Introduction

Anencephaly and exencephaly result from failure of anterior neural tube closure. While the term exencephaly literally means “an outside brain” and anencephaly refers to “the absence of brain,” these terms are often used in an interchangeable manner.1 In exencephalic fetuses, the cranial vault is not present and the neural tissue that is in contact with the external environment presents as an amorphous structure very rich in vessels, known as “area cerebrovasculosa,” that includes structures derived from the forebrain and skull.2 Some have reported that the area cerebrovasculosa comprises vascular tissue, glia, and few neurons surrounded by meninges.3,4 Others have described the presence of cavities surrounded by epithelium that we interpret as ventricles.4 To the best of our knowledge, the presence of the progenitor/stem cells has not been investigated previously. The lack of cortical precursor cells in exencephaly could explain the substantial lack of cortical tissue. Conversely, the presence of precursor cells in early gestation exencephaly would suggest that the potential to form a proper cortex was present. Establishing the extent of cortical precursor cells in a case of exencephaly will open new avenues for understanding the etiology of the exencephalic phenotype. Here we provide the first study on the progenitor cell cellular composition of cortical proliferative zones in area cerebrovasculosa of an exencephalic fetus.

Methods

Human samples

Tissue specimens were obtained from UC Davis and Shriners hospitals through consented autopsies with institutional review board approval.

Tissue preparation and immunostaining

We dissected out the area cerebrovasculosa from a female 17-week gestation fetus and from a control female 17-week gestation fetus, fixed them in 4% paraformaldehyde, and cryo-protected them in 30% sucrose in 0.1 M phosphate-buffered saline at 4 °C. The samples were coronally sectioned on a Leica cryostat at 16 mm and mounted on coated glass slides. Evenly distributed sections from exencephaly case were stained with cresyl violet (Nissl staining) to reconstruct the antero–posterior morphology. Enzymatic immunohistochemical staining was done after heat-mediated antigen retrieval (8 min at 110 °C) by placing the sections in blocking solution (10% donkey serum (ThermoFisher) and 0.1% Triton X-100 (#9002-93-1, Millipore) for 1 h at room temperature. Sections were then incubated overnight at 4 °C with the following primary antibodies: glial fibrillary acidic protein (GFAP; rabbit polyclonal 1:400, Z0334 Agilent), homeodomain-only protein homeobox (HOPX; mouse monoclonal 1:100, SC398703 Santa Cruz), IBA1 (rabbit polyclonal 1:500, 019-19741, Wako), Ki67 (rabbit polyclonal 1:300, ab15580 Abcam), microtubule-associated protein 2 (MAP2; rabbit polyclonal 1:100, ab5622 Millipore), OLIG2 (rabbit polyclonal 1:100, ab9610 Millipore), PAX6 (rabbit polyclonal 1:300, PRB-278P Biolegend), S100b (rabbit polyclonal 1:300, ab52642 Abcam), and TBR2 (chicken polyclonal 1:300, ab15894 Millipore). Sections were then incubated with the appropriate secondary antibody (biotin-conjugated donkey anti-rabbit, anti-mouse, and anti-chicken, 1:150, 711-066-152, 715-066-150, and 703-065-155, respectively, Jackson), amplified with avidin–biotin complex and developed with DAB substrate (SK-4105, VectorLab). In case of IBA1-PAX6 double immunostaining, alkaline phosphatase secondary antibodies (donkey anti-rabbit, goat anti-mouse, 1:150, 711-056-152, 711-055003, respectively, Jackson) were used to develop with VectorBlue (SK-5300, VectorLab). Nissl, Hematoxylin/Eosin (H&E), alizarin red for bone, and alcian blue staining were performed following the standard protocols. All sections were dehydrated and coverslipped with Permount (SP15-500, ThermoFisher) and stored at room temperature. Brightfield images were taken on an Olympus microscope equipped with high-resolution camera, using a ×40 oil objective, as Z-stacks of an average of four planes (1.8 μm spaced).

Statistical analysis

Z-stack projections of 1.8-μm-step Z-stacks images were created with the StackFocuser plugin and analyzed via Cell Counter plugin on the FIJI software. Three different cortical medio-lateral areas were quantified (from three different sections), and obtained data were averaged and standard deviations and standard errors were calculated. Quantified cells (control and exencephalic, respectively) are as follows: PAX6+ in the ventricular zone (VZ) = 255, 157; PAX6+ in the subventricular zone (SVZ) = 1475, 728; HOPX+ in VZ = 122, 135; HOPX+ in SVZ = 2182, 691; TBR2+ = 779, 151; MAP2+ = 344, 272; S100b+ = 43, 134; OLIG2+ = 50, 82. Results were compared among exencephaly case and a control 17-week fetus using t test. Statistical significance was set at 0.05.

