Zebrafish model of holoprosencephaly demonstrates a key role for TGIF in regulating retinoic acid metabolism.

Holoprosencephaly (HPE) is the most common human congenital forebrain defect, affecting specification of forebrain tissue and subsequent division of the cerebral hemispheres. The causes of HPE are multivariate and heterogeneous, and include exposure to teratogens, such as retinoic acid (RA), and mutations in forebrain patterning genes. Many of the defects in HPE patients resemble animal models with aberrant RA levels, which also show severe forebrain abnormalities. RA plays an important role in early neural patterning of the vertebrate embryo: expression of RA-synthesizing enzymes initiates high RA levels in the trunk, which are required for proper anterior-posterior patterning of the hindbrain and spinal cord. In the forebrain and midbrain, RA-degrading enzymes are expressed, protecting these regions from the effects of RA. However, the mechanisms that regulate RA-synthesizing and RA-degrading enzymes are poorly understood. Mutations in the gene TGIF are associated with incidence of HPE. We demonstrate in zebrafish that Tgif plays a key role in regulating RA signaling, and is essential to properly pattern the forebrain. Tgif is necessary for normal initiation of genes that control RA synthesis and degradation, resulting in defects in RA-dependent central nervous system patterning in Tgif-depleted embryos. The loss of the forebrain-specific RA-degrading enzyme cyp26a1 causes a forebrain phenotype that mimics tgif morphants. We propose a model in which Tgif controls forebrain patterning by regulating RA degradation. The consequences of abnormal RA levels for forebrain patterning are profound, and imply that in human patients with TGIF deficiencies, increased forebrain RA levels contribute to the development of HPE.

Distinct roles for hindbrain and paraxial mesoderm in the induction and patterning of the inner ear revealed by a study of vitamin-A-deficient quail.

The hindbrain and cranial paraxial mesoderm have been implicated in the induction and patterning of the inner ear, but the precise role of the two tissues in these processes is still not clear. We have addressed these questions using the vitamin-A-deficient (VAD) quail model, in which VAD embryos lack the posterior half of the hindbrain that normally lies next to the inner ear. Using a battery of molecular markers, we show that the anlagen of the inner ear, the otic placode, is induced in VAD embryos in the absence of the posterior hindbrain. By performing grafting and ablation experiments in chick embryos, we also show that cranial paraxial mesoderm which normally lies beneath the presumptive otic placode is necessary for otic placode induction and that paraxial mesoderm from other locations cannot induce the otic placode. Two members of the fibroblast growth factor family, FGF3 and FGF19, continue to be expressed in this mesodermal population in VAD embryos, and these may be responsible for otic placode induction in the absence of the posterior hindbrain. Although the posterior hindbrain is not required for otic placode induction in VAD embryos, the subsequent patterning of the inner ear is severely disrupted. Several regional markers of the inner ear, such as Pax2, EphA4, SOHo1 and Wnt3a, are incorrectly expressed in VAD otocysts, and the sensory patches and vestibulo-acoustic ganglia are either greatly reduced or absent. Exogenous application of retinoic acid prior to 30 h of development is able rescue the VAD phenotype. By performing such rescue experiments before and after 30 h of development, we show that the inner ear defects of VAD embryos correlate with the absence of the posterior hindbrain. These results show that induction and patterning of the inner ear are governed by separate developmental processes that can be experimentally uncoupled from each other.

Abnormal development of the sinuatrial venous valve and posterior hindbrain may contribute to late fetal resorption of vitamin A-deficient rat embryos.

BACKGROUND: Normal embryonic development and survival in utero is dependent on an adequate supply of vitamin A. Embryos from vitamin A-deficient (VAD) pregnant rats fed an inadequate amount of all-trans retinoic acid (atRA; 12 microg per g of diet or approximately 230 microg per rat per day) exhibit severe developmental abnormalities of the anterior cardinal vein and hindbrain by embryonic day (E) 12.5 and die shortly thereafter. METHODS: In the present study, we sought to determine whether supplementation of VAD-RA supported (12 microg per g of diet) pregnant rats with retinol (ROL) at the late-gastrula (presomite or rat E9.5) or early somite stages (E10.5), or provision of higher levels of atRA throughout this period could prevent abnormalities in the developing cardiovascular and nervous systems. RESULTS: A newly described defect in the sinuatrial venus valve along with enlarged anterior cardinal veins and nervous system abnormalities and the later death of embryos are prevented by supplementing pregnant animals with ROL on the morning of E9.5. If ROL supplementation is delayed by 1 day (E10.5), most embryos are abnormal and die by E18.5. Supplementation of VAD rats with atRA (250 microg per g of diet) between E8.5 and E10.5 also prevents the cardiovascular and nervous system abnormalities and a significant number of these embryos survive to parturition. Thus, high levels of atRA can obviate the need for ROL between E9.5 and E10.5. CONCLUSIONS: These results support an essential role for retinoid signaling between the late gastrula and early somite stages in the rat embryo for normal morphogenesis of the primitive heart tube and the posterior hindbrain. Further, these results suggest that embryonic death occurring at midgestation in the VAD rat may be linked to the abnormal development of one or both of these embryonic structures.

Vitamin A deficiency results in the dose-dependent acquisition of anterior character and shortening of the caudal hindbrain of the rat embryo.

