Genesis of teratogen-induced holoprosencephaly in mice

Abstract

Evidence from mechanical, teratological, and genetic experimentation demonstrates that holoprosencephaly (HPE) typically results from insult prior to the time that neural tube closure is completed and occurs as a consequence of direct or indirect insult to the rostral prechordal cells that induce the forebrain or insult to the median forebrain tissue, itself. Here, we provide an overview of normal embryonic morphogenesis during the critical window for HPE induction, focusing on the morphology and positional relationship of the developing brain and subjacent prechordal plate and prechordal mesoderm cell populations. Subsequent morphogenesis of the HPE spectrum is then examined in selected teratogenesis mouse models. The temporal profile of Sonic Hedgehog expression in rostral embryonic cell populations and evidence for direct or indirect perturbation of the Hedgehog pathway by teratogenic agents in the genesis of HPE is highlighted. Emerging opportunities based on recent insights and new techniques to further characterize the mechanisms and pathogenesis of HPE are discussed.

Figure 1

A scanning electron micrograph (a, dorsal view) and a histological section (b) illustrate the normal cranial morphology of Hamburger and Hamilton (H&H) Stage 7 and 8 chick embryos, respectively. The dashed line in (a) illustrates the location of the sagittal section in (b). Subjacent to the developing forebrain (FB) and midbrain (MB) are columnar cells comprising the prechordal plate [PP, which is continuous with the foregut endoderm (FGE)] and mesenchymal (fibroblastic/non-epithelial) cells comprising prechordal mesoderm (PM), respectively. Caudal to the latter is the notochord (N), which underlies the hindbrain (HB). FG=foregut.

Figure 2

Scanning electron micrographs illustrate the morphology of gestational day (GD) 7-7.5 (Theiler Stage 10, a-d; and 11, e-j) mouse embryos. As shown in (a) and at higher magnification in (b), at the tip of the ventral side of the cup-shaped embryo, cells having a relatively small surface area are intercalated into the endodermal layer. Small groups of these cells extend along the anterior midline of the embryo (arrows in b). Slightly later in development, these cells, which at higher magnification than shown, can be identified by the presence of a prominent mono-cilium, populate the anterior midline (arrows in c). A sagittal cut through an embryo of this stage (d) illustrates its cup-shaped form, with the ectodermal layer (including the neuroepithelium) lining the interior, and the amnion (open arrow) separating it from the extraembryonic components. The antero-ventral midline is indicated by arrows. The primitive steak and node (}) are located in the caudal midline. By GD 7.5, the position of the prechordal plate is notable on the ventral side of the embryo as a circular area (dashed arrow in e and white circle in f) that is located rostral to the notochoral plate (more caudally-positioned arrows). At this time, Shh is expressed in the cells of the antero-ventral midline, as indicated by the dark grey area in the schematic in (f), while gooscoid is expressed in the prechordal plate (white circular region in f). Shown in (g) is an embryo that was cut at the level of the prechordal plate (dashed line in e). Illustrated is the columnar neuroepithelium of the forebrain (FB) and its proximity, in the midline, to the prechordal plate (dashed arrows). A dorsal view of an embryo from which the amnion has been removed (h), shows the neural plate occupying the majority of the anterior half [region inside the dashed circle, including the forebrain (FB)]. As shown in (i), a midsagittal cut illustrates the relationships of the notochordal plate (solid arrows), the prechordal plate (dashed arrow), amnion (open arrow) and forebrain region (FB). At this developmental stage, mesodermal cells continue to be laid down as cells ingress through the primitive steak and node (}). Removal of the ventral cell layer on the left side of an embryo, including removal of the prechordal plate from the region shown by a dashed arrow, illustrates the subjacent cardiac and cranial mesenchyme (M) (j).

Figure 3

Scanning electron micrographs illustrate the morphology of mouse embryos at Theiler stage 12, when the embryos are at approximately 8 days of gestation, and when 1-7 pairs of somite pairs are evident. The embryos in (a & b) are younger than that in (c). In the former the optic sulci (arrows in c) are not yet visible in the developing forebrain (FB) region of the neural plate. As shown in (b), removal of the epithelium of the left side of the developing brain, including that in the rostral midline, illustrates the subjacent prechordal plate (dashed arrow) and mesenchyme (M). In (c) the portion of the cranial neural plate that is the developing diencepahlon is located between the two dashed black lines. The prechordal plate is subjacent to the median aspect of this brain segment, as indicated by the dashed white shape. T = telencephalon.

Figure 4

Images of H&H 10-11 chick (a-c) and Theiler stage 14-15 (GD 9-9.5) mouse embryos (d-i) illustrate relationships between the developing brain and subjacent tissues at the time of anterior neural tube closure (a-f) and shortly thereafter (g-i). In the chick, a sagittal cut through the anterior neuropore (open arrow) illustrates the notochord (arrowheads in b). At higher magnification, as shown in (c), the prechordal mesoderm (PM) can be identified rostral to the notochord; underlying the median diencephalon (D) is the prechordal plate (PP); the telencephalon (T) is rostral to the PP; and the ectoderm of Rathke's pouch (the progenitor of the anterior pituitary; arrow) is continuous with that of the buccopharyngeal membrane (dashed circle). The inner layer of the buccopharyngeal membrane is continuous with the endoderm of the foregut (FG). In the mouse, at the time of anterior neuropore closure, while the developing forebrain is more ventrally positioned with respect to the remainder of the neural tube than in the chick, the overall morphology in these species is comparable (d-f). By the time that 3 pharyngeal arches (I, II, III) are clearly identifiable in the mouse, a midsagittal view illustrates that the PP can no longer be defined (g-i). Arrowheads in i = notochord; E= eye.

