Antisense morpholino-mediated knockdown of Xdhcr7 severely compromised both placodal and neural development

In order to investigate the effect of Xdhcr7-morpholino on neurogenesis, we analysed the expression of Xsox3, Xn-tubulin. Xdlx3, Xgsh1 and Xnkx2.2. At neurula stage, the Xsox3 expression (Fig. 26a) was severely reduced (65 %, n = 13 / 20) in the neural plate and more severely in the presumptive lens placode (red arrowheads in Fig. 26a). The expression domain around the eye field was usually expanded on the injected side, (55

%, n = 11 / 20), but compared to the control side, the staining was clearly weaker (Fig.

26a). At tailbud stage, the expression was severely reduced in the brain and lens (55.55

%, n = 15 / 27), and in the branches (red arrows in fig. 26b and b´´) as well (70.3 %, n = 19 / 27). On transversal sections (Fig. 26b´´1 – b´´3), the expression appeared scattered (less organised as compared to the control side) in the forebrain, and the neural tube appeared reduced in size on the injected side (b´´1). When analysed for Xn-tubulin expression, the neurula stage Xdhcr7-morpholino injected embryos showed severe reduction of the transcripts level on the injected side (Fig. 26c; 72,4 %, n = 21 / 29), both in neural and placodal domains. At tailbud stage, 57.1 % (n = 20 / 35) of the analysed embryos showed a severe reduction of Xn-tubulin expression in the brain and cranial placodes (red arrowheads in fig. 26d and d´´). On transversal sections (Fig. 26d´´1 – d´´4), the expression appeared almost absent in the neural tube and neural retina of the injected side. Hence, with regard to the expression of Xsox3 and Xn-tubulin, no discrimination was observed between neural and non neural effect of Xdhcr7-morpholino. For Xdlx3 expression (Fig. 27), a severe reduction was observed at neural plate stage, mostly in the posterior domain (Fig. 27a). At this stage, 48 % (n = 12 / 25) of the analysed embryos displayed a severely reduced expression ofXdlx3. At tail bud stage (Fig. 27b – b´´) 55.9 % (n = 19 / 34) of the analysed embryos showed a severe reduction of the pharyngeal pouches expression domain of Xdlx3 (red arrowheads in Fig. 27b and b´´). On serial horizontal sections (Fig. 27b´´1 – b´´4), the expression appeared also reduced in the olfactory placode (op in fig. 27b´´1). The analysis of Xdlx3 expression then suggested that the Xdhcr7-morpholino´s effect was not restricted to neural structures only. Our next interest was then to answer the question as to whether Xdhcr7-morpholino could also interfere with brain patterning.

Figure 26: The antisense morpholino-mediated knockdown of Xdhcr7 severely interferes with neural and neuronal markers expression.

a and c: anterior views (dorsal up) of stage 14 embryos; the position of the midline is shown by white dashes. b, b´´, d and d´´: side views of stage 32 embryos. The frontal views are shown in b´ and d´.

Two-cell stage albino embryos were injected (in one of two blastomers) with 1pmol of Xdhcr7-morpholino, in combination with 200pg of n--Gal capped RNA as linage tracer. Injected embryos were cultured in 0.1 X MBS until opened neural plate (NF st. 13 / 14) or tailbud stage (NF st. 32 / 32), fixed in MEMFA, and hybridized with a Dig-labelled Xsox3 (a – b´´), or Xn-tubulin (c – d´´) antisense RNA, after X-gal staining (blue colour).

At neural plate stage (a), although the staining was less on the injected side, the anterior expression domain of Xsox3 was expanded as compared to the control side. The expression in the presumptive lens placode was completely abolished (red arrowheads in a). When analysed at tailbud stage (b – b´´), the expression of Xsox3 was severely reduced all along the neural tube, and showed a “scattered” feature in the forebrain (b´´1). The lens and branchial placodes expression domains were almost absent on the injected side (red arrowhead in b and b´). The expression of Xn-tubulin(c – d´´) was in general severely reduced, both at early and late stage. At early stage (c), the expression was almost abolished both in the placode and neural plate.

