In the present study, we show that human cortical interneurons, molecularly defined by the expression of NOS1, NPY, and SST, are either absent or dramatically reduced in the cortex of fetal and infant cases of HPE with severe ventral forebrain (striatal) hypoplasia (HPE-B). Furthermore, we provide evidence that PVALB-positive cortical interneurons, which normally are detected around the time of birth in the human neocortex, might be depleted from late fetal and infant HPE-B brains. However, because PVALB-positive interneurons appear in the cortex after other analyzed subpopulations and only small number of late fetal and infant cases have been analyzed in this study, additional analyses are necessary to substantiate this observation. Nevertheless, these results from late fetal and infant cases are consistent with our findings on NOS1/NPY/SST- and CALB2-positive cortical interneurons in HPE-B cases, as CALB1 is coexpressed by these 2 subtypes of interneurons, and PVALB-positive cortical interneurons arise from the MGE progenitors like NOS1/NPY/SST-positive interneurons. In contrast, CALB2-positive interneurons were present in appropriate locations and significant numbers in the neocortex and hippocampus of all HPE cases, whereas CALB1-positive cortical interneurons were present in late fetal and infant HPE cases, even though in apparently reduced numbers. In addition, we show that Cajal–Retzius neurons, SP neurons, and CP projection neurons are not as dramatically affected in these HPE cases. Thus, our results indicate that development of NOS1/NPY/SST-positive interneurons is consistently and dramatically affected in human HPE with severe striatal hypoplasia.
Several lines of evidence suggests that the subpopulations of cortical interneurons expressing NOS1(NADPH-d)/NPY/SST are most likely lost in the HPE-B cases. First, all 3 of these molecular markers are concomitantly absent from the cortex of HPE-B brains. Second, their expression in the few remaining interneurons of the hypoplastic ventral forebrain or at the corticostriatal border is observed. And third, their absence correlated consistently with ventral forebrain hypoplasia. It is possible, though less likely, that cortical interneurons are present in the HPE-B cortex, but have simultaneously lost NOS1, NPY, and SST expressions. A more likely scenario is that the ventral forebrain hypoplasia in these cases has led to decreased generation and disrupted cortical migration of this subpopulation of interneurons.
The consistent depletion of NOS1/NPY/SST-positive cortical interneurons in all fetal and infant HPE-B cases with severe striatal hypoplasia was concomitant with the dramatic reduction in ventral forebrain cells expressing TITF1
, whose mouse homolog is expressed by MGE progenitors and MGE-derived NOS1/NPY/SST interneurons (Wonders and Anderson 2006
; Fishell 2007
). Furthermore, these defects are reminiscent of those found in Titf1
-deficient mice, which lack this subtype of striatal and cortical interneurons but not CALB2-positive cortical interneurons (Pleasure et al. 2000
). Interestingly, the human TITF1
homolog has been suggested as a candidate gene for HPE (Devriendt et al. 1998
) and its haploinsufficiency causes benign hereditary chorea, a movement disorder associated with abnormal striatal interneurons (Breedveld et al. 2002
). In contrast, cells expressing DLX1/2
, markers of different interneuronal progenitor cells, were readily observed throughout the ventral and dorsal forebrain of all HPE and control midfetal cases, indicating that these putative interneuronal progenitors were less selectively affected in the same HPE cases. Interestingly, in mice, Dlx1/2
are necessary for the development of Calb2
-expressing cortical interneurons (Anderson et al. 1997
; Xu et al. 2004
), which in our HPE cases are not dramatically affected by severe ventral forebrain midline and striatal hypoplasia. Thus, our results show that NOS1/NPY/SST-positive cortical interneurons and related TITF1-positive progenitors are selectively and consistently more affected in HPE with ventral forebrain (striatal) hypoplasia. Furthermore, these findings are consistent with the possibility that at least a vast majority of NOS1/NPY/SST-positive interneurons are generated by TITF1-positive progenitors in the ventral forebrain and migrate dorsally into the cortex. Finally, this depletion of cortical inhibitory interneurons provides a possible pathophysiological mechanism for motor deficiencies and seizures often associated with HPE and characterize human HPE with severe ventral forebrain hypoplasia as a developmental “interneuronopathy” (Kato and Dobyns 2005
