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In Xenopus, the primary neurons form in three domains either side of the midline in the posteriorneurectoderm. At the late neurula stage there are approximately 120 primary sensory neurons on each side of the embryo. Co-injecting synthetic mRNA encoding retinoic acid receptor alpha (NR1B1) and retinoid X receptor beta (NR2B2) results in an increase in the number of primary neurons and this is further enhanced by the addition of retinoic acid indicating that elevated retinoid signalling promotes an increase in the number of cells undergoing primary neurogenesis. However, primary neurogenesis remains confined to the three domains that normally give rise to primary neurons indicating that not all regions of the neurectoderm respond equivalently to elevated retinoid signalling. The inhibition of retinoid signalling with a dominant negative retinoid receptor or treatment with citral, an inhibitor of retinoid metabolism, inhibits the formation of primary neurons. However, the lateral extent of the neurectoderm does not differ following these experimental manipulations suggesting that changes in primary neuron cell number, in response to changes in retinoid signalling, cannot be accounted for by significant gains or losses of neurectoderm. In addition, two lines of evidence are presented to suggest that retinoid signalling affects primary neurogenesis by acting directly on the neurectoderm. First, animal caps neuralized by noggin undergo primary neurogenesis in response to retinoid signalling and second primary neurogenesis is elevated in neural conjugates in which the ectodermal, but not the mesodermal, component has been co-injected with RAR/RXR mRNA.
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Fig. 1.
A quantitative analysis of Isl1+ primary sensory neurons in the Xenopus embryo. In all figures, embryos are shown with anterior to the right. (A). The Xenopus embryo expresses Isl1 in columns either side of the midline (m) predominantly in primary sensory neurons (black arrowheads). The Isl1+ primary sensory neurons (enlarged in insert) are found at equal density along the length of the posteriorneurectoderm. The anterior border for Isl1+ primary sensory neurons (black and white arrowheads) is a short distance caudal to rhombomere 5 shown by double labelling with the rhombomere marker krox-20 which identifies rhombomeres 3 and 5 (marked on the figure). Scale bars: main image 120 μm, inset 20 μm. (B). Time course of formation of Isl1+ primary sensory neurons. The mean number of Isl1+ primary sensory neurons per side of an embryo was determined for more than 10 embryos at each developmental stage. The bars represent the standard error of this calculation. At stage 22 there was a mean of 120 Isl1+ primary sensory neurons on each side of the embryo. (C). The effect of applied RA on the numbers of Isl1+ primary sensory neurons. The mean number of Isl1+ primary sensory neurons for one side of an embryo was calculated for at least ten embryos at stage 17 over a range of applied RA concentrations, bars represent the standard error of the calculation. In untreated embryos there was a mean of 82 (SE 3.5) and in embryos grown from the late blastula stage in 10−7 M RA there was a mean of 95 (SE 3.0) Isl1+ primary sensory neurons. (D). There is no asymmetry in the formation of Isl1+ primary sensory neurons. The ratio of the number of Isl1+ primary sensory neurons on the left compared to the right side of the embryo was determined from mid to late neurula stages for more than 8 embryos at each stage. There was no significant difference in the number of neurons on one side compared to the other.
Fig. 2. Elevated retinoid signalling increases the number of primary sensory neurons. (A–C). Isl1+ primary sensory neurons at stage 20 in (A) control, (B) RAR/RXR mRNA injected and (C) RAR/RXR mRNA injected + RA treated embryos. The number of neurons increases in embryos subjected to elevated retinoid signalling though the neurons are still found in restricted, if widened, domains and are not dispersed throughout the neurectoderm. Scale bar applies to each panel A–C and represents 60 μm. (D). The ratio of Isl1+ primary sensory neurons on the left compared to the right side of the embryo was calculated for uninjected controls (top panel). Each dot represents an individual embryo. The peak (dark yellow) and distribution ranges (light yellow) of the ratio of Isl1+ primary sensory neurons are indicated. Isl1+ primary sensory neurons were then counted in embryos unilaterally injected with RAR/RXR (middle panel) and RAR/RXR with 10−7 M RA (lower panel). The blue dots represent individual embryos injected with 250–500 pg and red dots individuals with 62-125pg of RNA. Following injection of 250–500 pg of RAR/RXR mRNA most individuals have increased numbers of Isl1+ primary sensory neurons on the injected side. Following RA treatment a larger proportion of embryos have increased numbers of Isl1+ primary sensory neurons on the injected side. In a small number of cases (marked with a †symbol) there is a significant decrease in the number of Isl1+ primary sensory neurons on the injected side of embryos receiving 250–500 pg of RNA. In these cases it appeared as if gastrulation was compromized on the injected side of the embryo. (E–G) Neural specific tubulin expression assayed by wholemount in situ hybridization in (E), control, (F), RAR/RXR mRNA injected and (G), RAR/RXR mRNA injected + RA treated embryos. The three domains of primary neurons either side of the midline are marked in (E), s, sensory, i, inter and m, motor neurons. The expression of NST in the trigeminal ganglion is also marked (tg). (F) the injected side is marked with a red dot and the horizontal bar marks the midline. There is increased NST expression in the sensory neuron and motor neuron domains (black arrows) compared to the uninjected side. However, these domains remain separated by a region of neurectderm that does not express NST (open arrow). (G). Increased NST expression is seen on the injected side of the embryo grown in 10−7 M RA but still does not extend significantly into the non-neural ectoderm or into the region of the neurectoderm between sensory and motor neuron domains (open arrow).
