Sun et al., 1999). However, Fg/8

expression was absent (n=5/7; ClassI/II type mutants) or very severely reduced (n=3/7; Class III type mutants) in the ANR of null mutants (Figure 6.15). TUNEL staining indicated that programmed cell death is slightly increased in null mutants (Class I/II > Class III > wild-type) although such embryos exhibited a slight increase in general apoptosis

Together these observations suggest that the anterior truncations observed in mD2LIC -/- embryos are likely to be caused by reduction of the ADE and interference with anterior signalling by the ANR.

6.2.10 Neural tube and cephalic mesenchyme associated cell death profiles are altered in null mutants and are likely to contribute to exencephaly

mD2LIC -/- embryos show a delay in neural tube closure, resulting in variable degrees of exencephaly. In Class I mutants neural tube closure has often not been initiated in embryos with 12 somites and their neural folds have not elevated giving the anterior neural plate a flattened appearance.

During development of the neural epithelium cell death plays an important role in

modelling the neural tube such that fusion along the dorsal midline occurs correctly (Weil et al., 1997). In wild-type embryos, neural tube closure in the region of the midbrain-

hindbrain is preceded by a high level of cell death in this area, and this cell death is crucial in order to achieve normal neural tube closure (Martinez-Barbera et al., 2001). In contrast, a comparable form of cell death does not occur in the neural epithelium of null mutants, providing a possible contribution to the failure in neural tube closure (Figure 6.13). In addition, supporting cephalic mesenchyme has been shown to be necessary for neural tube closure to proceed to completion (Weil et al., 1997). However, unlike wild-type embryos, ectopic cell death is observed in the cephalic mesenchyme surrounding the

notochord in the region of the midbrain-hindbrain region in mD2LIC null embryos (Figure

6.13).

These results suggest that failure of neural tube closure is due in part to altered cell death profiles. However it remains unclear whether this altered cell death is a direct or an indirect

consequence of loss of mD2LIC.

Notochord signalling and correctly patterned floor plate are believed to critical to achieve

fsrcl Lowl.

correct neural tube closure (Chen and Behringer, 1995; of nl_p 10^ ) , hence failure in

neural tube closure may be the indirect result of a disruption in the notochordal and floor plate signalling fields.

6.2.11 Expression of L-R axis associated nodal signalling components is altered or absent in null mutants

Heart and vascular development is delayed and defective in mD2LIC -/- null mutants.

Cardiac development proceeds to at least the linear heart tube stage in all classes of mutant, but only half of Class I mutants proceed beyond this stage. All embryos that develop beyond the linear heart tube stage (regardless of class) exhibit a randomisation in the direction of heart looping morphogenesis, indicating a L-R axis patterning defect. Null mutants also display defects in embryonic turning ranging from failure to initiate the turning process to reversal in the direction of turning. In those embryos that completed embryonic turning (Class III mutants only), 30% exhibited a reversal in the direction of

turning. Together these results indicate that mD2LIC -/- embryos are defective in left-right

patterning

Chapter 6: Functional analysis o f m P2LIC

1-2 somite stage

WT

ventral V dorsal