Figure 5

Ethanol-induced dysmorphology in GD 9.5 and 11 mouse embryos following acute ethanol exposure on GD 7. Frontal views of the face of control (a, b) and ethanol-exposed embryos (d, e; g, h), along with views of the forebrain interior of the embryos in b, e, and h (c, f, and i) illustrate diminished median tissue in the affected embryos, with median apposition of the olfactory placodes/nasal pits (dashed circle) and ganglionic eminences (arrows) (d-i). Modified from [Sulik and Johnston, 1983; Sulik, 1984]

Figure 6

Ethanol-induced dysmorphology in GD 14 mice following acute ethanol exposure on GD 7, and corresponding human phenotypes. Illustrated are children with Fetal Alcohol Syndrome (b), cebocephaly (d), and median cleft lip (f), whose features are represented in the spectrum of defects induced by acute maternal ethanol exposure in mice (a, c, d). Modified from [Sulik et al., 1988].

Figure 7

Retinoic acid-induced dysmorphology in mouse embryos. As compared to control embryos (a, d), those whose mothers are treated with retinoic acid on GD 7 present with defects consistent with those shown in Figs. 5 & 6. In addition, retinoic acid exposure causes mandibular hypoplasia (M; b, c, e), and forebrain/ midface deficiencies that are severe enough to result in proboscis formation (representing median union of the lateral nasal prominences; arrow in c), or complete absence of facial features (with the exception of remaining maxillary tissue, open arrow) and aprosencephaly (e). Bars in a-c = 10μm. Modified from [Sulik et al., 1995].

Figure 8

Ochratoxin A (OA)-induced dysmorphology in mouse fetuses. Illustrated are the holospheric forebrain (✳) in a cebocephalic fetus (a, b), extreme hypotelorism (c), and synophthamia (d), all of which followed acute maternal OA treatment on the 8th day of pregnancy. Note that the snout of each of these fetuses has several rows of hair follicles (arrows), which are indicative of maxillary prominence origin. Bars in a-d = 500μm. Modified from [Wei and Sulik, 1993].

Figure 9

Cholesterol-deficiency-induced dysmorphology in mouse embryos. Comparison of a sagittal cut through the head of a control (a-c; b & c are reciprocal halves), and an affected embryo (d-f; f & g are reciprocal halves) illustrates deficiency in the forebrain, i.e. the portion of the brain that is rostral to the mesencephalic flexure (black dashed arrow), a narrow aqueductal isthmus (white dashed arrow), and a thickened hindbrain floor (⋆) in the latter. Bars in a, b, d, e = 5mm; in c, f = 100μm. Modified from [Lanoue et al., 1997].

Figure 10

Anencephaly/HPE in the mouse and human. As shown in a mouse fetus with cholesterol deficiency-induced dysmorphia (a), and in a child with a similar presentation (b), facial features typical of HPE may be accompanied by anencephaly/ rostral neural tube closure failure (arrow). The arrowhead in (a) indicates a single nostril.

Figure 11

Cyclopamine-induced dysmorphology in mouse fetuses and corresponding human phenotypes. A wide spectrum of dysmorphology including cebocephaly (a) and cleft lip (b, c) result from drug exposure initiated on GD 8.5. Notably, while the fetuses in b & c and the corresponding children present with bilateral cleft lip (e & f), the size of the intermaxillary segment (prolabium; arrows) differs. In the cebocephalic child shown in (d), although the lip is closed, there is no intermaxillary segment. Modified from Lipinski et al, submitted.

Figure 12

Cyclopamine-induced CNS dysmorphology in fetal mice. As compared to frontal (coronal) histological sections made at the level of the eyes and pituitary in a control GD 16.5 fetus (a, b), abnormalities including union of the frontal lobes of the brain (open arrow in c) and complete pituitary aplasia are notable in a sections from the cebocephalic fetus shown Fig. 11 a (c, d), while a cleft palate (arrow in e), anterior pituitary agenesis and persistent posterior pituitary can be seen in sections from the fetus shown in Fig. 11c (e, f). Modified from Lipinski et al, submitted.

Figure 13

Reconstructed high resolution magnetic resonance images illustrate normal brain morphology (a,b) and HPE (c-f) in fetal mice. The specimen shown in a dorsal and frontal view in (c, d), respectively, is asymmetrically affected. As in this specimen, that shown in (e, f) presents with rostral union of the cerebral hemispheres, while the more caudal aspects of the brain appear relatively normal. Pink = olfactory bulbs, Red = cerebral hemispheres, Light green = diencephalon, Magenta = midbrain, Dark green/teal = cerebellum, Medium green = myelencephalon. Modified from [Godin et al., 2010].

Figure 14

Cerebral cortical heterotopias in fetal mice with holoprosencephaly. Histological sections of the cebocephalic fetus whose face is reconstructed from magnetic resonance images and shown in (a) illustrate cerebral cortical leptomeningeal heterotopias (arrows in b). The boxed area in (b) is shown at higher magnification in (c).