A detailed prole of uth expression was obtained, from the moment of egg formation in the queen's ovaries to the rst larval stage. The precise locali- sation of the transcript in the ovaries and embryos was then determined by
AmelCSP5 TcasCSP8 AgamCSP7 DmelCSP4 BmorCSP16 AmelCSP2 TcasCSP1 DpulCSP3 AmelCSP5 TcasCSP8 AgamCSP7 DmelCSP4 BmorCSP16 AmelCSP2 TcasCSP1 DpulCSP3 1 1 1 1 1 1 1 1 44 43 60 55 60 59 56 55 - - - M K I K I L L F F T I L A L I N V K - - - A Q D D I S K F L K D R P Y V Q K Q L H C I L D R G - - - M P L V K S L V V V V L L I G V V Y Q - - - V Q G Q L G - - L A G N N Y I E K Q L L C A L D K A - M S S K A L P N L F M L S A A V I V V M A A L V I V G P Q P A A A N D S Q N I N R L L N N Q V I V S R Q IM C V L E K S - - - - M L L L N K N R V I S L V V N F I F L I I L I S - - S S V Q A D E R N I N K L L N N Q V V V S R Q IM C I L G K S M I E W K R F K I L H F L S Y L G L L V L V V V C A A Q Q N - R P Q V T D T A L D E A L N D K R F IQ R Q L K C A L G E A - - M A S A I K A L L I V C A L F I Y T V T A E T E E G Q S G R S R V S D E Q L N M A L S D Q R Y L R R Q L K C A L G E A - - - M L I L Q I A H L C A Q F C L L A A I F T C V K P Q L - - T R I S D E A I E S T L N D R R Y L L R Q L K C A T G E A - - - M A R L Q L V W Q L A L V C V V L F L A G Q A Q A Q L P A S N A A V E A A I R N P R Y M R R Q I N C L L N E S 45 44 61 56 61 60 57 56 104 99 117 112 121 117 112 114 H C D V I G K K I K E L L P E V L N N H C N R C T S R Q I G I A N T L I P F M Q Q N Y P Y E W Q L I L R R Y K IM K Y Y - P C D A L G N Q I K G A L P E I I G K N C E R C D S R Q V A N A R R I A R Y V Q T K H P D V W N A L V K K Y S V - - - - - P C D Q L G R Q L K A A L P E V IQ R N C R N C S P Q Q A Q N A Q K L T N F L Q T R Y P E V W A M L I R K Y G A V - - - - E C D Q L G L Q L K A A L P E V I T R K C R N C S P Q Q A Q K A Q K L T T F L Q T R Y P D V W A M L L R K Y D S A - - - - P C D P I G K R L K T L A P L V L R G A C P Q C S P Q E T K Q IQ K T L S Y V Q R N F P Q H W A K L V R Q Y A G A V K G S P C D P V G R R L K S L A P L V L R G A C P Q C S P E E T R Q I K K V L S H IQ R T Y P K E W S K I V Q Q Y A G V S - - - P C D P V G R R L K S L A P L V L R G S C P Q C T P Q E M K Q IQ K V L A F V Q K N Y P K E W N K I L H Q Y A G - - - - - P C D N I G R T M R Q L V P A L I K G Q C P G C S P Q Q H Q Q A M K V M N V V S Q Q Y P Q E Y S R I Y Y T Y N Q Q Q G - -
Figure 5.1: Alignment of Unable-To-Hatch and its relatives in other Arthro- pods: the beetle T. castaneum (Tcas); the mosquito A. gambiae (Agam); the y, D. melanogaster (Dmel); the moth, B. mori (Bmor) and the water ea, D. pulex (Dpul).
in situ hybridization. The analysis of csp5 by Northern blot, shown in gure 2, conrms its expression in the ovaries and in the embryos, as reported in chapter 4. It addition, it reveals two distinct waves of transcriptional activ- ity. Following the initial expression in the ovary, the maternal transcript is found in the newly-laid eggs, but the message fades away during the blas- todermal stage and reappears later, around the germ band stage. The rst phase corresponds to a characteristic pattern of expression seen for mater- nal transcripts, which are eliminated from the embryo at the midblastula transition [158]. The second phase involves the production of a zygotic csp5 transcript in the developing embryo.
