The embryonic development of oenocytes

The PlacZ enhancer trap, recovered from the preceding screen, has proved

Table 4.1 A literature survey of genes expressed and/or displaying a function in oenocytes

Gene _expression Oenocyte Function in_oenocytes References

Notch (A/)

big brain (bib)

mastermind (mam) hepatocyte nuclear factor 4 {Hnf4) seven up (svp) lam in C [iamC) ventral veiniess {vvf} aa N-acetyltransferase (aaNATI) sprouty {sty) spalt {saf) Delta-aminole vulinate synthase {alas) dFKBP59 Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Hartenstein et al. (1992) Hartenstein et al. (1992) Doherty et al. (1997) Hartenstein et al. (1992) Hoshizaki et al. (1994) Hoshizaki et al. (1994) Riemer et al. (1995) Anderson et al. (1995) Hintermann et al. (1996) Hacohen et al. (1998) Chen et al. (1998) Barrio et al. (1999) Ruiz de Mena et al. (1999)

N)

Table 4.2 Oenocyte expressing lines

Data compiled from an ongoing in silico screen for oenocyte expressing lines using the Drosophila

databases Flybase (1999), Flyview (2000) and private enhancer trap collections of Cahir O ’Kane and Andrea Brand. GAL4 lines were crossed to flies carrying a UAS-n/s/acZ transgene. Oenocyte expression was assessed for each line either by immunostaining against 3-gal or by fluorescent microscopy in living embryos for GFP lines.

The stock names are given according to the source of the line. Homozygous phenotypes are listed where E and L refer to lethality in the embryonic and larval stages, respectively. The presence (+) or absence (-) of oenocyte expression in embryos (Emb) and third instar larvae (L3) is shown. High-level reporter expression in oenocytes is indicated by ++. A question mark is given if future double labelling experiments, with an oenocyte specific marker, are necessary to confirm or rule out oenocyte expression. st.x> refers to expression from stage x onwards during embryonic development.

GAL4 Drivers

lacZ traps

GFP trap

Line Chromosome Phenotype Emb Oeno L3 Oeno Source

P0197 II Lethal + - _Umea P0206 II Viable + + Umea 56B ? ? - + A. Brand OK72 ? ? - + C. O’Kane P0030 X(19B) Viable - + Umea P0093 X (18F03-04) Viable - + Umea P0103 X(18F) Viable 4- + Umea

P0110 X(19A-B) Viable - + Umea

P538 II (29E2-4 Hnf4) Lethal - 4- 1. Kiss, Bloomington

P706 II Viable ++ - Y. Jan, Bloomington

P880 III (69D mri) Lethal (L) _{++ (?)} 4- Y. Jan, Bloomington

P890 III Lethal (L) + + 4- Y. Jan, Bloomington

P898 II Viable _{+ (?)} - Y. Jan, Bloomington

P937 II (47A13-14) Lethal - 4- Bloomington

PI 340 II (32F1-2 saf) Semi-lethal ++ 4- A. Spradling, Bloomington

P1342 II (43D1-2 Aldh-\\\) Lethal - 4- A. Spradling, Bloomington

P1377 II (46B dap) Lethal _{+ (?)} - A. Spradling, Bloomington P1785 III (72D) Male sterile - + S. Wasserman, Bloomington P2103 III (75B1-2) Lethal - 4- G. Rubin, Bloomington

P2356 II (55F02-03) Lethal - 4- A. Spradling, Bloomington

1(2)05730 II (35D esg) Lethal _{+ (?)} - U. Nauber A405 II (32F1-2 saf) Lethal -n-(st.10>) - R. Schuh

first expressed at early stage 11 in dorsal stripes of cells in abdominal segments A1 to A8. Double-labelling with an anti-EN antibody revealed that these cells lie in the posterior compartment (Figure 4.1 A). During embryonic stage 11, a subset of these cells delaminate from each dorsal stripe and maintain 810^°'"^ expression