The results of this study offer insights into the developmental origins of different cortical interneuron cell types. Consistent with previous findings (Judas et al. 1999
; Ulfig 2002
; Meyer 2007
), we showed that molecularly defined cortical interneurons exhibit a distinct temporal sequence in their generation, migration routes, and differentiation during early human fetal development. For example, immature CALB2-positive interneurons with a migratory morphology were numerous in the lateral and caudal ganglionic eminences, paleocortical periventricular zone, and the subpial granular layer/MZ, and the subventricular zone (SVZ), the cortex (Meyer 2007
; data not shown). In contrast, immature NOS1/NPY/SST-positive neocortical interneurons with a migratory morphology first appear around 15 wg in the neocortical SP and are scarce in the MZ or cortical SVZ (Judas et al. 1999
; data not shown). These differences in the spatial distribution and molecular differentiation of these 2 main subtypes of human cortical interneurons are accompanied with differential distribution of markers associated with distinct subtypes of interneuronal progenitors. As previously described in the midfetal human and cynomolgus monkey, numerous ASCL1-positive putative progenitor cells and CALB2-positive interneurons are present in the dorsal pallial (cortical) proliferative zones (Letinic et al. 2002
; Zecevic et al. 2005
; Petanjek et al. 2008
). Interestingly, our analysis revealed that ASCL1-positive cells were present in all HPE brains including those lacking cortical NOS1/NPY/SST interneurons. This finding is consistent with the possibility that at least a portion of human cortical interneurons expressing CALB2
is derived from neocortical ASCL1 (MASH1)-positive progenitors as previously described (Letinic et al. 2002
). However, whether these ASCL1-positive progenitors, which in mice are known to give rise to both neuronal and glial cells (Kim et al. 2008
), give rise to interneurons in human within the cortical proliferative zones during midfetal ages remains to be determined. Importantly, the correlation between the selective absence of a particular subtype of cortical interneurons and the disruption of TITF1-positive MGE progenitors in human HPE with severe ventral forebrain hypoplasia, together with the spatial and temporal differences in localization and differentiation of molecularly defined cortical interneurons in normal human fetal brain, suggests that different interneuron subtypes are derived from distinct progenitors.
Our results also provide insights into how different interneuron cell types may become more abundant during the development of the human cortex. Humans and other primates have a higher proportion of neocortical GABAergic neurons compared with rodent species (Gabbott and Bacon 1996
; DeFelipe et al. 2006
). Several studies indicate that this higher proportion of GABAergic interneurons is accompanied with an increase in molecular and morphological diversity and complexity of cell types (Lewis and Lund 1990
; Hof et al. 2000
; Preuss and Coleman 2002
; DeFelipe et al. 2006
). Though not specific to primates, CALB2
-expressing double bouquet interneurons and tyrosine hydroxylase–expressing interneurons are especially abundant in the human neocortex, where they are thought to modulate the activity of a large variety of circuits (DeFelipe et al. 2006
; Benavides-Piccione and DeFelipe 2007
). Our findings are consistent with the possibility that developmental increases in cell number and spatial extension of specific interneuron progenitor pools, such as ASCL1-positive cells, have led to the selective amplification and dispersion of CALB2-positive, and perhaps other, interneurons in the developing human cerebral cortex.
Taken together, our findings indicate that the diversity of human cortical interneurons is established early during neurogenesis, with distinct subpopulations originating from spatially, temporally, and molecularly segregated pools of progenitors. Furthermore, our results are consistent with the possibility that the dorsal expansion of cortical CALB2-positive interneurons and the associated ASCL1-positive progenitor pool might contribute to the increased diversity of cortical interneurons and the formation of more elaborate cortical circuits in humans.