G ene E xpression in the developing brain E arliest docum ented expression* D rosophila H om ologue R eference
BF-1 Telencephalon B8.5 - 9 forkhead Xuan et a i , 1995
Coimexin 43 Telencephalon, midbrain-hindbrain junction B8 Belliveau and Naus, 1995
D ix -1,2, 4 and 5
Forebrain B10.5 Distal-less B u lfo n eeta /., 1993
Bmx-1 Forebrain B 9 .5 Empty spiracles Simeone er a/., 1992
Bmx-2 Fore and midbrain B 8-8.5 Simeone et a l , 1992
F g f8 Most rostral tip o f neural tube and midbrain hindbrain junction B8 - 8.5 TGF-P family related to Decapentaplegic
Crossley and Martin, 1995
G b x 2 Forebrain, rhombomeres 1 and 2 B 1 1 .5 Unplugged Bulfone ef a/., 1993
Gsh-1 Gsh-2
Diencephalon and hindbrain Fore and hindbrain
B8.5 Valerius et a/., 1995
Singh et a/., 1991
Lim 1 (Lhx-1) Diencephalon, hindbrain B 8 .5 B a m e se ta /., 1994
LHX-2 Forebrain (possibly midbrain) B 9 .5 Aperous Rubenstein and Puelles, 1994
Mash-1 Dorsal anterior midbrain Spreads to ventral forebrain
B8 B9
Achaete-Scute complex Guillemot and Joyner, 1993
Math-1 Trigeminal ganglion and dorsal neural tube B9.5 Atonal Akazawa et a/., 1995
M sx-l= H ox 7 M sx-2
Rostral neural crest and neural tube Similar to Msx-1
B8 Msh
Muscle segment homeobox
Davidson, 1995 Msx-3 Cephalic fold at level o f 4 * ventricle, not present in rhombomeres 3 and 5 B9 Shimeld et a/., 1996
Nkx-2.1 Forebrain B9.5 H6 V idovic et a/., 1994
Nkx-2.2 Fore and midbrain B9.5 Stadler et a/., 1995
Nkx5.1=HM X3 Forebrain B 8 .5 H6 Rinkwitz-Brandt et a l , 1995
Nkx5.2=H M X2 Forebrain B 10.5 Stadler et a/., 1995
N otch-1 Midbrain and ventral forebrain B8 Notch Guillemot and Joyner, 1993
Oct-6 Anterior head folds Restricted to hindbrain
B8 B9
Z w arteta/., 1996
otp Fore and hindbrain B10.5 Orthopedia Simeone et a/., 1994
Otx 1 O tx 2
Large region o f anterior neiu-al tube Fore and midbrain
Fore mid and hindbrain
B7.5 B8.5 - 9 B8.5 - 9
Orthodenticle Simeone et a/., 1992
Pax 2 Pax 5
Extensive expression in forebrain
Thin band in forebrain and at mid/hindbrain boundary
B8 (10 somites) B9 (25 somites)
Paired Rowitch and MacMahon, 1995
Pax 6 Forebrain and hindbrain B8 Eyeless Walther and Gruss, 1991
Pou 3 fl Pou 3f2,3 and 4
Fore and midbrain Forebrain
B 8 .5 Drifter Alvarez-Bolado et a/., 1995(Alvarez- B o la d o e ta /. 1995)
Pou 4 f l and 2 Mid and hindbrain B 14 Ipou He et a/., 1989
Rpx Anterior neural folds B 7 Hermesz et a/., 1996
* Earliest expression refers to documented expression within the developing neural tube or CNS. occurs earlier, during the period o f interest for the present study (i.e. B8 onwards).
and Gruss, 1992). Other differences are seen in the spinal region of the neural tube, where Pax2 is expressed in longitudinal columns along the entire anterior-posterior axis.
Although a similar pattern is seen with Pax5, Pax2 expression is both stronger and precedes that of Pax5 (Asano and Gruss, 1992; PÇschel et a l 1992). The expression pattern seen at the midbrain-hindbrain junction is similar for both Pax2 and Pax5 although the onset of Pax5 expression is later. Pax2 expression in this region precedes that of Wnt-1 and En-I and has been postulated to play a role in regulating the activation of these genes (Rowitch and McMahon, 1995). Pax2 and Pax5 are initially expressed in a broad domain in the midbrain, with a rostral limit at the forebrain-midbrain boundary. Expression becomes restricted to a band at the midbrain-hindbrain boundary by B9.5 and a discrete band of expression is seen in the forebrain region and optic vesicle by the 25 somite stage (Rowitch and McMahon, 1995).