Results

We obtained a 17-week-old fetus diagnosed as an anencephaly case. However, we determined that this case better fit the description for exencephaly because of the presence of an exposed anterior neural tube structure. This suggests the possibility that some cases that had been diagnosed as anencephaly may have been exencephaly. The fetus lacked the skull, eyes were prominent, and it lacked the forehead (Fig. 1b). The body and limbs were well formed. The area cerebrovasculosa that was dark red and resembled a blood coagulum appeared to be divided into two hemispheres. We extracted the tissue (Fig. 1c), cryopreserved, cut into 14-μm coronal sections, and stained a series of equidistant slides with Nissl to label all cells (Fig. 1a, d–h), H&E for erythrocytes, alizarin red for bone, and alcian blue for cartilage. The area cerebrovasculosa contained brain tissue that was surrounded by a collection of blood cells, rudimentary cartilage, and a reduced number of low-mineralized bone cells. The area cerebrovasculosa was approximately half the size, and the prosencephalon was one third the size of the same structure in age-matched control 17-week gestation fetal brain (Fig. 1d, g). The third ventricle was opened but the midbrain architecture appeared mostly preserved. The spinal cord appeared anatomically normal. The developing telencephalon contained two perceptible lateral ventricles and what appeared to be a ganglionic eminence and cerebrocortical tissue. The ventricles were closed at some points along the rostro-caudal axis but open at other levels. The developing cerebral cortex was very thin in the radial dimension but expanded in the tangential dimension. There were degenerating/dying cells in the tissue, primarily away from the ventricular cavity. The ventricular surface was undulated in some regions, and the perimeter of the lateral ventricles was increased (Fig. 1g).

Fig. 1: Exencephaly brain anatomy.
figure 1

a Coronal sections of the exencephaly brain, section interval 352 μm. b Exencephalic fetus. Dark gray = area cerebrovasculosa. c Area cerebrovasculosa. Top R rostral, C caudal, L left, R right. Arrowhead indicates midline. d, e Nissl-stained coronal section and reconstruction of an age-matched 17-week gestation cortex. f Nissl stain of inset shown in d. g Nissl staining of section in “a2”. h Nissl stain of inset in f. iz intermediate zone, cp cortical plate. Arrowheads = midline. Scale bars: a 3 cm, b, c 1 cm, d 2 mm, f 200 μm, g 1 mm, h 50 μm.

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Immunostaining for GFAP, a radial glia precursor cell (RGC) marker, revealed atypical, discontinuous RG fibers that coursed toward the pia (Fig. 2a, b).5 We found no M-phase cells at the ventricular surface and small number of M-phase cells away from the ventricle. However, there were Ki-67+ cells both near and away from the ventricle (Fig. 2j). The presence of Ki-67+ cells but lack of M-phase cells suggest that neural precursor cells were in the cell cycle but arrested in a stage such as G1 or G2. We quantified and compared the number of precursor cells in an area that was 1-mm wide parallel to the ventricular surface and that extended radially through the thickness of the neural tissue of both the exencephaly and age-matched control cases. This approach allowed us to compare the density of cell types within radial segments of the cortical tissue across cases. Three different cortical medio-lateral areas were quantified, and data compared among exencephaly case and a control 17-week fetus using t test. Statistical significance was set at 0.05. The number of PAX6+ cells (RGCs) was decreased by 38.4% in the VZ (p < 0.03) and by 50.6% in the SVZ (p < 0.02) of the exencephaly case compared to control (Fig. 2c–f, u’). The number of HOPX+ cells (RGCs) was increased by 24.7% in the VZ (p < 0.01) but decreased by 59.4% in the SVZ (p < 0.001, Fig. 2u’). We also compared the ratio of precursor cells in the SVZ and VZ (SVZ/VZ) and found that the ratio of PAX6+ cells was decreased by 19.7% (p < 0.03) and the ratio of HOPX+ cells was decreased by 71.5% (p < 0.01) in the exencephaly case compared to the control (Fig. 2u’’). These data are consistent with a general decrease in the number of precursor cells that was especially prominent in the SVZ. Consistent with this concept, we found that, while Tbr2+ cells were present and appropriately positioned superficial to the dense band of PAX6+ cells near the ventricle, the number of TBR2+ cells (intermediate progenitor cells (IPCs)) were decreased by 80.6% (p < 0.0001, Fig. 2g, h, u’). There was not an identifiable cortical plate, but MAP2+ neurons, S100b+ astrocytes, and OLIG2+ oligodendrocytes were present and intermingled in the SVZ and a potential intermediate zone. The number of MAP2+ neurons was also decreased by 20.9% (p < 0.003), the number of S100b+ astrocytes was increased by 246.6% (p < 0.02), and the number of OLIG2+ oligodendrocytes did not change (Fig. 2u’’’). We found that IBA1+ microglial cells were present in the proliferative zones, but these cells were largely amoeboid and lacked the complex morphology of microglia in the normally developing forebrain (Fig. 2q, r).6,7,8 IBA1+ cells had enveloped and phagocytosed precursor cells in the proliferative zone of the exencephaly case, as in the normally developing cortex (Fig. 2s, t).6,7,8