Fig. 3. Depressed retinoid signalling decreases the number of Isl1+ primary sensory neurons. (A,B). (A) Isl1 expression in the primary sensory neuron domain of a normal embryo. (B) Embryos injected with RARdn mRNA have a reduced number of Isl1+ primary sensory neurons. In this example the width of the domain is wider in the RARdn injected embryos than normal. Scale bar applies to (A) and (B) and is equal to 60 μm. (C) Analysis of the effect of unilaterally injecting the xRARα2dn mRNA on the ratio of Isl1+ primary sensory neurons on the injected compared to the uninjected sides of the embryo. The yellow background indicates the peak and distribution range of Isl-1+ primary sensory neurons in normal embryos (shown in Fig. 2D). Blue dots indicate embryos injected with 1.0 ng and red dots embryos injected with 0.5ng RARdn mRNA. In contrast to Fig. 2D there is now a shift towards reduced numbers of Isl1+ primary sensory neurons on the injected side of the embryo. (D) NST expression assayed by wholemount in-situ hybridization in an embryo unilaterally injected with RARdn mRNA. The injected side is marked with a red dot and the midline by a bar. The expression of NST in the motor, and inter neuron domains is undetectable, and is also missing from the trigeminal ganglion domain (open arrows). Expression in the sensory neuron domain is detectable (grey arrow) but much weaker than on the control side of the embryo.
Fig. 4. Citral, an inhibitor of retinoic acid synthesis, inhibits the formation of primary neurons. (A) Functional assay of embryo movement at the tailbud stage in response to external stimulation. Embryos were grown from the late blastula stage to the early neurula stage in control (0.1× MBS), 10−7 M RA or 60μM citral and then in normal media to the tailbud stage (stage 28). Samples of 25 embryos were then analysed by lightly touching the embryo with a fine needle adjacent, but posterior, to the otic vesicle. The number of tailflips was then counted. Each batch of embryos was stimulated 50 times. Whereas control and RA treated embryos displayed a range of responses to stimulation, the citral treated embryos gave only the an occasional and short response. (B–D). NST expression in embryos treated with citral or citral plus RA. (B), embryos grown from the late blastula to early neurula stage in 40 μM citral show NST expression that is essentially normal (black arrow) in some, but decreased NST expression (open arrow) in other individuals. (C), The addition of 10−7 M RA at the late blastula stage to embryos grown in 40 μM citral results in the recovery of NST expression (black arrow). (D). Embryos grown in 60 μM citral have little or no NST expression (open arrow) though it is sometimes possible to distinguish weak NST expression (small black arrow). The addition of 10−7 M RA to these embryos results in the widespread rescue of NST expression (black arrow).
Fig. 5. The level of retinoid signalling in the embryo does not affect the lateral extent of the neurectoderm. (A–C). sox-2 expression in embryos unilaterally injected with: (A) RAR/RXR, (B) RAR/RXR with the addition of RA and (C) RARdn mRNA. The injected side is marked by a red dot, the midline by a horizontal bar and the lateral extent of the neurectoderm by vertical bars. Although the morphology of the neural plate, particularly in the anteriorneurectoderm is altered in embryos treated with RA (B), the lateral extent of sox-2 expression is unaltered compared to the control sides in each of the manipulations. (D,E) The expression of sox-2 in normal (D) and citral-treated (E) neurula stage embryos. Although the citral treated embryos show morphological changes compared to the normal controls, the expression of sox-2 was unaffected. This suggests that although treatment with citral inhibits the primary neurogenesis it is not doing so by eliminating the formation of the neurectoderm.
Fig. 6. Retinoid signalling promotes the formation of primary neurons in neuralized animal caps. Animal caps were taken from control and experimental embryos injected with either noggin or RAR/RXR mRNA or a combination of these RNAs and grown from the late blastula stage in the presence or absence of 10−7 M RA. The animal caps were assayed at the equivalent of the late neurula stage for the expression of the primary neuron marker, NST. Widespread punctate expression of NST was seen only in animal caps injected with noggin and RAR/RXR mRNA and treated with RA (E). Both non-neuralized animal caps with elevated retinoid signalling (D) and neuralized animal caps with RAR/RXR but no ligand showed no NST expression. The addition of RA to noggin-neuralized animal caps (F) consistently showed a small amount of NST expression clustered to one end of the animal cap (arrowhead). This may reflect the observation that animal caps are heterogeneous and respond to neural induction more strongly on the dorsal side (Sharpe et al., 1987).
Fig. 7. Elevated retinoid signalling in the ectodermal component of neural-inducing conjugates results in the increased expression of the primary neuron marker, NST. (A–C) NST expression in conjugates of animal cap ectoderm and dorsolateral mesoderm, grown in 10−7 M RA and assayed at the late neurula stage for the expression of NST. The component injected with RAR/RXR mRNA is indicated in green print. Expression of NST is increased (black arrows) in area in conjugates that received RAR/RXR in the animal cap component (C) compared to controls (A). Conjugates with additional RAR/RXR transcripts in the mesoderm (B) showed less and reduced NST expression possibly because the retinoid signalling ventralizes the mesoderm (Ruiz i Altaba and Jessell, 1991) and makes it a poorer neural inducer. (D,E). A section through one of the conjugates shown in (C) seen under normal (D) and uv (E) light to correlate NST expression (blue patches indicated by arrows) in (D) with the animal cap ectodermal component of the conjugate (green fluorescence) in (E).