5.1.4 Fgf8 expression in the developing brain
The FGFs are a family of signalling molecules consisting of 9 members which share an approximately 120 amino acid core but vary in their N and C termini (reviewed by Baird, 1994). Fgf8 is the most complex member of the family to be identified, with 7 secreted protein isoforms (Crossley and Martin, 1995). Fgf8 is expressed prior to and during gastrulation and in the later embryo in the developing limbs (particularly the apical ectodermal ridge), the pharyngeal region and facial pnimordia and the developing neural tube (Crossley and Martin, 1995). From E8.0, Fgf8 is expressed at the most rostral limit of the neural plate, in the prospective forebrain region, in the lateral edges which eventually fuse during cranial neural tube closure. A second expression domain is seen at the
prospective midbrain-hindbrain junction, with expression in the isthmus until E l 2.5 (Crossley and Martin, 1995).
The critical role of Fgf8 in midbrain patterning was highlighted by the work of Crossley et a l, who showed that FGF8 coated beads are able to induce the development of an ectopic
midbrain in the forebrain region of the chick embryo (Crossley et a l 1996).
This suggests that Fgf8 plays a key role in patterning the developing midbrain and may also function to pattern other regions where it is expressed.
5.1.5 Do gene expression patterns define the forebrain-midbrain
boundary?
We have seen that the forebrain-midbrain boundary is much less well defined in terms of gene expression than the midbrain-hindbrain boundary. A study specifically aimed at identifying genes expressed in this region could not only identify genes that may pattern the forebrain-midbrain boundary, but also shed light on factors which may control the
morphology and development of the anterior neural tube. Chapter 4 introduced the concept of variation in the closure pattern of the anterior neural tube between normal mouse strains. The present Chapter describes the expression pattern of several genes in the forebrain- midbrain region, particularly in relation to any variation in expression pattern with the position of closure 2. Genes that show an altered expression pattern with the variation in closure 2 may play a role in defining the position of this closure site and hence regulate the
process of neural tube closure. Conversely, those genes whose expression pattern remains constant regardless of closure 2 positioning could be interpreted as genes which define
structures within the definitive forebrain and midbrain, rather than affecting the morphological process of neural tube closure.
The genes studied in this Chapter were selected from the information provided on their early expression in the published literature (Table 5.1). Pax2, Pax5 and Fgf8 are expressed in the region of closure 2 at the E8.5 stage and were used in the present study.
5.2 Methods
5.2.1 Collection of embryos for gene expression studies
The results of Chapter 4 established that certain normal strains of mice vary in the
positioning of closure 2. The DBA/2, GDI and NZW strains were taken as representative of caudal, intermediate and rostral closure positions respectively (described in relation to the forebrain-midbrain boundary. Figure 5.1). Embryos from each strain were collected at E8.5 prior to the onset of cranial neural tube closure. Embryos were dissected, keeping the yolk sac intact, and were examined to determine the degree of cranial neural tube closure. Embryos prior to closure 2 were cultured (Section 2.2), observed at regular intervals and removed from culture specifically at the stage when closure 2 occurred. Embryos were then dissected free from the yolk sac, washed and fixed in 4% PEA as described in Section 2.7.1.
5.2.2 Whole-mount in situ hybridisation with Pax2, Pax5 and Fgf8
Gene expression studies were carried out by the use of whole-mount in situ hybridisation with RNA probes. The production of probes and hybridisation protocol is described in Sections 2.6 and 2.7. Pax2 and Pax5 probes were obtained from Dr Peter Gruss and the Fgf8 probe from Dr Andrew McMahon. Sense probes were applied, under the same conditions as antisense probes, to GDI embryos in order to establish that the expression pattern seen was specific for the antisense probe. At least 5 embryos of each strain were hybridised under the same conditions with the same reagents to assess any variations between embryos.