The developing nervous system is particularly vulnerable to vitamin A deficiency. Retinoid has been proposed to be a posteriorizing factor during hindbrain development, although direct evidence in the mammalian embryo is lacking. In the present study, pregnant vitamin A-deficient (VAD) rats were fed purified diets containing varying levels of all-trans-retinoic acid (atRA; 0, 0.5, 1.5, 6, 12, 25, 50, 125, or 250 microg/g diet) or were supplemented with retinol. Hindbrain development was studied from embryonic day 10 to 12.5 ( approximately 6 to 40 somites). Normal morphogenesis was observed in all embryos from groups fed 250 microg atRA/g diet or retinol. The most caudal region of the hindbrain was the most sensitive to retinoid insufficiency, as evidenced by a loss of the hypoglossal nerve (cranial nerve XII) in embryos from the 125 microg atRA/g diet group. Further reduction of atRA to 50 microg/g diet led to the loss of cranial nerves IX, X, XI, and XII and associated sensory ganglia IX and X in all embryos as well as the loss of hindbrain segmentation caudal to the rhombomere (r) 3/4 border in a subset of embryos. Dysmorphic orthotopic otic vesicles or immature otic-like vesicles in both orthotopic and caudally ectopic locations were also observed. As the level of atRA was reduced, a loss of caudal hindbrain segmentation was observed in all embryos and the incidence of otic vesicle abnormalities increased. Perturbations in hindbrain segmentation, cranial nerve formation, and otic vesicle development were associated with abnormal patterning of the posterior hindbrain. Embryos from VAD dams fed between 0.5 and 50 microg atRA/g diet exhibited Hoxb-1 protein expression along the entire neural tube caudal to the r3/r4 border at a time when it should be restricted to r4. Krox-20 protein expression was expanded in r3 but absent or reduced in presumptive r5. Hoxd-4 mRNA expression was absent in the posterior hindbrain, and the rostral limit of Hoxb-5 protein expression in the neural tube was anteriorized, suggesting that the most posterior hindbrain region (r7/r8) had been deleted and/or improperly patterned. Thus, when limiting amounts of atRA are provided to VAD dams, the caudal portion of the hindbrain is shortened and possesses r4/r5-like characteristics, with this region finally exhibiting r4-like gene expression when retinoid is restricted even more severely. Thus, regions of the anterior hindbrain (i.e., r3 and r4) appear to be greatly expanded, whereas the posterior hindbrain (r5-r8) is reduced or absent. This work shows that retinoid plays a critical role in patterning, segmentation, and neurogenesis of the caudal hindbrain and serves as an essential posteriorizing signal for this region of the central nervous system in the mammal.

Defects in embryonic hindbrain development and fetal resorption resulting from vitamin A deficiency in the rat are prevented by feeding pharmacological levels of all-trans-retinoic acid.

Vitamin A is required for reproduction and normal embryonic development. We have determined that all-trans-retinoic acid (atRA) can support development of the mammalian embryo to parturition in vitamin A-deficient (VAD) rats. At embryonic day (E) 0.5, VAD dams were fed purified diets containing either 12 micrograms of atRA per g of diet (230 micrograms per rat per day) or 250 micrograms of atRA per g of diet (4.5 mg per rat per day) or were fed the purified diet supplemented with a source of retinol (100 units of retinyl palmitate per day). An additional group was fed both 250 micrograms of atRA per g of diet in combination with retinyl palmitate. Embryonic survival to E12.5 was similar for all groups. However, embryonic development in the group fed 12 micrograms of atRA per g of diet was grossly abnormal. The most notable defects were in the region of the hindbrain, which included a loss of posterior cranial nerves (IX, X, XI, and XII) and postotic pharyngeal arches as well as the presence of ectopic otic vesicles and a swollen anterior cardinal vein. All embryonic abnormalities at E12.5 were prevented by feeding pharmacological amounts of atRA (250 micrograms/g diet) or by supplementation with retinyl palmitate. Embryos from VAD dams receiving 12 micrograms of atRA per g of diet were resorbed by E18.5, whereas those in the group fed 250 micrograms of atRA per g of diet survived to parturition but died shortly thereafter. Equivalent results were obtained by using commercial grade atRA or atRA that had been purified to eliminate any potential contamination by neutral retinoids, such as retinol. Thus, 250 micrograms of atRA per g of diet fed to VAD dams (approximately 4.5 mg per rat per day) can prevent the death of embryos at midgestation and prevents the early embryonic abnormalities that arise when VAD dams are fed insufficient amounts of atRA.

Exencephaly and hydrocephaly in mice with targeted modification of the apolipoprotein B (Apob) gene.

Apolipoprotein B (apoB) is a key structural component of several lipoproteins. These lipoproteins transport cholesterol, lipids, and vitamin E in the circulation. Humans that produce truncated forms of apoB have low plasma concentrations of apoB, beta-lipoproteins, cholesterol, and often vitamin E. This condition has been modeled in mice by targeted modification of the apoB gene. Homozygous transgenic mice display all of the hallmarks of the human disorder. Unexpectedly, approximately 30% of the perinatal homozygotes are exencephalic and of those that have closed neural tubes, approximately 30% are hydrocephalic. The latter condition has also been noted in a relatively small proportion of the heterozygous mice. Vital staining of gestational day 9 (GD9) homozygous offspring has illustrated a striking pattern of excessive cell death involving the alar plate of the hindbrain. Histological and scanning electron microscopic analyses have confirmed this finding. We speculate that varying degrees of affect, as noted among GD 9 and 10 embryos, lead to the spectrum of malformations, including hydrocephaly, present in term fetuses. Analysis of vitamin E deficiency as a possible causative factor has illustrated that homozygous fetuses, indeed, show this deficiency. Amelioration of the defects through alpha-tocopherol supplementation of the maternal diet has been explored. Further analyses of this transgenic mutant promise to provide significant information relative to the role of deficiency of vitamin E and other apoB dependent compounds in dysmorphogenesis.