(data not shown). In A1 to A7, the positive cells adopt a characteristic

shape and time-course experiments provide strong evidence that they will later become oenocytes (see below). However, in A8 oenocytes are not formed. Instead, 310'^°''^ positive cells delaminate and migrate in a posterodorsal direction to become associated with the posterior spiracle (data not shown). In A1 to A7, delaminated oenocyte precursors are EN-negative (Figure 4.1 E), while the residual non-delaminated epidermal cells in the 310"^°"^ stripe remain EN-positive (Figure 4.1 D). By stage 15, oenocytes are found clustered in their final position (Figure 4.1 F), and it is evident that these cells have migrated ventrally with respect to the residual S70^°"VEN double-positive epidermal stripe. Also, despite having arisen in

en territory, mature oenocytes are located anterior to the EN stripe. This suggested the possibility of both anterior and ventral components of cell migration. Both of these movements will be discussed further within this chapter.

spalt (sal) was one of the genes previously reported to be expressed in oenocytes (see table 4.1), and two enhancer trap inserts in sal were isolated in the screen for oenocyte expressing lines (see Table 4.2). Here I use an antibody

against the zinc-finger protein encoded by sal to follow the early stages of

oenocyte development. Anti-SAL moderately labels a dorsal domain (referred to as the dorsal SAL domain) that straddles abdominal and thoracic segments from embryonic stage 9 onwards (data not shown). By early stage 11, SAL is upregulated in characteristic sickle-shaped nuclei (Figure 4.1 B) which form whorls

Figure 4.1 Oenocyte development during D ro so p h ila embryogenesis

Embryos carrying the PlacZ enhancer trap are shown in panels A and D-F

where (3-gal is in green and EN in red. Panels B and C display wildtype embryos immunostained with anti-SAL (red).

(A) An early stage 11 embryo showing expression overlaps (yellow) with

EN in the most dorsal cells of the EN stripe. 810^°'"^ is also expressed in the embryonic CNS from this stage onwards (not shown).

(B) A dorsolateral view of two parasegments on the curve of the extended germband in a stage 11 embryo. Low level SAL is observed in a dorsal domain, with a sharp boundary between expressing and non-expressing cells. SAL is upregulated in sickle-shaped oenocyte precursor nuclei that form a whorl, positioned at the ventral extent of the dorsal SAL domain.

(0) Following delamination the oenocyte nuclei become rounded and during stage 12 oenocytes are observed migrating ventrally as strings of SAL-positive nuclei. (D-E) Two confocal planes through the same stage 13 embryo. Cells remaining in the 310^°"^ stripe are observed in the plane of the epidermis, and they continued to show an overlap with EN (D). Beneath the epidermis the oenocytes are beginning to cluster and have switched off en (E).

(F) By stage 15, oenocytes are clustered in their final position and lie ventral to the remains of the 310^°"^ epidermal stripe (yellow overlap) and anterior to the EN stripe.

M

i

Ü U

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y.>* -J~

at the ventral extent of the dorsal SAL domain. These sickle-shaped nuclei also display upregulated 310'^°''^ expression (data not shown). Both pointed {pnt) and

ventral veiniess {vvf) are also expressed in cells of the whorl prior to their expression in mature oenocytes (Elstob et al., 200V; Rusten et al., 2001 and data not shown). These expression data strongly suggest that the sickle-shaped cells are oenocyte precursors, and further evidence is provided in a later section where lineage-labelling experiments are described. By stage 12, the delaminated oenocyte nuclei have rounded up and are observed in strings migrating ventrally (Figure 4.1 C). Anti-SAL continues to label oenocytes throughout the rest of their embryonic development, and also labels some components of the lateral chordotonal organs of the PNS, with which oenocytes are associated in the mature embryo (see below).

4.2.3 Ventral migration of oenocytes: a role for seven up

Out of more than 10 unknown P-element inserts displaying oenocyte expression,

3 1 0^°^^ was selected for investigation because homozygous embryos displayed an oenocyte phenotype (Figure 4.2B). This was a variable phenotype, observed in 50% of homozygotes, with single misplaced clusters in primarily the DV, but also the AP and z-axis (Figure 4.2A-B and data not shown), through to the most severe embryos in which oenocytes were completely absent in some segments whilst

Figure 4.2 The d o w n -a n d -o u t phenotype

Panels A and B show homozygous embryos immunostained for p-gal. All

other panels display X-gal stains of imprecise excision lines, in which the

lacZ reporter gene remains active. Unless otherwise stated all embryos are at