In situ hybridization on coronal (20 µm thick) and sagittal (30 µm thick) brain sections of P5 wild type and Bhlhb5^lacZ/lacZ (nulls) for subcerebral projection neuron markers, (A1–A4) S100A10, (B1–B4) mu-crystallin and (C1–C4) Crim1, reveals markedly reduced expression of these markers in caudal but not rostral motor cortex (wild type n=3; Bhlhb5-nulls n=3). Scale bars: 500 µm.

In situ hybridization for various area-specific genes on sagittal wild type and Bhlhb5^lacZ/lacZ (null) brain sections. Asterisks delineate area of gene expression change. In Bhlhb5-nulls, there is:
(A) Ectopic expression of Cadherin 8, in layers II/III of somatosensory cortex.
(B) Lack of COUP-TF1 expression in layer IV of somatosensory cortex.
(C) Decreased expression of ephrin-A5 in layers IV/V of somatosensory cortex.
(D) Lack of Lmo4 expression in layer IV of somatosensory cortex.
(E,F) Lack of RORβ expression in layer IV of caudal motor cortex (E), medially (M), but not laterally (L) in somatosensory cortex (F).
(G,H) Lack of Id2 expression in layer IV of caudal motor cortex (G), medially (G), but not laterally in somatosensory cortex (H).
(I) Lack of EphA7 expression in layer IV of occipital cortex.
Ages: P3 (A, C, E, F, G, H, I); P5 (B,D). For each gene, wt n=3; Bhlhb5-nulls n=3. Scale bars: 1 mm.

(A–Q) Genetic lineage axon tracing in Bhlhb5^cre/+; Z/EG/+ heterozygote controls (cre/+) and Bhlhb5^cre/lacZ; Z/EG/+ nulls (cre/lacZ).
(A–D) At P0, the cerebral peduncle appears normal in Bhlhb5-nulls via endogenous GFP fluorescence in side view of whole mount brains (A and C), and via GFP HRP-immunohistochemistry of sagittal brain sections (B and D).
(E–H) At P0, no Bhlhb5-expressing fibers are detected in the medullary pyramidal tract in Bhlhb5-nulls via endogenous GFP fluorescence in ventral views of whole mount brainstem (E and G), or via GFP immunohistochemistry of sagittal brainstem sections (F and H).
(I–M) In adults, very few Bhlhb5-expressing fibers are observed in the medullary pyramidal tracts of 10 of 17 Bhlhb5-nulls via endogenous GFP fluorescence (I and L). Bhlhb5-expressing fibers can be visualized in the other 7 (K) via GFP immunohistochemistry (J and M).
(N–Q) Bhlhb5-expressing fibers are observed in the medullary pyramidal decussation (N and P) but not in the ventral dorsal funiculus of the spinal cord (O and Q) in any Bhlhb5-nulls via GFP immunohistochemistry.
CPD, cerebral peduncle; PT, medullary pyramidal tract; D, decussation; CST, spinal segment of the corticospinal tract. P0 (controls n=4; Bhlhb5-nulls n=10); Adults (controls n=6; Bhlhb5-nulls n=17). Scale bars: 500 µm.

(A and A’) Ventral views of whole mount adult brainstem show dramatic reduction of the medullary pyramidal tract (PT) in Bhlhb5^lacZ/lacZ (null) adults (wild type n=5; Bhlhb5-nulls n=5).
(B–C’) PKCγ immunohistochemistry reveals CSMN axons in the medullary pyramidal decussation (B and B’) but not in the ventral dorsal funiculus of the spinal cord (C and C’), suggesting loss of the spinal segment of the corticospinal tract (CST) in Bhlhb5-null adults (wild type n=5; Bhlhb5-nulls n=5).
(D–G’) DiI anterograde tracing of the CST from P10 rostral motor cortex (wild type n=4; Bhlhb5-nulls n=4). Sagittal views show DiI labeled axons entering (D and D’) the cerebral peduncle, (E and E’) medullary pyramidal tract, and (F and F’) pyramidal decussation, but not in the ventral dorsal funiculus of the spinal cord in Bhlhb5-nulls. Thus, rostral CSMN axons stop at the pyramidal decussation in Bhlhb5-nulls (G and G’; schematics).
(H–K’) DiI anterograde tracing of the CST from P10 caudal motor cortex (wild type n=3; Bhlhb5-nulls n=3). DiI labeled axons enter (H and H’) the cerebral peduncle, but (I and I’) stop at the base of the pons with no fibers entering the medullary pyramidal tract, (J and J’) pyramidal decussation, or ventral dorsal funiculus of the spinal cord. Thus, caudal CSMN axons do not extend beyond the base of the pons in Bhlhb5-nulls (K and K’; schematics).
Insets in: (D,D’,H,H’) show DiI injection site; (E,E’,I,I’) are ventral views of whole mount brainstem.
CPD, cerebral peduncle; PT, medullary pyramidal tract; D, decussation; CST, spinal segment of the corticospinal tract.
Scale bars: 500 µm; Insets not to scale.

