Papalopulu N et al. (1991), Retinoic acid causes abnormal development and s...

XB-ART-24306

Development 1991 Dec 01;1134:1145-58.

Show Gene links Show Anatomy links

Retinoic acid causes abnormal development and segmental patterning of the anterior hindbrain in Xenopus embryos.

Papalopulu N , Clarke JD , Bradley L , Wilkinson D , Krumlauf R , Holder N .

???displayArticle.abstract???
Retinoic acid is a very potent teratogen and has also been implicated as an endogenous developmental signalling molecule in vertebrate embryos. One of the regions of the embryo reliably affected by exogenously applied RA is the hindbrain. In this paper, we describe in detail the hindbrain of Xenopus laevis embryos briefly treated with various levels of RA at gastrula stages. Such treatments lead to development of embryos with loss of anterior structures. In addition, RA has a general effect on rhombomere morphology and specific effects on the development of the anterior rhombomeres. This effect is demonstrated using neurofilament antibodies, HRP staining and in situ hybridisation using a probe for expression of the Xenopus Krox-20 gene. Anatomically it is evident that the development of the hindbrain normally anterior to the otocyst (rhombomeres 1-4) is abnormal following RA treatment. Sensory and motor axons of cranial nerves V and VII form a single root and the peripheral paths of V and VII and IX and X are also abnormal, as is the more anterior location of the otocyst. These anatomical changes are accompanied by changes in the pattern of expression for the gene XKrox-20, which normally expresses in rhombomeres 3 and 5, but is found in a single band in the anterior hindbrain of treated embryos which standardly fail to generate the normal external segmental appearance. The results are discussed in terms of both the teratogenic and possible endogenous roles of RA during normal development of the central nervous system. We conclude that low doses of RA applied during gastrulation have specific effects on the anterior Xenopus hindbrain which appear to be evolutionarily conserved in the light of similar recent findings in zebrafish.

???displayArticle.pubmedLink??? 1811933

Species referenced: Xenopus laevis
Genes referenced: egr2
???displayArticle.antibodies??? Neuronal Ab4 Somite Ab1

???attribute.lit??? ???displayArticles.show???

