Introduction

Embryological manipulations and mutational analyses have identified a number of tissues crucial for body axis establishment and early embryonic patterning (as described in Chapter 1). Studying the genetic and molecular properties o f these tissues would improve our understanding of the roles they play during embryogenesis.

A variety of strategies have been employed to assess gene function. These include: (i)

large-scale random mutagenesis screens such as those performed in Drosophila sp.

(Nusslein-Volhard and Wieschaus, 1980), C.elegans (Clark et al., 1990; Bucher and

Greenwald, 1991) and Zebrafish (Haffter et al., 1996; Driever et al., 1996) and underway in mouse (Meneely and Hermann, 1997); (ii) gene trapping in mouse. (Skames, 1993; Gossler et al., 1989), (iii) targeted mutations using homologous

recombination in mouse and Drosophila, (Doetschman et al., 1987; Joyner et al., 1989;

Kong and Golic, 2001), (iv) the overexpression of proteins and mRNA and (v) the administration of specific antibodies and dominant negative technologies. More recent

techniques used to assess gene function include RNA interference {KNAi\ Fraser et al.,

2000; Gonczy et al., 2000), RNA anti-sense interference (Cooke, 1999) and morpholino anti-sense oligo nucleotides (Heasman et al., 2000).

Whether the strategy employed involves random mutagenesis or a more targeted approach, it is necessary to isolate the gene of interest. cDNA libraries provide easy access for the isolation of specific genes. These libraries can be made from practically any tissue type and even from single cells. Therefore it is theoretically possible to isolate all the genes that are expressed in one’s cell or tissue of interest at a specific time. In addition techniques such as normalisation and subtractive hybridization can be used to improve and enrich the library to suit the needs of the user. For example

following normalisation both rare and abundant clones are more equally represented in the library, although the experimental manipulation of the library may result in the loss of rare transcripts.

A number of cDNA libraries have been generated from various species, and various methods have been used to screen them. One successful approach involves expression screening by whole-mount mRNA in situ hybridisation (WISH) (Gawantka et al., 1998; Christiansen et a l, 2001; Neidhardt et a l, 2000; Kudoh et a l, 2001). WISH provides the

Chapter 3: The Endoderm library screen

advantage over other screening methods that one may follow both the spatial and temporal expression pattern of a gene of interest during embryogenesis. Once a gene of interest has been isolated it can be used in functional analyses in order to address specific biological questions such as ‘what is the molecular basis o f body axis establishment and early embryonic development?’

3.1.1 The Beddington Endoderm library and clone selection

Previously in our laboratory, Harrison et al. (1995) constructed and characterised a

series of conventional, directionally cloned and non amplified cDNA libraries from the germ layer tissues of 7.5 dpc mid to late gastrula mouse embryos in order to identify and study developmentally important genes.

Libraries were made from each of the individual germ layer tissues, ectoderm,

endoderm, mesoderm, as well as a primitive streak library. The Endoderm library is

derived from a number of cell and tissue types, namely the visceral endoderm, ventral node, notochordal and prechordal plates and definitive endoderm, all of which have been implicated in axis establishment and maintenance (discussed in Chapter 1).

Consequently the Endoderm library represents genes expressed in each of these tissues

at 7.5d pc. Thus, in an attempt to search for genes whose expression patterns implicate them in axis specification, two other students (Ross Kettleborough and Rita De Sousa

Nunes) and I screened the Endoderm library by whole mount mRNA in situ

hybridisation of mouse embryos aged between 6.5 and 9.5 dpc.

We employed a strategy that took advantage of sequence data provided through the collaboration of our laboratory and the Max Plank Institute of Molecular Genetics, Ihnestrasse, Berlin, Germany (see Chapter 2: Methods and Materials). Briefly, clones were gridded and sequenced, like sequences were clustered, they were subjected to BLAST searches and these data were used to select genes of interest for WISH (180 clones in total). In effect we were performing a manual normalisation, in which we were not risking the loss of rare transcripts. Moreover, as we knew the identity of the clones selected, we did not repeat WISH of the same clone unnecessarily and we were able to choose previously unstudied clones. Both my individual results and the combined results of my co-workers and I will be presented here.

3.2 Results

3.2.1 An efficient strategy for an expression screen

This Study explored but a fraction of the Beddington Endoderm library; just 2% had been gridded with only half (4000 sequences) used in the clusteral analysis and this produced a total of 1957 independent clusters. Of these 180 clones being selected for WISH screening.