When analysed at later stage (d – d´´), the placodal expression domain was severely reduced, (red arrowheads in d and d´´). On transversal sections, the neural tube expression domain appeared severely reduced on the injected side (d´´1 – d´´4).

a b b´ b´´

Figure 27: The antisense morpholino-mediated knockdown of Xdhcr7 resulted in a severe reduction ofXdlx3expression.

a : anterior view (dorsal up) of stage 14 embryos; the position of the midline is shown by white dashes. b, b´´and b´´: side views of stage 32 embryos. The frontal view is shown in b´.

Two-cell stage albino embryos were injected (in one of two blastomers) with 1pmol of Xdhcr7-morpholino, in combination with 200pg of n- -Gal capped RNA as linage tracer. Injected embryos were cultured in 0.1 X MBS until opened neural plate (NF st. 13 / 14) or tailbud stage (NF st. 32 / 32), fixed in MEMFA, and hybridized with a Dig-labelled Xdlx3 antisense RNA, after X-gal staining (blue colour). Xdhcr7 morpholino caused a severe reduction ofXdlx3expression both at early (a) and late (b – b´´) stages of development. At neurula stage, almost noXdlx3staining could be detected on the injected side (is, red arrowhead in a). The same reduction was observed at tailbud stage, but the pharyngeal pouches expression domain appeared more severely reduced (see red arrowheads in b and b´´, and is in b´´2 – b´´4).

When embryos were analysed for Xgsh1 and Xnkx2.2 expression (Fig. 28), the results were more in line with the gain-of-function activity.

The forebrain expression domain of Xgsh1 (Fig. 28a – a´´) was reduced for 63.3 % (n = 19 / 30) of the analysed embryos. On transversal sections (Fig. 28 b´´1 – b´´3), this reduction appeared more pronounced in the forebrain. Moreover, the expression domains on the injected and control side were not symmetrical, suggesting an effect on the dorso-ventral patterning of the brain. When hybridized with the ventral brain marker Xnkx2.2 (Fig. 28b – b´´), 59.4 % (n = 19 / 32) of the embryos displayed a reduction of the expression. Same as for Xgsh1, the reduction was more severe in the forebrain, and the expression domain was ventrally shifted on the injected side (Fig. 28b´´1 – b´´2).

In summary, the antisense morpholino-mediated knockdown of Xdhcr7 severely compromised eye development, and interfered with the expression of both eye and neural marker genes.

Figure 28: The antisense morpholino-mediated knockdown of Xdhcr7 resulted in the reduction of the anterior expression domain ofXgsh1andXnkx2.2.

a, a´´, b and b´´ : side views of stage 32 embryos. The frontal views are shown in a´ and b´.

Two-cell stage albino embryos were injected (in one of two blastomers) with 1 pmol of Xdhcr7-morpholino, in combination with 200pg of n- -Gal capped RNA as linage tracer. Injected embryos were cultured in 0.1 X MBS until tailbud stage (NF st. 32 / 34), fixed in MEMFA, and hybridized with a Dig-labelled Xgsh1 or Xnkx2.2antisense RNA, after X-gal staining (blue colour).

The expression domain of Xgsh1 (a – a´´) appeared reduced on the injected side. On transversal sections (a´´1 – a´´3), the dorso-ventral patterning of the brain seemed to be affected, as the expression domain of Xgsh1 appeared ventrally shifted on the injected side. Same as forXgsh1, the expression domain ofXnkx2.2 (b – b´´2) was reduced and ventrally shifted (see b´´2). However, a general observation was that the reduction of bothXgsh1andXnkx2.2 expression was more pronounced in the forebrain.

a a´ a´´

1 2 3

a´ ´1 a´ ´2 a´´3

nt nt nt

NF st. 32

Xg s h 1

cs

cs cs cs

is

is is is

1 2

b b´ b´´

b´´2 b´´2

nt nt

X n kx 2. 2

cs

cs cs

is

is is

II.3.2.4. Antisense morpholino-mediated knockdown of Xdhcr7 mildly interfered