	Fig. 1. Dose-dependence of RA phenotypes. Xenopus embryos at stage 9 were treated for 30 min with increasing concentrations of RA and allowed to develop to stage 40. There is a truncation of anterior pattern and concurrent failure of tail development which increases with the dose of RA. The embryo treated with 0.5/XM has two small eyes and the least apparently altered morphology, while the embryo at \jiu has a single median cyclopic eye. Eyes do not develop in embryos treated with 2.5 im RA or higher concentrations, and development of the heart and visceral arch skeleton is also impaired.
	Fig. 2. Early appearance of RA phenotypes. The typical morphology of RA-treated embryos is shown at neural stage 16 in dorsal (A) and anterior (B) views. Embryos were both derived from a synchronous fertilisation of the same egg batch and one group of embryos at stage 9 was exposed to a 30min pulse of 0.5 or 1 /IM RA. Only the embryos treated with 1/JM are shown. In A note the thicker appearance of the neural folds (arrows) and elongation of RA-treated embryos in comparison to sibling control (C). In B note also neural fold thickening and the absence of a cement gland, indicated by an arrow in the control (C) embryo.
	Fig. 3. Whole-mount staining of control and RA-treated tadpoles with anti-neurofilament and anti-muscle antibodies. (A-D) Staining with 68 K anti-neurofilament antibody. A is a dorsal and C a side view of a control embryo. B and D represent the dorsal and side views respectively of embryos treated with 0.5 fm RA. For all panels anterior is to the left and posterior to the right. Note the anterior shift of the otic vesicle (OV) in the RAtreated embryos (B and D). In A and B white arrows mark the limits of the hindbrain with the midbrain and spinal cord, and in the RA-treated embryos (B) while the overall length of the hindbrain is similar to controls it has a broadened appearance. In C (controls) the small arrows mark the three exit points of the Vth, Vllth/VIIIth and EXth cranial nerves, and in RA-treated embryos (D) only two exit points are clearly distinguishable as marked by the arrows. Spinal nerves are indicated by solid arrowheads in A and C. (E-G) Staining with anti-muscle antibody 12/ 101. In E, side view of control tadpole (top) and 0.5 ^M RA-treated embryo (bottom). Eye is indicated by black arrowhead. Dorsal views of the same embryos are shown (F, control; G, RA-treated). White arrows (F and G) point to the position of the narrowing of the IVth ventricle (obex) and indicate that the posterior juncture of the hindbrain with the spinal cord occurs at the same position in control and RA-treated embryos, relative to the first somite.
	Fig. 4. Altered morphology of rhombomeric segments in the hindbrain RA-treated embryos. Control (A, C) and 0.5/iM RA-treated embryos (B, D) were whole-mount stained with an anti-neurofilament antibody. Optical sections through the hindbrain were obtained by confocal microscopy (A, C) and similar embryos were embedded in wax and sectioned for normal histological analysis (B, D). The normal number and patterning of the rhombomeres is clearly visible in control (stage 40) embryos. White arrowheads mark the rhombomeric boundaries or constriction in C. In the RA-treated embryos (B, D) the uniform pattern of periodic bulges is not apparent, but there are a few irregular, large, poorly defined constrictions which are visible in some embryos. The arrows mark the borders of the hindbrain (hb) between the midbrain (mb) and the first spinal nerves and indicate the similar overall size but broadened appearance of the hindbrain in RA-treated embryos. See also in Fig. 3 A and B. In all panels anterior is to the top.
	Fig. 5. Molecular segmentation in the hindbrain of RA-treated embryos. In situ hybridization of control (A-C) and 0.5 JJM RA-treated embryos (D-F) at stage 20 using a Xenopus Krox-20 probe. (A,D) shows a low power bright-field sagittal sections of the embryos. Bright-field (B,E) and dark-ground (C,F) of respective embryos. Anterior is to the left and ventral to top in all panels. Control embryos show two strong stripes of Krox-20 expression which correspond to the future rhombomeres r3 and r5. In RA-treated embryos, only a single stripe of Krox-20 expression is detected throughout all serial sections. This illustrates that despite the absence of overt morphological segments, some aspects of segmental organization are preserved in the hindbrain of RA-treated embryos.
	Fig. 6. RA alters regionalization in the hindbrain. Sections of stage 45 tadpoles through the hindbrain of control (left) and 0.5/iM RA-treated embryos (right, +RA) stained with anti-neurofilament antibody. White arrows mark the three exit points of the Vth, Vllth/VIIIth and IXth cranial nerves from top to bottom respectively in control embryos. Only two discrete exit points are observed in RA-treated embryos (right). From these sections it is not possible to determine if one of the nerves is missing or is superimposed upon another. However, the distance from the most posterior exit point and the first spinal nerve is identical in the two embryos. Note the more anterior position of the otic vesicle (OV) spanning the isthmus (i, open arrow) between the midbrain (mb) and hindbrain (hb).
	Fig. 7. Confocal analysis of RA-induced cranial nerve alterations in whole-mount embryos. The same embryos stained with anti-neurofilament antibody in Fig. 4 were examined by optical sections in lateral views. Anterior is to the right and posterior to the left in all panels. The RA-treated embryo is shown in A-C, and the control in D-F. OV marks the otic vesicle. In the control (D), the Vllth nerve appears as a flat band in front of the otocyst and the serial sections (D-F) clearly define the Vth, Vllth, Vlllth and IXth/Xth nerve complexes. In the RA-treated embryos, only the Vllth, VHIth, IXth/Xth nerves are apparent and no staining for the distinctive Vth nerve (trigeminal) is observed, h hindbrain; m midbrain.