The amount of the transcript is high during the initial cleavage (around four hours post-oviposition (POV), but decreases at the beginning of blas- toderm formation (eight hours after POV). The transcript completely disap- pears towards the end of the blastodermal stage (24 hours POV). Around 48
hours POV, at the germ band stage, a new wave of abundant csp5 transcrip- tion takes place (gure 5.2). Shortly before hatching (72 hours POV), the transcription level drops to an undetectable level, and the transcript does not reappear in the newly hatched larva, as shown in gure 5.6. It is note- worthy that csp5 is not present in the drones' genitalia, implying that all csp5 mRNA seen in the newly oviposited egg is of maternal origin.
1 2 3 4 5 6 7
Loading OBP11
Figure 5.2: csp5 in eggs, in the drone testis and in the queen ovaries. 1: 4 hours POV, 2: 8 hours POV, 3: 24 hours POV, 4: 48 hours POV, 5: 72 hours POV, 6: drone testis, 7: queen ovaries. The relative amount of rRNA is shown in the lower panel as an indicator of loading.
The ovaries of the honey bee, as those of other Hymenoptera, belong to the polytrophic meroistic category [82]: the oogonia produce both tropho- cytes (nurse cells) and oocytes (eggs), and the ovarioles consist of eggs in- terspersed with nurse cells. In situ hybridization assays show that the uth transcript is not detectable in the upper part of ovariole tubules. The rst weak staining appears in that part of ovariole the where oogonia multiply and dierentiate into large central cells and small follicle cells (gure 5.3.a). Staining becomes stronger further down the ovariole tubule (where central cells have become egg-cells and nurse cells). The strongest staining is visible in the area where nutritive chambers contain nurse cells with highly polyploid
nuclei (gure 5.3.b). The signal in egg cells is weak, but increases as they grow. Finally, in parallel with the nurse cells' degradation, the signal be- comes stronger in the egg cells, which acquire their cytoplasm from the nurse cells. Egg cells, just before arriving at the oviduct, show strong staining (not shown). After oviposition, the maternal transcript is usually localised in the posterior part of the eggs (gure 5.4.a), but can also be found in other ar- eas, or can be uniformly distributed throughout the egg (gures 5.4.b, 5.4.c). Since the eggs used for these in situs were 0-4 hours old, this variation is likely to be age-related. The high expression typically found at the posterior end part of the eggs might be related to the process of adhesion, as the honey bee eggs are glued to the cell base in which they develop.
1 mm (a) Antisense 0.5 mm (b) Antisense 1 mm (c) Sense
Figure 5.3: Expression of uth in the queen ovaries.
The second wave of strong expression appears in the ventral and anterior parts of embryos at the germ band stage. The expression is detectable across
the whole embryo with very a strong signal in germ band area and in the buds of head appendages (gure 5.5.a). This pattern can have two explanations; the gene may be expressed in both the ectoderm and the mesoderm, and may thus give a stronger signal ventrally where both layers are present and a weaker signal dorsally, where only the ectoderm is present. Alternatively, the transcript may be expressed only in the ectoderm, thus causing the labelling to be weaker dorsally, because the ectodermal cells of that region are more stretched and attened than the ventral ones, which are denser ([112]). The latter hypothesis is preferred, as it is somewhat more parsimonious, and sucient to explain the stronger expression at the anterior pole. The head appendages and the cerebral lobes, which are strongly labelled, contain only ectodermal tissue at that stage ([112]).
(a) Antisense (b) Antisense
(c) Antisense
1 mm
(d) Sense Figure 5.4: Expression of uth in 0-4 hours-old eggs.
In the later stage of fully matured embryo, the expression is narrowly localized to the head and thorax, and some weak staining is visible in the hind intestine (gure 5.5.c). Weaker staining is visible in the rudimentary
appendages of the thorax. Newly emerged and 1 hour-old larvae don't show any expression (gure 5.6).
(a) 44-48 hours (antisense) (b) 44-48 hours (sense)
(c) 66-70 hours (antisense)
1 mm
(d) 66-70 hours (sense) Figure 5.5: Expression of uth in the embryo.
1 mm
Figure 5.6: csp5 in rst instar larvae.