Fig. 2: Progenitor and neural cell identity in the cerebral cortex of exencephaly and age-matched control feti.
figure 2

a, b RGC (GFAP). c, d RGC (PAX6) e, f RGC in SVZ (HOPX). g, h IPC (TBR2). i, j Proliferative cells (Ki67), in inset prophase mitosis in SVZ (Nissl). k, l Neurons (MAP2) are indicated with an arrowhead. m, n Astrocytes (S100b). o, p Oligodendrocytes (OLIG2). q, r Microglia (IBA1). s Microglia in the proliferative zones (IBA1, brown)/Pax6+RGC (blue). t Microglia in the VZ (IBA1, blue)/Pax6+RGC (brown). Lower left panel: cellular processes of a microglia extending into the ventricle. u’ Number of cells expressing cell-specific precursor cell markers. u’’ Ratio of cells expressing PAX6 and HOPX in the SVZ/VZ of exencephalic (dark gray) and control (light gray). u’’’ Number of cells expressing cell phenotype markers in exencephalic (dark gray) and control (light gray) cortex. an. exencephaly brain, vz ventricular zone, svz subventricular zone, cp cortical plate. Scale bars: c, e, g, q, s = 200 μm; r = 40 μm, t = 20/40 μm, others = 100 μm. * means p value <0.05.

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Discussion

The combined evidence showing an undulated ventricular surface, a decreased ratio of SVZ/VZ precursor cells, a decreased number of TBR2+ cells, and a decreased number of neurons suggest a lack of neuronal differentiation or an interruption in the normal sequence of neural differentiation. The data showing a marked lack of M-phase cells indicates cell cycle arrest and is consistent with an interruption of cortical development. The data collected from this case suggest that the underlying condition did not initially preclude RGCs from undergoing the asymmetric divisions that produce TBR2+ IPCs9,10 but halted developmental progression. This reduced the number of precursor cells and severely cut production of cortical neurons. The reduced size of cortical tissue is also consistent with a marked reduction of symmetric proliferative divisions that would normally expand the precursor cell pool early in development.11 The increased number of S100b+ astrocytes and amoeboid microglial cells may represent gliosis in response to the abnormal proliferative environment in this case. The excessive cell death we observed may have resulted from a lack of proper signaling,12 such as a failure of bone morphogenetic or Sonic hedgehog pathways or other as yet unidentified factors. Nevertheless, the presence of PAX6+ and TBR2+ cortical precursor cells in the area cerebrovasculosa demonstrate that the fundamental building blocks of cortical formation had been established and that a normal sequence of developmental steps had been initiated early in this case.

Our data demonstrate that primary and secondary cortical precursor cells are present in exencephalic cortex at 17-week gestation. Thus our data are consistent with the concept that the lack of cortical tissue in exencephaly does not result exclusively from failure of neural tube closure but also from altered precursor cell differentiation. On the other hand, the abnormalities in precursor cells described may be consequence of gross geometrical abnormalities evoked by a classical neural tube closure defect. Thus cortical precursor cells in exencephalic cases, as presented here, are present and arranged in an appropriate laminar fashion, yet lack the capacity to properly generate the cellular constituents of cerebral cortex. These data do not rule out effects derived from classical neural tube closure defect, but they rather expand our understanding of the etiology of exencephaly, highlight the status of cortical progenitor cells that may be linked to the disorder, and will initiate new avenues of research into the role of progenitor cells in failure of neural tube closure in human.