(A,A’) HRP-immunohistochemistry for SERT on P7 tangential cortical sections. Thalamocortical afferents segregate into vibrissal specific “hollows”, but with less intense staining and indistinct borders in Bhlhb5-nulls (wild type n=7; Bhlhb5-nulls n=7).
(B,B’) HRP-immunohistochemistry for SERT on P6 coronal sections reveals normal thalamocortical afferents in the internal capsule (black arrowheads), but not in barrel cortex (asterisks) of Bhlhb5-nulls (wild type n=3; Bhlhb5-nulls n=3).
(C,C’) In situ hybridization for RORβ on P5 coronal sections reveals absent expression medially in caudal motor cortex (asterisks), but disorganized expression laterally in the barrel region of the somatosensory cortex (black arrowheads; magnified views in insets) suggesting a post-synaptic disruption of vibrissal barrels in layer IV of Bhlhb5-nulls (wild type n=3; Bhlhb5-nulls n=3).
(D,D’) Nissl staining on tangential sections confirms robust disruption of cytoarchitectonic barrels with only a faint vibrissal pattern in Bhlhb5-nulls (wild type n=3; Bhlhb5-nulls n=3).
(E–G and E’–G’) Confocal microscopy on SERT immunolabeled sections counterstained with Sytox Green nucleic acid stain (Sytox Grn) reveals near absence of septa (white arrowheads) in Bhlhb5-nulls (wild type n=3; Bhlhb5-nulls n=3).
Scale Bars: 250 µm (A,A’), 1 mm (B,B’,C,C’), 100 µm (D–G and D’–G’). Insets in (C,C’) not to scale.

(A) Emx2 is expressed in a high caudal to low rostral gradient in progenitors. Bhlhb5’s post-mitotic high caudal to low rostral gradient at E15.5 resolves into a sharp border between rostral motor and caudal sensory cortices by P0. Post-mitotic neurons inherit areal information specified in progenitors due to radial migration.
(B–C) The same molecular-genetic markers can indicate roles of a regulatory gene in specification or acquisition of area identities.
(B) Cad8 is normally expressed in layers II/III of rostral motor and occipital cortices, but not somatosensory cortex. In Emx2-nulls, a caudal shift of Cad8 expression pattern in the cortical plate (CP) suggests respecificiation of areal identity in a caudal direction, in progenitors (red arrow). In Bhlhb5-nulls, specification of areal identity is unaltered in progenitors (see Figure S5). In Bhlhb5-nulls, disruption of Cad8 pattern in CP (orange arrow) due to ectopic expression in somatosensory cortex (pink) suggests failure in acquisition of one somatosensory property in layers II/III.
(C) Id2 is expressed in layer V of the occipital and caudal motor cortices, but not rostral motor cortex. In Emx2-nulls, a caudal shift of Id2 expression pattern in CP suggests re-specification of areal identity in a caudal direction, in progenitors (red arrow). In CP (orange arrow) of Bhlhb5-nulls, disruption of Id2 expression in caudal motor cortex (but not occipital cortex) suggests a failure in acquisition of one caudal motor property in layer V.
(D–E) Combinatorial codes of regulatory molecules for generating abrupt areal borders of patterned genes in neocortex.
(D) Somatosensory suppression by Bhlhb5 generates Cad8 rostral motor-somatosensory and somatosensory-occipital transitions. Regulators of Cad8 expression in rostral motor and occipital cortex are currently unknown.
(E) Bhlhb5 generates the rostral motor-caudal motor Id2 transition, by controlling Id2 expression in caudal motor cortex. Regulators of Id2 expression in occipital cortex and putative repressors in rostral motor cortex are currently unknown.
M(R), rostral motor cortex; M(C), caudal motor cortex; S, somatosensory cortex; O, occipital cortex; X, Y, Z are undiscovered transcriptional regulators.