	Fig. 8. Camera-lucida drawings of facial and trigeminal cranial nerve root pattern in control and RA-treated embryos. (A) Ventrolateral view of a whole-mounted, control Xenopus brain. HRP was applied to the preotic ganglion and has labelled the roots and sensory tracts of the trigeminal (V) and facial (VII). The Vth root is more rostral than the Vllth root, and extends further caudally. Outlines of the positions of motor nuclei of III, IV, V, VI and VII are shown dotted. The ventral midline is dashed. (B) Ventrolateral view of a whole-mounted, RA-treated Xenopus brain. HRP was applied to the preotic ganglia as in A. Most of the axons entering the Vth and Vllth sensory tracts now do so at the same rostrocaudal level; a few are seen to enter the Vllth tract more rostrally. The dorsoventral position and rostrocaudal extent of the tracts are normal. Dotted outlines show position of labelled motor neurons in the positions of nuclei III, V, VII and VI. Midbrain-hindbrain border (mh); scale bar 100um.
	Fig. 9. Summary drawing of the motor nuclei and root positions of cranial nerves III, IV, V, VI, VII, IX and X in the Xenopus tadpole hindbrain. Normal (right) and RAtreated (left) specimens. Information on controls was collated from 24 stage 39-46 embryos, 7 of which had the preotic ganglia of V and VII as well as the roots of IV and VI filled with HRP. A set of 14 embryos had the tracts and nuclei of IX and X filled by a postotic ganglion HRP application and the remaining three embryos had both preotic and contralateral postotic ganglia HRP applications. The description of the relevant neuroanatomy of control embryos was derived from complete whole-mounted specimens and horizontally or transversely sectioned embryos. The observations are in agreement with previous neuroanatomical descriptions based on architectonics and neuronal tracing (see Matesz and Szekely, 1978; Will, 1982; Nikunwe and Nieuwenhuys, 1983). The RA data is derived from 24 treated embryos of comparable stages to controls. Of these, 11 were preotic HRP applications, 5 were postotic and 8 were combination fills with preotic on one side and postotic on the other. 16 of these specimens were sectioned (horizontally or transversely) and 8 were viewed as whole-mounts.
	Fig. 10. Camera lucida drawing of the central patterning of cranial nerve nuclei in control and RA-treated Xenopus brains. (A) Horizontal section showing the normal positions of cranial nerve motor neurons. The neurons of nuclei III, IV, V, VI and VII were filled from a preotic HRP application, and the neurons of the IXth/Xth complex from a postotic application. Data from two specimens have been superimposed. (B) Horizontal section through a RA-treated brain. HRP was applied to the preotic ganglion. Neurons occupying the positions of Vth and Vllth nuclei are seen to have a common exit point for their axons. Neurons of the Vlth nucleus occupy their normal position just caudal and medial to the Vllth motor neurons. One neuron in the contralateral IVth nucleus, and three cells in the IXth/Xth complex were also labelled. (C) Horizontal section through a RA-treated brain. HRP was applied to the postotic ganglia on one side and to the preotic ganglia on the other. The preotic fill has labelled only a few neurons in the Illrd, Vlth and IXth/Xth nuclei. Many more cells were labelled by the postotic application and most of these occupy the normal position of IXth/Xth neurons. A few neurons in the Vlth and one in the Vllth nucleus are also labelled. (D) Ventral view of a wholemounted, RA-treated Xenopus brain. HRP was applied to the preotic ganglia on one side only. This specimen had only one median eye and was clearly compressed in the rostrocaudal axis. Nonetheless the positions of neurons in the nuclei of III, V, VI, VTI, IX and X are relatively normal. The neurons within the IXth/Xth nuclei and the few within the contralateral Vlth and Vllth nuclei were labelled due to disrupted peripheral pathways of their neurons. Midline is dashed throughout, m/h is the midbrain-hindbrain border. Scale bar is 100 um.
	Fig. 11. Photomicrograph of a horizontal section through an otocyst in a RA-treated Xenopus. HRP was applied to a preotic ganglion (just off bottom left of micrograph). Note how axons (arrows) are wrapped around the otocyst and peripheral neurons lie adjacent to its lateral wall (curved arrows). These abnormal axon and neuron positions in RA-treated animals result in the anomalous labelling of postotic neurons from preotic HRP applications, and vice versa. Scale bar is 50um.
	Fig. 12. Photomicrographs of HRP-labelled cranial motor neurons in RA-treated brains. These transverse sections show that this dorsoventral position of the motor neurons and the sensory tracts is indistinguishable from normal. (A) Section through midbrain showing the normal dorsal looped trajectory of trochlear axons (curved arrows) and a single ipsilateral oculomotor neuron (III). Some neurons in the HRP-labeUed preotic ganglion are also seen in this section. (B) Section through anterior hindbrain showing neurons of the Vth/VIIth complex and a few VI neurons. HRP was applied preotically on the right and postotically on the left. (C) Ventromedial abducent neurons (VI) are labelled from a preotic HRP application, together with the sensory tracts of V and VII, and the contralateral IXth/ Xth tract is labelled from a contralateral postotic application. (D) Neurons of the IXth/Xth complex are labelled together with their associated sensory tracts on the same side. The contralateral Vth tract has also been labelled. Scale bar is 100 um.
	Fig. 13. Photomicrographs of neurons labelled from rostral spinal cord HRP applications. (A) Horizontal section through midbrain and hindbrain of a normal Xenopus larva. Ventral midbrain cells and vestibulospinal neurons are clearly labelled. Some more medial hindbrain reticulospinal neurons are more faintly labelled. (B) Horizontal section through midbrain and hindbrain of a RA-treated Xenopus. Eyes are present at the truncated anterior midbrain. As in controls, ventral midbrain cells and vestibulospinal neurons are labelled, but the distance between these two cell groups is reduced. Some reticulospinal neurons are also apparent. (C-E) Transverse sections of RA-treated embryos. (C) TS through midbrain showing large ventral midbrain neurons. The ectopically placed otocysts are apparent in this section. (D) TS showing one Mauthner's neuron (arrowed), and two Mauthner's axons (curved arrow) crossing the ventral floor plate at the level of the rostral vestibulospinal neurons. (E) TS through vestibulospinal complex, showing its characteristic location dorsolateral to the reticulospinal neurons, vm, ventral midbrain neurons; vsp, vestibulospinal neurons; rsp, reticulospinal neurons; oto, otocyst. Scale bar is 100 nm.