Of the 180 clones selected, probes could not be generated for 13, leaving 167 used for WISH (see Table 3.1)

Table 3.1-Breakdown of problems encountered with selected clones, which were unable to be used for WISH

Problem with selected clone Number of problematic

clones out of the total 180 clones selected

Number of problematic clones out of the 78 clones

that I selected

No bacterial growth 2 0

Did not linearise 2 2

No insert detected or insert very small

7 4

Transcription failure 2 0

Total 13 6

Number of remaining clones used for WISH screening

167 72

The results of the expression screen are summarized in Table 3.2. The outcome of each WISH was empirically classified according to the following scheme: (i) ubiquitous, where the corresponding gene was expressed in all tissues at equal levels at all stages, (ii) widespread, where the corresponding gene was expressed at often differing levels in a wide range of tissues at all stages (iii) restricted, where expression was detected in a small number of specific tissues in at least one of the stages examined or (iv) undetected expression.

A total of 30 out of the 167 clones (18.0%) screened by WISH were expressed in a restricted manner during embryogenesis (my contribution was 8/72 (11%) restricted expression patterns detected). Ubiquitous expression patterns were the most common comprising 42% of all the expression patterns obtained (and 49% of my results) followed by ‘widespread’ expression patterns which comprised 36% of the expression

Chapter 3: The Endoderm library screen

patterns (and 38% of my results). Interestingly, 4% of genes (3% of my results) did not appear to be expressed using WISH.

It is often easier to study the functions of genes that are expressed in a restricted manner. When analyzing complex phenotypes (due to a combination of primary and secondary defects), a knowledge of the spatial and temporal localization of the gene of interest helps one determine in which tissues and at what stage the primary defect is occurring. Therefore, emphasis will now be placed on the clones expressed in a restricted manner in the following results and discussion sections.

Table 3.2-Summary of the numbers of expression patterns obtained by WISH

Expression pattern Combined results My results

No, of clones % of total No. of clones % of total

Ubiquitous 70 42 35 49

Widespread 60 36 27 38

Restricted 30 18 a 11

No detected expression 7 4 2 3

Total 167 72

3.2.2 Isolation of 30 genes with restricted expression patterns

Table 3.3 presents a breakdown of the type of clones giving rise to restricted expression patterns. This table also provides comparative data showing the number of restricted patterns per total number of clones represented in each category.

The majority of the clones exhibiting restricted expression patterns belonged to groups other than the novel category, suggesting that a gene was more likely to be expressed in a restricted manner if homologs existed in other species. Indeed, three out of five uncharacterized ESTs that contained conserved ORFs provided restricted expression patterns.

Our prescreening strategy ensured that we did not select clones that had been previously studied in development and that expression patterns of potential homologs of our selected clones had not been published at the time of clone selection. As a result, 29 of the 30 restricted patterns were from distinct and developmentally uncharacterized

Table 3.3-Classification of clones giving rise to restricted expression patterns

Combined results My results

Non developmentally studied m ouse hom ologues or g en e s containing dom ains corresponding to; No. of clones selected No. of restricted patterns No. of clones selected No. of restricted patterns

Transcription regulators and proteins involved in chromatin structure

26 2(8%) 11 0 Splicing factors and proteins involved in RNA

binding and transport

12 2(17%) 2 0

Signaling molecules 22 5(23%) 6 0 Cell cycle regulators 11 3(27%) 2 1 Cytoskeleton and extracellular matrix

com ponents 11 3(27%) 1 0 Human d ise a se g en e s 15 3(20%) 1 0 Others 3 2(67%) 1 2 C onserved ORFs 5 3(60%) 3 2 Novel g e n e s 62 7(11%) 51 3 Total 180 30 78 8

Total screen ed by WISH (total selected minus problem atic clones)

167 30 72 8

Table 3.4 Classification of clones with expression restricted to one or more tissues represented in the Endoderm library.

Tissue

Visceral endoderm

Node and axial m esendoderm Definitive endoderm

Combined result Number of clones with

My results

Number of clones with

expression restricted to tissue expression restricted to tissue

(note this represents a total of (note this represents a total of

19 clones) 14 5 4 4 clones) 3 1 1 87

Chapter 3: The Endoderm library screen

clones. This compared well with other screens such as that of Niedhardt et al. (2000) where only 45% of genes with restricted expression patterns were uncharacterized. Finally, 19 of the 30 clones with restricted expression patterns (4/8 for my results) exhibited expression patterns restricted to the tissues represented in the endoderm library: that is,, tissues implicated in axis establishment (see table 3.4). Most of these clones were restricted to the visceral endoderm (14/19 combined results; 3/4 my results) followed by the node and axial mesendoderm (5/19 combined results; 1/4 my results). Some genes were expressed in more that one of the tissues of the endoderm library. For

example some genes such as mDlLIC (see below) were expressed in the node and later

in the definitive endoderm.

3.2.3 Descriptions of 8 new expression patterns isoiated

Of the 72 clones that I screened by WISH 8 exhibited restricted patterns of expression. These results are presented in table 3.5 and figures 3.1-3.8. Appendix 1 details a summarized breakdown of the results taken from my contribution to the WISH screen.