(A–J) HRP-immunocytochemistry for Bhlhb5 on coronal brain sections.
(A and B) At E12.5 (A) and E13.5 (B), Bhlhb5 is expressed in the cortical plate (CP) but not the ventricular zone (VZ).
(C and D) At E15.5 (C) and E17.5 (D), Bhlhb5 is strongly expressed in the CP, weakly expressed in the subventricular zone (SVZ), but not in the VZ.
(E–J) Bhlhb5 starts downregulating at the border of neocortex (NCX) and cingulate cortex (Cg) at P0 (E). At P4, downregulation proceeds into NCX anteriorly (F), and becomes more pronounced at the NCX-Cg border posteriorly (G,H). Bhlhb5 starts downregulating throughout the NCX by P7 (I) with markedly reduced expression by P14 (J). Black arrowheads in (E–H) mark extent of downregulation.
(K–M) Bhlhb5 does not colocalize with the S-phase marker BrdU (K; 30 min pulse), or the M-phase marker PH3 (L) in the VZ or SVZ (boxed region in C), but colocalizes with the post-mitotic neuronal marker, Tuj1 (M). Inset in (K) is magnified view of area adjacent to white arrowhead; shows lack of Bhlhb5 and BrdU colocalization in SVZ.
(N) Bhlhb5 is expressed by a subset of glutamatergic neurons.
(O) Bhlhb5 is not expressed by GABAergic interneurons.
LV, lateral ventricle. Scale bars: 250 µm (A–E), 500 µm (F–J), 50 µm (K, L, N and O), 25 µm (M). Inset in (K) not to scale.

(A–D) Triple immunolabeling of Bhlhb5 (A) with CTIP2 (layer V) (B), and Tbr1 (layers II, III, VI and subplate) (C), reveals Bhlhb5 expression in layers II, III, IV, and V at P4.
(E–F) Bhlhb5 is not expressed by a majority of Tbr1+ neurons (layer VI and subplate) at E13.5 (E) and E15.5 (F).
(G) Bhlhb5 is expressed in a subset of CTIP2+ neurons (layer V; more superficial) and layers superficial to the CTIP2-expressing layer at E15.5.
(H–J) In situ hybridization (H), immunocytochemistry (I), and X-Gal staining (counterstained with eosin) (J) on adjacent sagittal sections from a Bhlhb5^lacZ/+ heterozygote brain, reveals faithful recapitulation of Bhlhb5 mRNA and protein expression by Bhlhb5-lacZ reporter.
(K–P) Dorsal views of X-Gal histochemistry on whole mount brains. (K) At E12.5, Bhlhb5 is expressed highest medially in cingulate cortex and weakly in neocortex. (L) At E15.5, Bhlhb5 is expressed in a high caudomedial to low rostrolateral gradient in neocortex. (M,N) The Bhlhb5 gradient transforms into a sharp border (N; arrowhead) between the rostral motor (M) and sensory (S) cortical domains by P0. (O and P) Bhlhb5 expression is further restricted to primary sensory areas at P4 (O) and P7 (P) such as the primary somatosensory cortex (S1) and primary visual cortex (V1).
VZ, ventricular zone; SVZ, subventricular zone; CP, cortical plate; A, anterior; P, posterior; B, vibrissal barrel field. Scale bars: 50 µm (A–G), 500 µm (H–J), 1 mm (K–P).

(A–C) Schematic of the areal distribution of CSMN and CTPN. Medially, along the rostrocaudal axis, fewer CSMN (green) are located in rostral motor cortex (M(R)), more CSMN are located in caudal motor cortex (M(C)), and CTPN (red) are located in occipital cortex (O).
(D and E) Bhlhb5 colocalizes with SCPN marker, CTIP2, at E15.5 but not P4 in caudal motor cortex (arrows).
(F) Microarray expression profiling of Bhlhb5 in purified CSMN shows steep decline in expression between E18 and P14.
(G) Schematic of the genetic lineage tracing strategy to permanently mark cells of Bhlhb5+ lineage via continued GFP reporter production.
(H) Dorsal view of whole mount Bhlhb5^cre/+; Z/EG/+ heterozygote adult brain reveals cumulative spatiotemporal expression of Bhlhb5 with high GFP fluorescence caudally and low fluorescence rostrally in the neocortex. Yellow circle delineates approximate location of caudal motor cortex.
(I and J) Double immunolabeling of GFP (Bhlhb5) and CTIP2 on P5 sagittal section of Bhlhb5^cre/+; Z/EG/+ heterozygote brain. Bhlhb5 is expressed by a majority of presumptive CTPN in occipital cortex, CSMN in caudal motor cortex but not CSMN in rostral motor cortex. (J) Magnified view (arrowhead in I) shows very few GFP+ CTIP2+ neurons in rostral motor cortex.
F, Frontal cortex; P, Parietal cortex, T, Temporal cortex; O, Occipital cortex; M1, primary motor area; M2, secondary motor area; M(R), rostral motor cortex; M(C), caudal motor cortex; S1, primary somatosensory area; S2, secondary somatosensory area; A1, primary auditory area; V1; primary visual area; OB, olfactory bulb; CB, cerebellum. Scale bars: 200 µm (D,E), 1 mm (H), 500 µm (I), 50 µm (J).